Accepted Manuscript

A review on injectable chitosan/beta glycerophosphate hydrogels

for bone tissue regeneration

Sekaran Saravanan, Selvaraj Vimalraj, Palanisamy Thanikaivelan,

Sivanantham Banudevi, Geetha Manivasagam

PII: S0141-8130(18)33182-9
DOI: doi:10.1016/j.ijbiomac.2018.10.014
Reference: BIOMAC 10665
To appear in: International Journal of Biological Macromolecules
Received date: 26 June 2018
Revised date: 20 September 2018
Accepted date: 1 October 2018

Please cite this article as: Sekaran Saravanan, Selvaraj Vimalraj, Palanisamy
Thanikaivelan, Sivanantham Banudevi, Geetha Manivasagam , A review on injectable
chitosan/beta glycerophosphate hydrogels for bone tissue regeneration. Biomac (2018),
doi:10.1016/j.ijbiomac.2018.10.014

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 ACCEPTED MANUSCRIPT

A Review on Injectable Chitosan/Beta glycerophosphate Hydrogels for Bone Tissue

Regeneration

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Sekaran Saravanan1, , Selvaraj Vimalraj2, Palanisamy Thanikaivelan3, Sivanantham Banudevi1,

and Geetha Manivasagam4

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1- Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), Department
of Bioengineering, School of Chemical and Biotechnology, SASTRA University,

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Thanjavur- 613 401, Tamil Nadu, India.

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2- Department of Biotechnology & AU-KBC Research Centre, Madras Institute of
Technology (MIT), Anna University, Chrompet, Chennai - 600 044, Tamil Nadu, India.
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3- Advanced Materials Laboratory, Central Leather Research Institute (Council of
Scientific and Industrial Research), Adyar, Chennai- 600 020, India.
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4- Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore, India

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*- Correspondence

S. Saravanan, Ph.D.
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Assistant Professor
Centre for Nanotechnology & Advanced Biomaterials (CeNTAB)
Department of Bioengineering
School of Chemical and Biotechnology
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SASTRA University
Thanjavur, Tamil Nadu 613401

Email: saravanan@scbt.sastra.edu; ranklopg@gmail.com

Phone: +91-9080633567

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Highlights

 Chitosan-β-GP hydrogel system is injectable and with gelation at 37°C.

 Chitosan-β-GP hydrogels are widely explored for release of bioactive molecules.

 Hybrid or modified form possesses enhanced bioactive and mechanical properties.

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Abstract

Tissue engineering (TE) is a promising approach for repairing diseased and damaged

bone tissue. Injectable hydrogel based strategies offer a wide range of applications in

rapid recovery of bone defects by acting as filler materials and depots for delivering

various bioactive molecules and averting the need for surgical intervention. Chitosan (CS),

a natural polysaccharide, forms a thermosensitive injectable hydrogel through the addition of

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beta-glycerophosphate (β-GP). This hybrid hydrogel possesses numerous advantages namely

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mimicking native extracellular matrix (ECM) and providing an amenable microenvironment

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for cell growth. In this review, a brief insight into the gelation mechanism of CS/GP

hydrogels, modifications, bioactive additives and their applications in treating bone defects
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are presented.
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Keywords: Chitosan, beta-glycerophosphate, thermoresponsive hydrogel, bone, tissue

engineering.
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List of Abbreviations

3D- Three dimensional

ADSCs- Adipose derived stem cells

ALN- Alendronate

ALP- Alkaline phosphatase

antimiR- Anti-microRNA

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ASC- Acid soluble collagen

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BBS- Bovine bone substitutes

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BG- Bioglass

BMP-2- Bone morphogenetic protein-2

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BMSCs- Bone marrow stem cells

BSA- Bovine serum albumin

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BSP- Bone sialoprotein

BTE- Bone Tissue Engineering

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Ca- Calcium
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CHC- Carboxymethyl-hexanoyl chitosan

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CMC- Carboxymethyl chitosan

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CNT- Carbon nanotubes.

COL-1- collagen type 1

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CP- Calcium phosphate

CS- Chitosan

CS NP’s- Chitosan nanoparticles

CS-TBA- Chitosan-4-thio-butylamine

Dex- Dexamethasone

DNA- Deoxy ribose nucleic acid

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E.coli- Escherichia coli

ECM- Extracellular matrix

FDA- Food and Drug administration

FGF- Fibroblast growth factor

f-MWCNTs- Functionalized multiwalled carbon nanotubes

GA- Gallic acid

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GAGs- Glycosaminoglycans

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GP- Glycerophosphate

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HAp- Hydroxyapatite

hBMSCs- Human Bone marrow stem cells

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hDPSCs- Human dental pulp stem cells

HEPM- Human embryonic palatal mesenchymal cells

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miRNA- MicroRNA

MSCs- Mesenchymal stem cells

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Na-GP- Sodium glycerophosphate

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nBGC- Nano bioglass ceramics

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nHAp- Nanohydroxyapatite
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OCN- Osteocalcin

OM- Osteogenic medium

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OP- Osteopontin

OSX- Osterix

PCL- poly-ε-caprolactone

pDNA- Plasmid DNA

PEI- Polyethyleneimine

PEO/PLGA/PEO- Poly(ethylene oxide)-b-poly(D,L-lactic acid-co-glycocylic acid)-

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poly(ethylene oxide)

PEO/PPO/PEO- Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)

PG- Phloroglucinol

PLA- Poly-lactic acid

PLGA- poly(D,L-lactic acid-co-glycocylic acid)

PMMA- Polymethylmethacrylate

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PNIPAAM- Poly-N-isopropylacrylamide

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rBMSCs- Rat bone marrow derived stem cells

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rh-BMP-2- Recombinant human bone morphogenetic protein-2

Runx2- Runt-related transcription factor 2

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S.aureus- Staphylococcus aureus

SBF- Simulated body fluid

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SDF-α- Stromal cell derived factor alpha

TCP- Tricalcium phosphate

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TE- Tissue Engineering

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TMC- Trimethyl chitosan

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WS- Wollastonite
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Zn- Zinc

β-TCP- Beta tricalcium phosphate

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1. Introduction

Bone is a highly dynamic connective tissue that forms the structural framework of the

body and is involved in locomotion, mineral homeostasis and protection of internal organs.

Most cases of bone trauma destroy the natural bone healing ability resulting in functional

and structural aberrations thereof [1]. Biomaterial-based tissue engineering approach

upholds excellent promise for treating bone loss over the conventional gold standard

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method of autografting and other metallic prostheses. Preformed scaffolds and injectable

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hydrogel matrices are the principle components of bone tissue engineering (BTE)

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encompassing cell-based and cell-free strategies [2–6]. Hydrogels are crosslinked three

dimensional (3D) polymeric networks that possess a high density of hydrophilic groups with
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the affinity for water and capable of absorbing a significant amount of water. They are

marked with the remarkable properties including swelling, permeation, surface

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characteristics and ease for tuning physicochemical and biological properties [4,7]. Natural

and synthetic polymers are employed in the generation of several hydrogels with defined
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properties. Hydrogels responding to the temperature are known as thermosensitive

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hydrogels. They are of great interest due to the gelation at body temperature, which
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presents large applications including carrying cells, thermolabile drugs, and bioactive
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molecules and can fill into any irregular shaped bone defects. Natural polymers exhibit

more significant advantages over synthetic polymers including high biocompatibility and
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structural resemblance with natural bone components [7,8]. CS, a natural polymer along

with a polyol salt, glycerophosphate, is profoundly explored for thermosensitive gelation

properties in BTE applications [8–11]. However, low mechanical strength and poor

control on the release of encapsulated compounds have necessitated various modifications

to enhance these features. Despite the availability of various CS/GP based hydrogel

systems, concerned scarcity upon perfect hydrogels with desired properties is still a

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primary concern that needs to be addressed. In this review, CS/GP hydrogel systems for

BTE applications are briefly reviewed in order to provide directions for fabricating suitable

CS/GP injectable hydrogel with superior properties.

1.1. Bone and Bone Defect

Bone is a vascularized connective tissue, which provides adequate mechanical

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strength and structural support to the body. It is classified into cortical (dense), and trabecular

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(porous) bone and both these subclasses possess an orchestrated 3D architecture with high

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structural complexity (Fig.1).
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Figure 1: Schematic illustration of the architecture of bone tissue. Bone harbors its

constitutive elements at macro, micro and nano scale dimensions. Cortical and

trabecular bone represents the macro structures with osteons comprising of concentric

collagen bundles (lamellae) at microstructure dimensions. Individual collagen fibers

exhibit nanoscale morphology with anisotropic alignment. Bone remodeling is

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controlled by two vital key cellular players namely; osteoblasts (bone forming cells) and

multinucleated osteoclasts (bone resorbing cells). Osteoprogenitors differentiate into

osteoblasts, and senile osteoblasts termed as osteocytes form structural bridges in the

osteon.

Architectural arrangement of bone contains structures at macro, micro, and nano level

[12]. Osteons are the repeated units ranging from several microns build the cancellous bone.

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On the contrary, trabecular bone is filled with free spaces for housing bone marrow. The

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individual unit of osteon contains concentric bundles of anisotropically arranged collagen

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nanofibres called lamellae surrounding the central canal [13]. Calcium phosphate crystals are

arranged along the axis of collagen bound with various organic proteins at nano dimensions
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[14]. Bone tissue is continuously remodeled, and a balance is maintained by the critical

cellular constituents namely osteoblasts (bone forming cells) and osteoclasts (bone resorbing
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cells). Perturbations in this balance lead to bone loss and subsequent irreversible bone

damages in various pathological conditions. The mechanical properties of the bone are highly
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determined by the orientation and organization of the bone extracellular matrix (ECM).
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Impedance in normal biomechanics and destabilizing structural organization of the healthy

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bone tissue are the pathological risks associated with bone defects [15]. Cavity defects and
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segmental defects are the two major classes under which bone defects are classified. Both the

cases often require multiple reparative surgeries, and the treatment regimen depends on the
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site and size of the defect. Bone grafts and metallic prosthetic implants are the current forms

of treatment for correcting bone defects [16,17]. Autologous grafting harnesses the natural

healing capacity of bone harvested from the donor body and is still considered to be the gold

standard treatment in the clinical arena [18–21]. Wide-ranging aseptic surgical procedures

often augment the bone defect correction utilizing grafts; however, it does not guarantee the

complete healing of the defect. Graft tissue viability, vascularization, tendon functioning and

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maintaining nerve physiology are the crucial parameters, which are not completely fulfilled

by most of the surgical procedures [22]. Hence, achieving an utterly functional syncytium

with highly vascularized and biomechanically strong bone is a challenge to be addressed with

other alternative strategies.

1.2. Bone tissue engineering strategies

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BTE holds the promising role for reconstruction of bone defects following severe

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trauma. BTE integrates the principles of engineering and cell science to augment bone loss

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via cells, biomaterials, and a combination of both these elements to replace defect site with

biologically functional tissue [23].

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The BTE paradigm involves various irreplaceable vital players which include (i)

osteogenic cells, (ii) osteoconductive matrices (scaffolds and hydrogels), and (iii) osteogenic
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growth cues [23–25]. Mostly, BTE is amended by the use of 3D matrices as a template for

new tissue ingrowth. These 3D matrices aim to recapitulate the natural bone architecture
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thereby providing additional mechanical support to the defect site [26–28]. Biomaterial and
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cell-based strategies offer viable tissue equivalents through orchestrated processes of

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combining the osteogenic potential of cells and biomaterials [29,30]. Collective pieces of
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evidence from preclinical studies have proven the effectiveness of using live cells for bone

tissue regeneration with most of the in vivo studies focusing on mesenchymal stem cells
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(MSCs) as ideal candidates [31,32,32,33]. Investigations on optimizing cell numbers, type,

passage numbers and other questions related to cell-based strategies are still a puzzle to be

answered [32,34–36]. Next to the cell selection, choosing appropriate carrier materials for

cell and biochemical signaling molecule delivery is critical to ensure the survival of graft

upon transplantation.

Scaffolds are 3D matrices with adequate porosity gearing a geometry, which provides

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structural support to the defect site allowing the body’s intrinsic healing mechanism to heal

the bone defect [6,26,28,37–40]. Scaffold acts as temporary matrices for bone regeneration

with the outcome depending on the macro, microstructure and material properties. Various

biocomposite scaffolds (fibrous, foam, capsule) have been widely investigated for BTE

applications. Hydrogels represent a major and unique class of scaffolding materials with 3D,

hydrophilic networks of crosslinked polymeric chains.

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1.3. Injectable hydrogels

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Hydrogels can absorb several folds of water into their structures without

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disintegrating thereby mimicking the natural tissue environment [4]. In the recent studies,

injectable in situ forming hydrogels have been widely reported for orthopedic applications
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[7,8,41–43]. Unlike the use of pre-fabricated scaffold which requires surgical implantation,

hydrogels can be injected into the defect site to confront any geometrical deformities and
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with minimally invasive procedures. Hydrogels are remarkable platforms used to fill the

bone defects in non-load bearing sites, which do not require high mechanical strength.
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Moreover, injectable hydrogels are used to reinforce the injured bone tissue and act as a
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carrier for delivering therapeutic agents and cells (Fig.2). Injectable hydrogels reach the
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site of the defect and deliver loaded biomolecules/cells and then form a gel through
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transitional properties when a response to a stimulus changes the physical and/or

chemical properties.
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Figure 2: Schematic representation of injectable hydrogel based approaches for

repairing bone defects. Bone marrow mesenchymal stem cells isolated from the patient
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are maintained under normal medium or under osteogenic stimulants for

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osteodifferentiation. Both cell-free construct and cell-based construct are utilized for
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treating bone loss at both non-load bearing and load bearing sites.
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They are gaining greater attention in the field of BTE owing to their minimum
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invasiveness and the ability to repair and regenerate bone tissue successfully. Apart from

these properties, hydrogels harbor network of hydrophilic groups, which are responsible for

fluid retention and imparting elastic force responsible for stress resistance. Natural and

synthetic polymers having stimuli-responsive behavior have been extensively studied for

their use as thermosensitive hydrogels in BTE applications due to their ability to deliver cells,

biomolecules, and drugs. Natural polymers include CS, cellulose, gelatin, xyloglucan,

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alginate, gellan gum, hyaluronic acid, chondroitin sulfate, carrageenan, dextran, pullulan,

ulvan, etc., with their respective derivatives. Poly-N-isopropylacrylamide (PNIPAAM),

poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO/PPO/PEO)

block copolymers, poly(ethylene oxide)-b-poly(D,L-lactic acid-co-glycolic acid)-

poly(ethylene oxide) (PEO/PLGA/PEO) triblock copolymers, and amphiphilic triblock

copolymers, composed of PEO and poly-ε-caprolactone (PCL) (PEO/PCL/PEO) represent the

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synthetic polymeric materials. Despite the choice of different kinds of polymers for

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hydrogels, natural polymers possess higher biocompatibility and structural similarity to

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the ECM components and can be modified to obtain a specific biological response [44–

47]
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Injectable hydrogels are fabricated and grouped as physical [48,49] or chemical

hydrogels [48,50–52] based on the mechanism of gelation and formulated structure upon
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the cross- linking. Physically cross-linked hydrogels are of great interest due to their

simplicity in preparation and most importantly it doesn’t show any cytotoxicity, whereas,
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the chemical gel has covalently bonded polymeric units in the system. Lack of
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functional group’s stability, reduced coupling agency, and involvement of cytotoxic cross-
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linkers have limited its use; however, various methods have been introduced to reduce the
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toxicity by preserving the biological nature of the polymers. Physical gels respond to

changes in environmental conditions including pH [8], light [53,54], electric field,

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temperature [9], magnetic field and ions [55] and also called as smart polymers. Polymers

responding to temperature are widely studied due to their simplicity and their ability to exert

only minimal adverse effects on living tissues compared to other stimuli such as pH or

electric field. Amongst the different polymers employed for producing thermo-responsive

hydrogels, CS, a natural polymer holds great advantages and widely explored for BTE. Here,

we discuss the gelation perspective and applications of CS-based thermoresponsive gelling

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systems in BTE. This review also intends to provide an update on various thermosensitive

hydrogels based on CS currently developed for BTE applications.

2. CS/GP hydrogels

CS is a linear polysaccharide and deacetylated product of chitin, a structural

element found in the exoskeleton of crustaceans [56–58]. It is composed of randomly

arranged β-(1,4)-linked D-glucosamine and N-acetyl- D-glucosamine units. CS exhibits

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high biocompatibility, biodegradability, structural resemblance with GAGs and

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antimicrobial activity, which are explored for tissue engineering applications. CS based

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thermoresponsive systems are investigated in aiding regeneration of bone [9], cartilage [7],

periondotium [59], liver [60], myocardium [61]and neuronal regeneration [62].

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2.1. Mechanism of gelation

CS is not a thermosensitive polymer, and thermal gelation is achieved by the addition

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of GP in the CS solution. [63,64] have clearly elucidated the gelation mechanism. CS is

insoluble in neutral and basic solutions but is soluble at a pKa of 6.5 due to the
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protonation of free amino groups in its structure. Neutralization of CS solutions to pH

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value beyond 6.2 results in the immediate formation of hydrated gel-like precipitate. The
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phase separation of CS occurs only at pH > 6 to form a hydrogel. Maintaining CS solution

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below room temperature within a physiological relevant pH range of 6.8 to 7.2 in the

presence of GP polyol salt transforms the system into a thermo-responsive gelling system
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of being liquid at room temperature and transformation into gel as the temperature is

increased to physiological temperature (cloud point of ≥ 37°C). The inclusion of GP salt to

CS solution modulates the electrostatic, hydrophobic interactions and hydrogen bonding

during gel formation. GP added into the system plays three major roles such as (a)

increasing the pH to a physiological range (7.0 to 7.4), (b) inhibiting immediate

precipitation of hydrated gel and (c) imparting thermogelling character at 37°C. The

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molecular mechanism and major steps occurring during gel formation include: [i] addition of

GP results in reduction of electrostatic repulsion and increase in hydrogen bonding between

CS chains, [ii] occurrence of electrostatic interactions between phosphate groups of GP

and amino groups of CS, and [iii] enhanced CS-CS hydrophobic interactions by the action

of glycerol on water. Rheological investigations indicated that the temperature dependence

of gelation originated from hydrophobic interactions of CS chains and glycerol groups. CS

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chains aggregation is prevented by CS-water interactions at low temperatures, and upon

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heating, the water molecules are removed by the glycerol moieties, which promote CS

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chains association leading to gel formation. Besides the modulation of electrostatic forces

by the rise in temperature, hydrophobic interactions also account for a significant role in
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CS/GP gelling system. Rise in temperature limits the orientation of dipolar water molecules

enclosed around the CS polymeric chains via enhancement of vibrational and rotational
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energies of water molecules. Energized water molecules are removed around the CS

chains resulting in the association of dewatered hydrophobic segments with each other. At
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low temperature, CS adopts a coiled configuration due to the presence of intramolecular

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hydrogen bonds and masking of physical junctions that is essential to form a gel. The
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number of intramolecular hydrogen bonds is reduced b y a n increase in temperature

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thereby allowing CS molecules to unfold and favoring gelation. Hence, it is noteworthy in

mentioning the role of pH and temperature in this thermosensitive system.

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2.2. Biological properties of CS/GP for BTE applications

CS/GP based hydrogels have been widely studied for efficient delivery of wide

range of bioactive molecules, drugs and growth factors, and provide structural

organization for creating a multilayered system with cells and tissues (Fig. 3A &3B).

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Figure 3: (A) Thermal gelation of chitosan solution in the presence of β-

glycerophosphate (GP). The solution exhibits liquid state at 25°C and solidifies to form
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a gel at 37°C. Cationic chitosan chains are linked closer to the negatively charged GP

molecules. The decrease in electrostatic repulsion among CS-CS chains, increase in

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electrostatic attraction between CS-GP and formation of hydrogen bonds are

responsible for gelation without precipitation. (B) CS/GP hydrogels possess neutral pH,
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which is ideal for encapsulating various growth factors, cells, drugs and nucleic acids
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(pDNA, miRNA) for augmenting bone healing.

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Such strong supportive aspects of CS/GP system have put forward its candidature for BTE

applications. CS possesses various biological properties, which widen its applicability for
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tissue engineering applications [60,65]. The structural similarity with GAGs [66] has

promoted its use for cartilage and bone regeneration. Numerous studies have shown the

wound healing potential of CS by modulating the function of various inflammatory cells

such as neutrophils, fibroblasts, endothelial cells, and macrophages [67–71]. CS is

biocompatible with a minimal immune response, and the cationic nature of CS is

attributed to most of its vital biological properties such as hemostasis, antimicrobial

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activity, mucoadhesiveness and osteoconductive properties [72]. The presence of amino

and hydroxyl groups in its structure is exploited extensively for conjugating various

drugs, growth factors and other polymeric additions thereby endorsing its applications in

TE [73].

Apart from CS, GP itself is one of the components of osteogenic medium, which

promotes the differentiation of MSCs into osteoblasts [74]. The major reason for its

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implication in BTE is its unique ability of osteoconduction, which is required for bridging

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bone defects in vivo and easily modifiable to various forms like porous sponges,

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hydrogels, films, and fibers [65]. CS also has the property of attracting various

proteoglycans [75]. CS polymeric units are easily degraded in vivo by the hydrolytic
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scission mediated by lysozyme [76,77]. The degraded monomeric units, mostly amino

sugars, exert no toxicity and are incorporated into metabolic pathways [76]. CS films
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extensively support osteoblasts attachment, proliferation, and differentiation with no

discernible cytotoxicity. Interestingly, CS hydrogels have the ability of selectively

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supporting cell attachment. In a recent study, it was found that CS preferentially support
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the osteoblast’s adhesion compared to fibroblasts [78].

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Apart from these properties, CS has been utilized to treat bone defects in load-
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bearing sites in a combination with electrostatically opposite polymers, ceramic particles

and in non-bearing sites as single standing polymeric scaffolds and hydrogels [75,79–82].
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In situ forming CS hydrogels exhibits dynamic characteristics such as gelling at body

temperature, maintaining neutral pH that imparts the ability of loading cells, nanoparticles

and other therapeutic cues intended for tissue healing and bone formation. As reported for

the first time, CS/GP hydrogel was able to maintain greater than 80% cell viability of

various cell types including Cos-7, L929, Rat-1 in vitro [83]. The applicability for cell

transplantation for tissue repair was investigated by encapsulating chondrocytes in CS/GP

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gels subcutaneously in athymic mice and the encapsulated cells secreted matrix similar

to cartilage. However, this study has no insight into the control of gelation time.

An ideal hydrogel for bone tissue regeneration should exhibit relatively short

gelation time, controlled degradation and good pH stability. The degradation and gelation

time is controlled by varying the concentration of β-GP, an increased β-GP addition

provides shorter gelation time. However, increased β-GP levels were found to cause toxicity

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to human HS68 and mouse embryonic fibroblasts [84]. An alternative approach is to

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utilize NaHCO3 addition which resulted in reduced gelation time from 10 min to 3 min

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with pH of 7.25 [5]. Increased concentration of gelling agents results in hypertonicity and

immediate death of cells. NaHCO3 at 0.4 M concentration along with 150 μl of 50% GP
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improved thermal stability, mechanical strength and minimized cytotoxicity. Therefore,

adjusting its concentration has yielded a non-toxic combination with optimal gelation time
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and controlled degradation.

3. Need for modifications in CS/GP hydrogels

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CS/GP hydrogels are flexible and have the property of acquiring bone defect of any
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geometry but possess reduced mechanical strength and low osteoinduction properties
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rendering them with less pronounced ability of binding to the host bone tissue (poor
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osseointegration). Bone defects are often associated with other complications such as

poor osteoconduction across the defect, poor vasculature, hypoxia, microbial infections
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and other causatives, which require a combinatorial approach involving hydrogels. To

circumvent these issues, different strategies have been proposed to improve the

physicochemical and biological characteristics of the hydrogel formulation by addition

of the second polymer, the inclusion of ceramic particles, modification of CS, t h e

inclusion of bioactive elements, and/or the addition of secondary carriers for tailoring drug

release either individually or in combination. Although numerous reports have been presented

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based on CS/GP hydrogel system for various tissue engineering applications, we have reviewed

its applicability only on BTE applications in this work.

3.1. Improved cell adhesion, prolife ration and differentiation upon collagen inclusion

Hydrogel for biomedical applications should support cell growth with appropriate

degradation rate and possess short gelation time with pH stability simultaneously. CS/GP

hydrogels exhibited longer persistence post-transplantation due to longer degradation time

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up to several weeks, which is undesirable for new tissue formation [85]. Recent studies

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have indicated that the gelation rate was short upon increasing the concentration of GP

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but exhibited detrimental effects on the exposed cells [83,85]. Thermal gelation often

results in the decrease in pH during sol-gel transition and pH destabilization is observed

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in most of the preparations, which are unfavorable for housing cells. The amine groups

present in CS, protonate in acidic solution and impart a net positive charge. Hence,
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anionic polymers can easily interact with CS thereby minimizing the intensity of amino

groups to be neutralized by GP [65]. In this aspect, negatively charged acid soluble collagen
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(ASC) was added to CS/GP system to strengthen the system as well as creates a biomimetic
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environment similar to bone [86]. Type 1 collagen (COL-1) is the major constituent of
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bone ECM, provides structural framework of the bone tissue and involves in t h e cascade
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of temporal events leading to a new bone formation [87]. It also mediates

osteodifferentiation of human bone marrow derived stem cells (hBMSCs) via Integrin
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dependent mechanism [88]. Addition of COL-1 to CS/GP hydrogels was extensively

investigated and found to improve both biological and physical properties of CS/GP

hydrogels.

These hybrid hydrogels exhibited good plasticity and the predominant interactions in

this system were (a) intra and intermolecular hydrogen bonds between CS/ASC and GP,

CS/ASC and water, (b) electrostatic interactions between amine groups of CS and

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carboxyl groups of ASC, between guanidyl groups of ASC, phosphate groups of GP and

amine groups of CS, (c) Van der Waals interactions and (d) hydrophobic interactions.

Upon t h e rise in the temperature and pH by the addition of GP, collagen fibrillogenesis

was initiated, which aided gel formation. The inclusion of ASC improved the geometrical

micro architecture of the hydrogel to an open porous and fibrous structure with high

interconnectivity. Fig. 4A indicates in vitro fibril-forming ability of the collagen molecules as

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evident from the fibrous structure formation with high collagen content [89]. Furthermore,

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pH stability and hemocompatibility were enhanced by ASC addition to the system.

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Culturing of L929 cells in the hydrogel exhibited no appreciable toxicity and supported

better cell proliferation, which is attributed to the presence of collagen in the porous
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structure assisting in adherence and proliferation [84]. A similar study reported that MSCs

showed rapid proliferation, growth with well-spread morphology and extensive intercellular
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connections upon collagen addition to CS/GP hydrogel [90]. In vivo, the collagen based

CS/GP hydrogel promoted neo-vascularization and ectopic bone formation. In

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concurrence to the previous study, the addition of COL-1 to CS/GP hydrogel significantly
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enhanced the spreading of hBMCs embedded in the gel matrix (Fig. 4B). COL-1 containing
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hydrogels exhibited increased gel compaction resulting in the stiffer matrix. At 12th day of
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culture, cells were well spread with spindle morphology indicating extreme

biocompatibility; while collagen-free hydrogel had cells in a rounded morphology with

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aggregates and very less interaction with ECM indicating compensation due to loss of

cell adhesion [91]. Additionally, the DNA content in CS gel decreased by more than

50% while in CS gel containing collagen, there was an increase up to 70% (Fig. 4C). Cell

adhesion to extracellular matrix protein initiates cellular morphogenesis, proliferation and

differentiation. Human bone marrow derived stem cells cultured on COL-1 containing

hydrogel proliferated and differentiated to osteogenic lineage by the upregulation of

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osterix (OSX), bone sialoprotein (BSP) and increased alkaline phosphatase (ALP) activity

with higher calcium deposition [91].

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Figure 4: (A) Scanning electron micrographs of CS/GP/COL-1 hydrogels exhibiting

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highly porous interconnectivity. The inclusion of collagen formed a fibrous network in

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the hydrogel due to the in vitro fibrillogenesis of collagen molecules. This morphological
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difference will aid in cell adhesion, spreading and subsequent cellular events. (B)

Enhancement of cell adhesion and spreading upon collagen addition. Cells were round
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and with no attachment in the naïve CS/GP hydrogel. Collagen inclusion promoted the

cells to exhibit spindle morphology when cultured for a period of 12 days. (C) Collagen

containing hydrogels showed an increased DNA content compared to collagen free

hydrogel at day 12. Reprinted with permission from [91], Elsevier Publishing Group.

Isolation, proliferation, and osteo-differentiation of BMSCs are limited due to the

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restriction in the number of cells isolated and clinical discomfort. Adipose-derived stem

cells (ADSCs) prove to be promising owing to its ease in isolation, strong proliferative

capacity, and multi-lineage differentiation. ADSCs exhibited high potential for its role in

BTE along with biomaterial based constructs [92–94]. These cells grown in collagen

containing CS/GP hydrogel supported the in vitro expansion over 7 days. The inclusion of

collagen improved the porous morphology from fairly loose, irregular pores with a larger

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diameter t o intense, uniform and many coherent pores with high interconnectivity.

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Presence of collagen had enhanced the stabilization of hydrogel pore architectures due to

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filling in the interspaces and encapsulation with the crosslinked CS structure leading to

many stable and cohesive porous structures. Increased collagen also accelerated the
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degradation of hydrogel due to the faster degradation rate of collagen compared to CS.

Collagen is not soluble in aqueous solution, but it maintained the osmolality of the hydrogel
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suitable for cell growth.

Yet another study exposed the advantage of adding collagen to CS/GP system. Dog-
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BMSCs grew well in the hydrogel with collagen and maintained a well-spread morphology
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after a 7-day culture period [95]. T h e a bsence of collagen in the hydrogel led to cells with
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rounded morphology, and in contrary, the cells showed spindle morphology with
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unidirectional alignment attributing to the supportive nature of collagen inclusion. After an

extensive culture in osteogenic medium for 28 days, cell-loaded hydrogel construct with
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collagen addition altered the pore morphology from honey comb-like structures to a

regular and spongy structure. Increased porosity with intense pore interconnectivity

preferentially favored cell crawling, oxygen, and nutrient diffusion. High magnification

images revealed the presence of cells with intercellular connections and high deposition of

mineral nodules only in the presence of collagen suggesting the osteoconductivity of the

construct favoring bone regeneration. Upon in vivo assessment in nude mouse model,

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where dog BMSCs were grown in osteogenic medium (OM) for a week before seeding into

the collagen containing hydrogel and implanted in vivo, indicated the pre-

osteodifferentiated cells inside the hydrogel continued to maintain an osteogenic phenotype

and synthesized ectopic bone without other osteogenic stimulants. Corroborating various

research assessments on CS/GP hydrogel system with collagen addition, we can summarize

that collagen addition improved gelation characteristics, enhanced biocompatibility,

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supported cell adhesion and maintained vertical alignment patter n of cells, promoted

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differentiation and mineralization in vitro, and exhibited ectopic bone formation in animal

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models [91,95].

3.2. Recreating inorganic mineral phase through tailore d addition of ceramics

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Amongst various bone tissue reconstruction strategies, hydrogels have been explored

in many clinical settings due to its minimal invasiveness and ability to fill small and
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irregular shaped defects. These systems are structurally similar to ECM of natural bone

tissue with high biocompatibility. Despite several advantages of using hydrogel for bone
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reconstruction, inability to bond with natural bone without fibrous capsule formation, lack
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of osseointegration and poor mechanical strength are the potential pitfalls of hydrogel-
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based treatment approaches [96,97]. One of the ideal improvements in strengthening the
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above-mentioned setbacks is the inclusion of mineral phase in the hydrogel. Different

bioresorbable ceramics such as tricalcium phosphate (TCP), nano-hydroxyapatite (nHAp),

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nanobioglass ceramics (nBGC) and wollastonite (WS) have been instigated for crossing over

these drawbacks, and this subtle change is expected to impart similar properties of mimicking

the mineral part of the natural bone [98–101]. Moreover, ceramics and bioglasses bind to

host bone tissue without the induction of fibrous capsule in vivo [102]. These materials

possess high apatite-forming ability when in contact with human plasma mimicking

simulated body fluid (SBF) allowing them for osseointegration. In recent years,

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nanoparticles of ceramics are employed over conventional ceramics due to decreased grain

size enhancing cell anchorage, improving osteoblast proliferation, differentiation and

mineralization [103]. Although injectable ceramic materials are proposed f o r orthopedic

applications, the inability to carry cells, bioactive molecules and drugs due to high processing

temperature narrowed its application and restricted its use only as filler materials [104]. In a

study, CS/GP/Collagen hydrogel was fabricated with the addition of different weight

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percentage of nBGCs (0-2 w/w%) and investigated for BTE [100]. Addition of collagen to

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CS/GP system had relatively changed the pore dimension from 100-300 μm to 100-900 μm

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and addition of nBGCs did not alter the pore size in the hydrogels. nBGC particles were

uniformly distributed in the matrix with no aggregation. Furthermore, rheological assays

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indicated no appreciable difference in the gelation time even after nBGC addition to the

hydrogel. Both nBGC and collagen addition improved the mechanical properties post
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gelation. nBGC at 2 wt% addition increased the stiffness to 39% and upon adding collagen

to 30 wt% improved the stiffness by almost 95%. The hydrogel combinations were
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biocompatible to SAOS-2 and HEK 293T cells. Bioglass particles incorporated inside the
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CS/GP hydrogel would dissolute faster compared to CS degradation and support during
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the first stage of implantation followed by remodeling of the bone on its own as the CS
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is progressively degraded. Bioactive properties of bioglass particles arise majorly from the

dissoluted ions, which in turn govern various cellular processes including gene activation
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and mineralization [105–107]. CS/GP hydrogel with BG particles demonstrated a slight

decrease in the gelation time with increasing concentrations of nBGC (0-50%) and deposited

apatite layer upon immersion in SBF. Apatite deposition was dependant on concentration of

nBGC and time of immersion in SBF [108]. In vivo studies clearly showed an increase in

bone formation upon implantation of nanoceramics by altering the fibroblast to osteoblast

ratio.

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In an attempt to resemble natural bone hierarchy, composite biomaterial based

constructs have been designed by several researchers by combining polymeric and

inor ganic phase. Apart from bioglass ceramics, β-TCP has gained considerable attention in

BTE for its excellent biocompatibility, bioactivity, osteoconductivity, and ability of

osseointegration with high thermodynamic stability [109,109–113]. CS/GP system has low

ability to bond with natural bone due to their chemico-physical differences from bone

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tissue [99]. Addition of ceramic particles may circumvent this setback. Incorporation of

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β-TCP to CS based composites reduced the degradation rate and enhanced the bone

regeneration. In fact, the presence of β-TCP did not inhibit the CS interaction with the

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polyols. The phosphate groups of β -TCP interacted with ammonium groups of CS.
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Additionally, the β -TCP would increase osteoblasts adhesion, proliferation and

differentiation. Upon implantation of β -TCP in vivo, it is gradually dissolved and absorbed

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without compromising the healing of bone defects, which proceeds with ingrowth of

new bone tissue [99].

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Hydroxyapatite (HAp) possess a close resemblance to the inorganic component of the

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natural bone matrix, slow degradation in situ and has led to extensive investigations to
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synthesize HAp as a bone substitute and replacement for BTE applications [114–119].
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Synthetic HAp possesses strong affinity to natural bone tissue and shows greater advantage

compared to other bone substitutes such as allografts and metallic implants [119]. HAp is
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characterized by greater osteoconductive and osteoinductive properties with excellent

biocompatibility making them ideal candidates for bone repair as coating and fillers in

bone or teeth reconstruction [120–123]. However, poor mechanical strength is the major

setback associated with conventional HAp.

Sculpturing down the conventional hydroxyapatite to nano dimensions yields nano-

hydroxyapatite (nHAp) wi t h improved mechanical properties and many other biological

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actions due to decreased grain size. Improvements in protein adsorption and osteoblast

adhesion are other advantages of nHAp [9,119,121,124]. Addition of nHAp improves

stiffness, osteoconductivity and pore interconnectivity in the collagen-based scaffold for BTE

applications [125]. [9] concluded that 0.1 w/v% nHAp addition to Zn-CS/GP hydrogel was

not altering the gelation time and pore interconnectivity. In this study, CS was doped with

zinc to improve the osteoconductive and antibacterial properties. nHAp was deposited along

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the pore walls and struts, which would facilitate nucleation of mineral, protein adsorption,

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cell and cell attachment. Water retention ability of the hydrogel increased upon addition of

nHAp due to the presence of free –OH groups, which provided hydrophilicity thereby

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promoting fluid retention and improved protein adsorption. Whereas the degradation
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property of the hydrogel was not affected by nHAp addition, e xogenous biomineralization

on the nHAp containing hydrogels was prominent compared to plain CS/GP hydrogel.
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nHAp acted as nucleation sites for initiating crystal deposition and significantly improved

the crystallinity of the hydrogel. As expected, the hybrid hydrogels exhibited antibacterial
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activity against E. coli and S. pyogenes with no alteration in the activity on nHAp
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inclusion. The composite hydrogel showed excellent biocompatibility towards mouse

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mesenchymal stem cells and promoted calcium deposition under osteogenic conditions
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indicating the osteoconductive nature. O u t o f t h e m o u s e MSCs grown in the

conditioned medium obtained from both the hydrogels, nHAp containing hydrogels promoted
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significant osteoblastic differentiation under osteogenic conditions by the upregulation of

various osteoblastic marker genes such as ALP, runt-related transcription factor-2 (Runx2),

COL-1 at 7th day and a significant enhancement in mRNA level of osteocalcin (OCN) was

seen at 14th day of incubation. Similar to the increased mRNA levels of Runx2, the protein

expression also followed a similar trend upon nHAp containing hydrogel treatment. Upon in

vivo implantation in rat tibial bone defects, the hydrogel promoted bone bridging with a

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significant amount of new bone formation (Fig 5A). The radiographs suggest significant

periosteal reaction with negligible signs of radiolucent gap in nHAp containing hydrogel

group in comparison to the control group 2 weeks post-surgery.

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Figure 5: (A) Radiological assessment of bone healing in rat tibial defect. In vivo

injection of thermosensitive zn-CS/GP/nHAp hydrogel into rat tibial critical-sized bone

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defect healed significantly with new bone formation compared with the control and
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hydrogel without nHAp. Reprinted with permission from [9], Biomed Central

Publishing Group. (B) Micro-CT evaluation of bone formation in rat cranial defect. The
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3D construction images indicated an increased bone formation in hydrogels containing

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SDF-1α incorporated CMC nanoparticles compared with empty group and hydrogel
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containing free SDF-1α. The bone volume/total volume is represented in the bar graph.

Reprinted with permission from [126], Elsevier Publishing Group.

Natural bone is composed of an intricate hierarchal arrangement of nanostructured

HAp crystals arranged along the axis of the collagen fibrils [127,128]. Mimicking this unique

structure,[129] designed a composite hydrogel of CS/nHAp/Col and assessed its ability for

cell delivery in vivo. HA/Col powder was prepared by self-assembly and later incorporated

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into the hydrogel. The system showed thermal gelation at body temperature with acceptable

pH and possessed higher elastic modulus. Interestingly, the cells grown in normal medium

for 15 days did not differentiate into osteogenic lineage even though HA particles are

present in the system. As osteogenic differentiation requires one or more factors such as

bone morphogenetic protein-2 (BMP-2) and the normal medium is devoid of such factors,

osteogenesis was not observed. Moreover, rat bone marrow derived stem cells (rBMSCs)

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cultured in the hydrogel under normal medium retained the self-renewal and proliferative

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ability for up to 20 days.

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An ideal hydrogel should support cell adhesion, proliferation, 3D spatial

organization, and differentiation. Apart from inducing mineralization and resembling

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inorganic phase of bone, HA ceramics were found to alter cellular morphology extensively

[130]. nHAp containing CS/GP hydrogel pores were coherent, intensive with uniformity and
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range from 10-20 μm. The hydrogel without nHAp showed loose pores with larger diameters.

nHAp addition stabilized the structure of the hydrogel. Upon nHAp addition, at 3rd day of
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culture, human dental pulp stem cells (hDPSCs) exhibited outstretched shape and the
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proliferation was high with a fully extended shape at 7th day. Tenuous cellular morphology
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with reduced growth was observed without nHAp addition [131].

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CS/GP hydrogels are flexible, but they have low osteoinduction properties rendering

them less pronounced binding with the host bone tissue. Combinations with osteoinductive
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materials and growth factors facilitate its use for most of the BTE applications. From

various reports, it is clear that nHAp, TCP and calcium phosphate (CP) ceramics offer

unique ability to impart osteoinduction to CS/GP hydrogels. Addition of calcium

containing ceramics to bone tissue engineering matrices is of great interest due to the

resemblance in their chemical composition to the inorganic component of naïve bone matrix

[131]. One such compound containing calcium in its structure is calcium glycerophosphate

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(GPCa), variant of Na-GP. It is the calcium salt of glycerophosphoric acid, which improves

the bioactivity of the combined materials and stimulate bone regeneration. GPCa addition to

polyurethane scaffolds greatly enhanced the calcification and biocompatibility [132]. Its

inclusion into CS has been reported for the first time to prepare CS hydrogel with superior

properties by tailoring the addition from 0.2 t o 0.4 g of GPCa [133]. Increasing the content

of GPCa leads to a decrease in pore size from 25±5.8 to 17.8±2.8 µm and 9.9±2.4 µm.

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Addition o f GPCa at a higher concetnration (4% w/v) reduced cell viability due to

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structural changes of the hydrogel which limit osteoblasts proliferation, nutrient diffusion,

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and metabolic by-products exchange. Hence, GPCa addition at 2% w/v was found to be the

best of the examined concentrations. After a complete assessment of these studies, it can be
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summarized that bioceramics addition to CS/GP hydrogel system significantly improved the

biocompatibility, bioactivity, osteoconductivity and ability of osseointegration via apatite

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deposition.

3.3. Modifications of CS for enhancing the properties of CS/GP system

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Enhancement of the physicochemical and biological properties of CS/GP system

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can also be achieved by various modifications to CS backbone and preserving the intact
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thermosensitive property [134,135]. Amino groups in the CS become protonated and

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efficiently complexes with metal ions, proteins, DNA, lipids and other negatively charged

polymers [134]. Previous studies reported a thermosensitive hydrogel system employing zinc
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doped CS-GP hydrogel for BTE applications [9,11]. Zinc has been reported to exert its role

in osteoblasts mineralization through Zn trafficking involving storage of proteins and

transporters [136–139,139]. The gelation time decreased by 1 min and swelling ability

also decreased significantly in the Zn-doped CS based hydrogel compared to undoped CS.

Higher swelling potential will often result in disassembly of the injected hydrogel from the

defect site and induce stress to the surrounding tissue ultimately leading to implant failure.

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Henceforth, the controlled swelling ability after Zn doping is proving to be advantageous.

Chelation of Zn by the protonated amino groups decreases the availability of hydration and

hence, a decreased swelling was observed. As expected, Zn doping significantly improved

the broad spectrum antibacterial activity against E. coli and S. aureus. Biocompatibility and

osteoconductivity were other properties improved by the hydrogel [11].

Various investigations on modifications of CS certainly exposed the advantages of

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such alterations in the context of biological applications. Amino groups of CS is the target

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site for various modifications to produce derivatives of CS such as carboxymethyl-

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hexanoyl chitosan (CHC), carboxymethyl chitosan (CMC), trimethyl chitosan (TMC),

polyethyleneimine-chitosan (PEI-grafted CS) and polyethylene glycol (PEG)- grafted CS

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[81]. One such study investigated the effect of thiol group addition to CS structure and

produced a water-soluble CS which opened many insights into drug and biomolecules
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delivery [140]. Thiolated CS has found to be soluble at neutral pH. Furthermore, the thiol

groups act as bridge points for forming disulfide bonds with other proteins, which result in
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mucoadhesion. A novel thermoresponsive hydrogel based on chitosan-4-thio-butylamine

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(CS-TBA) incorporated with GP and nHAp, CS-TBA/GP/nHAp, exhibited gelation time of

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about 10 min and with an average pore size of 40-80 µm and nHAp particles were uniformly
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distributed. Irrespective of the modification to CS structure, there was no significant

difference in the degradation rate between two hydrogels were observed. Slow hydrolysis of
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disulfide bonds and degradation of CS backbone structure are vital in the sustained protein

release (BSA) compared to the unmodified hydrogel. Indirect toxicity measurements using

hBMSCs and Caco-2 cells indicated the cytocompatible nature of the prepared hydrogel. In

a nutshell, the modified hydrogel is suitable for drug delivery, BTE applications with low

cytotoxicity and appropriate degradation rate.

3.4. Enrichment of CS/GP hydrogels

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The inclusion of bioactive components to CS/GP hydrogel promotes its bioactivity

and aids in bone regeneration. Bone injury creates hypoxic environment and generates

oxidative stress that inhibit osteoblastic differentiation and promote bone resorption in vivo

[141–143]. Previous investigations have put forward that cells have been well protected

from oxidative stress by tannins, plant-derived polyphenols with anti-oxidant and

antibacterial properties [144,145]. Gallic acid (GA) found in terrestrial plants and

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Phloroglucinol (PG), a major component of terrestrial plants and seaweeds respectively have

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been incorporated into CS/GP/ALP hydrogels [146]. The cellular damage mediated by free

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radicals often lasts for a short period but the effects are irreversible. Hence, bone

regeneration materials with anti-oxidant activity are of high benefits to negate the oxidative
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stress-mediated damages. The hydrogel showed antioxidant activity, and CS itself

possessed scavenging activities, dependant on time and degree of deacetylation.

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Increased scavenging activity was observed with a high degree of deacetylation [147].

A n action of nitrogen at the C2 position in CS attributed to its scavenging property. Other

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mechanisms postulated to describe the antioxidant activity are: (i) formation of stable
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macromolecule radicals and absorbing hydrogen ions from the solutions by amino groups on
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CS, (ii) hydroxyl groups of CS exhibits scavenging activities via hydrogen abstraction.
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PG and GA enrichment showed antioxidant activities in unmineralized state and

mineralized state, and there was diminished activity in GA containing hydrogel while PG
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containing hydrogel retained its activity. The possible mechanism behind this altered activity

is the attraction of Ca2+ ions towards the COO- groups, which increased the mineral growth

around GA causing steric hindrance resulting in diminished activity. The hydrogel is

biocompatible to MG-63 cells, and they exhibited well spread morphology on PG

containing hydrogels compared to naive hydrogels where they formed cell aggregates which

indicate insufficient cell-matrix adhesion compensated with cell-cell adhesion. Whereas

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GA containing hydrogels exhibited cytotoxicity and diminished cell growth thus, careful

selection of antioxidant is essential for fabricating a non-toxic hydrogel.

Selection of appropriate combinations for fabricating CS/GP hydrogels is a crucial

step and will determine the success of the injected hydrogel. One or more combinations

will compensate for properties that are not satisfied by the polymers/other additions in the

hydrogel. In this context, electrical conductivity has proven to be highly beneficial for BTE

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applications. Conductive substrates are used for electrical stimulation of cells which in turn

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accelerate bone formation and regeneration [125,148–151]. It also increases the osteoblast

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markers and promotes secretion of ECM proteins and growth factors synthesis.

Piezoelectricity and high mechanical strength are two vital properties of bone tissue that are
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often ignored while constructing hydrogel [152,153]. Despite carbon nanotubes (CNT)

application in BTE scaffolds [154,155], only one study reported the use of f-MWCNT
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inclusion to CS/GP hydrogel and assessed its suitability for BTE [156]. CNT at different

concentrations ranging from 0.05-1% was incorporated into CS/GP hydrogel and exhibited a
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porous architecture; upon increasing the CNT addition the average pore size and porosity
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were increased. Higher porosities result in a high surface area, which influences cell
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attachment and migration. The inclusion of higher content of CNT might increase the
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distance between CS chains and disrupting crosslinking thereby promoting increased

water retention and porosity. Similarly, an i ncr easi ng trend in electrical conductivity of
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the hydrogels was also reported. Amongst the CNTs incorporated hydrogels, 0.5% CNT

inclusion was found to have superior t ensile strength (38.9±1.4 MPa) and compressive

strength of 1236±9.9 MPa), and met the specific biodegradability requirements. f-MWCNTs

at 0.1, 0.5 and 1% inclusion into the hydrogel were able to improve ALP production by

MG-63 cells. As expected, the electrical stimulation of MG-63 cells grown in 0.1 and

0.5% f-MWCNT showed higher cell proliferation compared to unstimulated samples.

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Overall, this investigation provided various insights into creating conductive hydrogel

platforms for bone tissue regeneration.

Despite various strategies for improving the bioactivity of CS/GP hydrogel through

the addition of polymers, bioceramics, and nanoparticles, introducing CaP phase in CS/GP

hydrogels rem ai ns elusive. T o a ddress this issue, incorporation of the enzyme, alkaline

phosphatase ( ALP), is a more promising strategy for hydrogel calcification. ALP is

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responsible for mineralization of bone, and it has been employed in bone implants as

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coating and covalently linked to scaffolds to improve mineralization in vivo [156–158]. In

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order to promote mineralization for better osseointegration, ALP was included in the CS/GP

preparations at 0.11 and 0.23 mg/ml concentrations [157]. This study compared the CS
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preparation of same degree of deacetylation of 83% with different weight average molecular

weight (Mw) and number average molecular weight (Mn). ALP release was unaltered in all
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the preparations. Addition of ALP accelerated the gelation time in all these preparations. ALP

mediated the hydrolysis of beta-glycerophosphate into glycerol and phosphate. The released
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free phosphate interacts effectively with the deprotonating –NH3+ groups on CS which masks
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the electrostatic repulsion and promotes interchain interactions. While the free glycerol
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dehydrates CS chains promoting hydrophobic interactions, negatively charged ALP also

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interacts with positively charged CS to promote gelation. This is the first and only work to

prove the feasibility of incorporating ALP in CS/GP hydrogels for inducing mineral
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formation.

4. CS/GP hydrogels as depots for delivering cells, drugs and bioactive molecules

Hydrogels influence the therapeutic outcome of drugs and biomolecules (growth

factors, peptides, and miRNAs) delivery intended for clinical use. Controlled degradability,

unique tunable physical properties and ability to protect liable drugs are the major advantages

promoting the use of hydrogels for drug and biomolecules delivery [55,159–161]. The

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presence of porous structures, ability to hold water and supporting cell growth makes them

ideal candidates for carrying cells inside for tissue engineering applications. Traditional drug

administration requires repeated and a high dose of the drug to produce a therapeutic effect,

which lowers the efficacy of treatment and patient compliance. Hydrogels are attractive

owing to their minimal invasiveness, biocompatibility, and ease for encapsulating hydrophilic

drugs [4,50].

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In most of the cases, degradation of the hydrogel is coordinated to match tissue

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regeneration for appropriate delivery of drugs and biomolecules. Presence of micro and

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macropores in the hydrogel makes them an ideal candidate for controlled delivery

applications (as depots). The inclusion of bioactive molecules and therapeutic peptides that
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augment in bone healing in bone defects is widely studied, and many reports have

emphasized the use of BMPs in collagen carriers, absorbable collagen sponge, TCP, PLGA
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microspheres, hyaluronic acid, gelatin sheet and various polymeric scaffolds. BMPs are a

member of transforming growth factor-beta superfamily involved in the regulation of bone

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morphogenesis through chemotaxis, bone cell mitosis, and differentiation [162–164]. BMP-2
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and BMP-4 are already employed in spinal fusion treatments, approved by FDA. However,
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poor release pattern and requirement of multiple doses of BMP-2 to compensate for the loss
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of BMP-2 during material handling and burst release are the limitations to be answered.

Despite the release of BMP-2 by the hydrogel, rapid release of active materials mainly by the
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phenomenon of swelling and degradation lead to insufficient maintenance of drug release for

a longer period.

An interesting study reported the release profile of BMP-2 from CS/GP hydrogel

system, which would act as an implantable delivery system for BMP-2. The release

profile for BMP-2 at two different loading concentrations was found to be 41% for 2 ng/ml

and 48% for 20 ng/ml over a three week period. Preosteoblast mouse stromal cells (W-20-

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17) and human embryonic palatal mesenchymal (HEPM) cells were viable and

proliferated for 3 days in the hydrogel. The BMP-2 loaded hydrogel improved the ALP

activity by 3.6 fold in W-20-17 cells and 2.8 fold increases in calcium deposition by HEPM

cells after 14 days of incubation. OC synthesis was found to be higher at day 7 for W-20-17

and at both 7 and 14 days for HEPM cells [165]. This investigation elucidated the effect of

hydrogel on controlling the release of BMP-2 and also indicated that the effects of BMP-2

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released from hydrogel are dependent on the cell types.

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Apart from utilizing CS/GP hydrogel as a reservoir for the release of BMP-2, nHAp

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addition to CS/GP hydrogel served as a carrier for controlled release of recombinant

human bone morphogenetic protein-2 (rh-BMP-2). In addition to this, dexamethasone (Dex)

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was also incorporated to study the effects on proliferation and differentiation of MSCs.

Dex and rh-BMP-2 were loaded into nHAp at 1.6×10-3 and 7.8×10-3 g/g, respectively.
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MSC proliferation was intuitive in the hydrogel/nHAp loaded with both Dex and rh-

BMP-2. Dex is a synthetic glucocorticoid, which promotes Ob differentiation by

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upregulation of ALP, OP and OC genes. rh-BMP-2 acts similarly, and along with Dex in
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the hydrogel, a synergistic effect of differentiation o f MSCs i n to osteoblasts was

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observed even at day 15 [166].

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Although BMP-2 was incorporated in CS/GP hydrogel, unmodified CS is only

soluble under acidic conditions which render the ability to carry pH-sensitive proteins or
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peptides. Thiolated CS is water soluble, with improved mucoadhesiveness is suitable for

delivering cysteine-rich proteins or peptides. CS-TBA based GP hydrogel carried a

cysteine terminated peptide 24 (P24) containing residues 73–92 of the knuckle epitope of

BMP-2 (N → C: KIPKA SSVPT ELSAI STLYL SGGC) and investigated [167]. After

encountering an initial burst release, almost 96% P24 was released from unmodified CS

hydrogel, whereas the release rate for CS-TBA/HA/GP hydrogel was much slower and

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sustained with a cumulate release of only 43% at day 16. T h e sustained release pattern of

P24 is associated with the CS network degradation and the presence of reactive thiol

groups (reacts with P24).

Application of CS/GP hydrogel system is limited owing to its low mechanical

strength, faster in vivo degradation, decreased dimensional stability and loss of spatial

support, and poor diffusion of O2. A study utilized CS/dextran/PLA/GP hydrogel for

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superior mechanical strength and prolonged degradation endurance [168]. It also

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investigated the sustained delivery and retaining the bioactivity of BMP-2. BMP-2 was

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loaded directly before the gelation, encapsulated in alginate microspheres and embedded in

the hydrogel. Both gel and alginate microspheres will exert a combinatorial regulation over
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the release of BMP-2. Addition of microspheres had no change in the gel forming ability

at physiological temperature and pH. Surprisingly the gelation time was 2 min, which
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has the property of arranging itself into defect geometry rapidly. Higher gel density from

adding microspheres and presence of remaining hydroxyl group in the alginate microspheres
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post-crosslinking with CaCl2 might take part in the hydrogen bond interactions in the gelling
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system. The system also showed no blocking of the pores by microspheres and distributed on
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the pore walls while sparsely encased inside the pores. BMP-2 release from the hydrogel
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followed an initial burst and gradual release over time with 37% released on the first day

and 50% at 7th day. Loading of BMP-2 guide them to be entangled with themselves or other
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components in the gel and available freely dispersed in the pores. BMP-2 is hydrophilic

thus involve in various interactions which restricts the freedom [166]. Molecules having

freedom easily diffuse into the releasing medium, which causes burst release and

followed by the BMP-2 release due to break down of gel components leading to a slow and

sustained release.

In the microspheres embedded gel, BMP-2 released for over a period of 4 weeks

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due to double resistance caused by microsphere and gel matrix. Released BMP-2

induced ALP activity in vitro in C2C12 myoblasts and induced ectopic osteogenesis in mice

models. Bone destruction from Osteomyelitis, an inflammatory bone disease arises from

existing disease condition or direct contamination form contagious site or implant.

Despite aseptic surgical interventions for treating bone loss, open surgery possesses the

potential for infection involving air exposure. Infection treatment involves the removal of

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necrotic tissue and administration of broad-spectrum antibiotic therapy for 3-6 weeks,

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which results in potential hazardous side effects.

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Localized treatment at the site of implant would minimize the toxic effects and

reduce the level of therapeutic dose. Antibiotic-loaded polymethylmethacrylate (PMMA)

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cement is available for treating osteomyelitis but associated with a major disadvantage of

rapid elution of antibiotic from the implant thereby no control on the maintenance of
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minimum inhibitory concentration (MIC) [169–171]. CS/GP hydrogels offer the unique

advantage of negating the risk of multiple surgeries due to its injectability, combine
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osteoinductive and osteoinductive factors, and acting as a depot for antibiotic release.
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Gentamicin release was investigated from CS/GP hydrogel combined with bovine bone
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substitutes (BBS), made of decellularized bone matrix matching the composition of nHAp
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[172]. The prepared hydrogel (with 4 mg Gentamicin) released gentamicin above the MIC

value (0.002 mg/ml) and showed antibacterial activity against E. coli. The work also
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concluded that the prepared antibiotic-loaded hydrogel could be applied as a coating to

various implant prosthesis before implantation in the site of bone loss.

Alendronate (ALN) is a BCS III bone Resorption inhibitor administered for the

treatment of post-menopausal osteoporosis, Paget’s disease, malignant hypercalcemia,

primary hyperparathyroidism and metastatic bone diseases. It has poor oral bioavailability

and causes jaw osteonecrosis, oesophageal irritation and cancer, atrial fibrillation and atypical

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fractures [173]. Intravenous injection cause nephrotoxicity, the transdermal route using

lipophilic adhesives resulted in ALN precipitation. ALN coated bone fixation plates have

limited bone resorption during fractured bone healing. ALN is lost in most of the

encapsulation process in the aqueous phase [61,174,175]. Hence, CS/GP hydrogel was

investigated to act as a depot for sustained ALN release [173]. ALN addition did not affect

the thermoreversibility of the hydrogel, and higher ALN content reduced the pore size and

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produced a much-condensed architecture. Gelation occurred within 15 min in vivo thereby

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minimizing the chances for undesired leakage. CS acted as permeability enhancer and opened

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tight junctions to improve uptake of ALN from hydrogels. Therefore, the injectable depot

system would increase the patient compliance by maintaining therapeutic levels of ALN for a
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sustained period thereby avoiding the necessity of frequent ALN administration. Drugs and

other biomolecules are released from the hydrogel at physiological pH through the swelling
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(water absorption) and degradation of the CS matrix thereby exerting therapeutic effects.

Certain inflammatory condition of the bone generates acidic microenvironment,

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which can be exploited for delivering biomolecules via protonation of CS chains leading
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to mechanical relaxation of coiled CS chains resulting in the release of entrapped

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molecules. One such investigation explored the feasibility of modified CS included with
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Naringin, a naturally derived antioxidant, to treat periodontitis a n d found that it was

successfully released to produce therapeutic effects. Extracellular acidosis is marked by

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an acidic pH 5.5 due to low O2 tension and from the end products of neutrophils,

macrophages. Under this pH, the CS is protonated leading to the release of naringin in

bursts during the acute phase. Also, the hydrogel continued to release naringin for 120 h

[176].

Spatiotemporal regulation is achieved by combining multiple biochemical cues in

drug delivery systems along with scaffolds to promote bone regeneration at a greater pace

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[177,178]. Homing of stem cells to the site of a bone defect will significantly promote

healing of bone tissue with appropriate vascularization to a greater extent [33,179–181].

Stromal cell-derived factor-1α (SDF-1α), a vital chemokine involved in homing of MSCs

possess limited half-life and easily degraded by enzymes [182,183]. Hence delivery in a

biologically active form is an alarming challenge and often requires a carrier for sustained

release. CS based carriers have been effectively explored for carrying FGF, BMP-7, SDF-1

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α, pDNA and anti-cancer agents [66,184–186]. Carboxymethyl-chitosan (CMC), a water-

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soluble derivative possesses net negatively charged functionalities suitable for forming

nanoparticles upon complexing with CS and SDF-1α; a basic protein that can bind to CMC.

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The formed C S / CMC nanoparticle with SDF-1α was incorporated into CS/GP
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h y d r o g e l system for sustained delivery [187]. About 85% SDF-1α was released from

hydrogel without nanocarrier in the first 4 days with a large initial burst. At day 12, almost
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90% were released. Whereas, release rate from the nanocarrier embedded hydrogel was

regulated with an initial burst release of 20% and cumulative release of 40% even after 28
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days. In vivo investigations showed significant healing of the rat cranial defects with 38.5%
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new bone formation compared to 26.3% in the hydrogel without NPs and empty group with
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8.64% (Fig. 5B). Detailed investigation evidenced that more host MSCs were recruited to
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the cranial defect and participated in the new bone formation.

CS/GP system holds a promising advantage f o r t h e multifactor delivery system. CS

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NP’s possess unique ability to protect and control the release of miRNA, pDNA, and siRNA.

AntimiR-138, a short non-coding RNA, promoted osteogenesis by upregulating Runx2,

ALP, OX and BMP-2 mRNA in MSCs, and stimulated ectopic bone formation in vivo,

when entrapped in CS/hyaluronic acid/tripolyphosphate NPs embedded CS/GP hydrogel

system containing SDF-1α. Fast release of SDF-1α and sustained release of antimiR-138

from the above nanoparticle/hydrogel composite system was also hypothesized [126]. SDF-

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1α homes the MSCs from bone marrow to the site of bone defect and antimiR-138

promotes osteo-differentiation. T h e inclusion of SDF-1α and antimiR-138 based NPs did

not affect the porosity of CS/GP hydrogel. But, addition of NPs clearly produced rough

structures on the pores due to the presence of NPs. The release rate of SDF-1α was lowered

after the addition of NPs into the hydrogel. However, the release of NPs from the

nanoparticle/hydrogel composite system was not influenced. SDF is a highly basic molecule,

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which forms interactions through electrostatic forces with hyaluronic acid and tripolyphosphate

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in NPs resulting in the decline of SDF release rate. Dual release of SDF-1α and antimiR-

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138 had no significant synergistic effects on MSCs migration compared to NPs group.

Nanoparticle/hydrogel composite system comprising both the SDF-1α and antimiR-138 based
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NPs promoted the expression of COL-1, OPN, OCN at 3, 7, 14 and 21 days in comparison

to the hydrogel group. Hence, sequential release of SDF-1α and antimiR-138 is essential for
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enhancing MSC migration at early stage and promotion of osteogenesis at a later stage. SDF-

1α and antimiR-138 based NPs were released from the hydrogel in a burst manner where
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the diffusion of SDF was faster, which recruited host MSCs and surrounded the hydrogel.
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Later the released antimiR-138 promoted the osteogenesis in the recruited cells. SDF was
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released for few days while antimiR-138 was slowly released for several weeks. It created a
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localized niche for osteogenic differentiation thereby healing rat cranial defect in vivo.

Cell-free strategies for treating recalcitrant and poorly vascularized bone defects often
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are not promising and hence, cell laden material carrier is actively pursued to augment

bone defects. Delivery and maintaining cells in a viable state accounts for the success of cell-

based carrier strategies in treating bone defects. The appropriate strategy could be based on

the composition of biomaterials t o encapsulate stem cells that creates microenvironments,

which can regulate t h e i r survival and differentiation. Upon initial assessment, CS/GP

solution maintained in a gel state for 4 weeks in vitro with no changes in the structural

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integrity. rBMSCs were able to grow in high number in the CS/GP h y d r o gels for 7 days

and most of them located inside the gel. Attached cells were round in shape and flipodial

extensions anchored to the gel surface suggesting the biocompatibility and suitability of

the hydrogel for maintaining cell viability without any structural deformation. Among

the in vivo injected gel with rBMSCs cells, gelation occurred almost immediately after

injection and maintained the shape even after 28 days [188].

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Hence, it is clear that CS/GP hydrogels control the release of various bioactive cues

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and exhibits a sustained release pattern, which is necessary for maintaining the therapeutic

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concentration at the site of defect to promote cascade of biological events. The list of

bioactive cues released from the CS/GP based hydrogels and their significant effects are
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summarized in Table 1. Fabrication of CS/GP hydrogel with defined parameters and control

over the physicochemical characteristics often requires careful tailoring of various additives
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for achieving a biocompatible formulation.

Bioactive cues Carrier for Dosage Significant effects Reference

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incorporated bioactive cues

 rh-BMP-2 Nanohydroxyapat  120 μg/ml  Rapid proliferation of [166]
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 Dexamethasone ite (nHAp)  10-7M MSCs

 Increased ALP activity
at 3, 7, 11, 15d
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BMP-2 cysteine Thiolated CS 20mg  Sustained release of P24 [167]

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terminated peptide hydrogel with for 16d

24 (P24) nHAp  Inhibited SaoS-2
proliferation and
promoted osteoblastic
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differentiation
 Promoted ectopic bone
formation
BMP-2 CS hydrogel 2 and 20 ng/ml  Sustained release for 3 [175]
weeks
 Enhancement in ALP
activity of W-20-17
 Increased calcium
deposition by HEPM
 Greater osteocalcin
synthesis
BMP-2 Alginate 9.7 μg/mg  Release of BMP-2 was [168]

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microspheres over a period of 4 weeks

 Controlled release due to
double resistance by
microsphere and gel
matrix
 Induced ALP activity in
C2C12 cells
 Promoted ectopic
osteogenesis
SDF-1α CS-CMC 500ng/ml  Sustained release of [186]
nanoparticles SDF-1 α (40% release

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after 28 d)
 Increased new bone
formation (38.5±4.5%)

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after 8 week
implantation

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 SDF-1α  CS hydrogel  100μg/ml  Exhibited Sequential [126]
 antimiRNA-138  CS/hyaluronic  1000 pmol delivery
acid/tripolyph  Induced homing of host
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osphate MSCs
nanoparticles  Promoted osteogenic
differentiation

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Enhanced calvarial bone

regeneration
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 ALP CS hydrogel  25 mg/ml  Exhibited antioxidant [146]

 Phloroglucinol  0.5 mM or activity

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 Gallic acid 1mM Facilitated MG-63 cells

adhesion and spreading
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Naringin CHC hydrogel 0.85%  Reduced inflammation [176]

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and halted periodontal

damage
Gentamicin CS hydrogel 4 mg/ml  Antimicrobial effect on
E.coli [172]
Alendronate CS hydrogel 2-10 mg/ml  Prolonged release for [173]
more than 60 d
 Reduced inflammatory
response

Table 1. List of bioactive molecules delivered by CS/GP based hydrogel in BTE

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Table 2 depicts the information on concentration CS/GP hydrogel constituents from

various reports on CS/GP hydrogel; As can be seen, there were subtle changes in the amounts

of CS, GP, polymers, and bioceramics, which dictated various biological properties and

controlled the gelation time. This would help the readers in fabricating a CS/GP based

hydrogel system with desired properties for various tissue engineering applications, in

particular for BTE.

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Chitosan Additions β-GP Gelation References
concentration (polymers/bioceramics/bioactive concen time
(%) molecules) tration (minutes)
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[86]
2% Acid soluble collagen 2% 5% -NA-
(w/v)
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[91]
2% Bovine type 1 4 mg/ml 7.5% > 5 min
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collagen (w/v)

[[100]]
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2% Type 1 collagen 2 mg/ml -NA- 3 min 35s to

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3 min 59s

Bioactive glass 1 and 2%

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Nanohydroxyapatite 5 mg/ml
2% (nHAp)
12% -NA-
(w/v) [166]
-7
Dexamethasone 10 M

rhBMP-2 120 μg/ml

Rat tail type 1 [189]

2.2% collagen 1 mg/ml 10% 12-14 min

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[155]
3% f-MWCNT 0.5% 7.5% -NA-

Zn-CS 2% - - 5.8% 5 min [11]

Zn-CS 2% nHAp 0.1% 0.35 M < 10 min [9]

CS-TBA 2% nHAp 2% 5.6% 10 min [140]

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CS-TBA 2% nHAp 2% 5.6% 10 min [167]

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BMP-2 peptite (p24) 2 mg/ml

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2.5% ALP 25 mg/ml 10% -NA- [146]
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PG (or) 0.5 mM
GA 1 mM
4% ALP 1.25, 2.5 mg/ml 10% < 15-5 min [190]
2% nBG 10, 20, 30, 40, 0.74 29.3-38.3 [108]
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50% mol/l
1.4% β-TCP 1.5 and 1% 5.8% 5 min [99]
2% Type 1 collagen 2 mg/ml 10% -NA- [90]
2% Alendronate 2, 5, 10 mg/ml -NA- 3.3 – 4 min [173]
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2% Type 1 collagen 3.5 mg/ml 8% 8 min [95]

2% nHAp -NA- 5.6% 15 min [131]
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2.4% Bovine bone 4 mg 10% -NA- [172]

substitute
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gentamicin
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2% SDF-1α /CS/CMCS 500 ng/ml 5.6% 10 min [187]

NP’s

2% SDF-1α 100 μg/ml 5.6% 10 min [126]

CS antimiR-138 NP’s 1000 pmol

Table 2: Formulations of various CS/GP hydrogel systems proposed for BTE

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6. Conclusions and future pe rspectives

Injectable CS hydrogels offer numerous advantages for BTE due to its minimal

invasiveness and tunable properties. In this review, we corroborated the research o u t c o m e

of several investigators on the role of thermosensitive injectable CS/GP hydrogels for

bone tissue regeneration. First, the mechanism behind thermal gelation of the hydrogel was

reviewed. Secondly, various properties of CS supporting their candidature for BTE

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applications along with the improvements were then briefly discussed. Despite the

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injectability and thermal gelation properties of CS/GP hydrogels, poor mechanical strength

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and loss of osseointegration limited their potential utilization. Finally, we highlighted the

role of polymeric addition, bioceramics inclusion, conjugation of drugs and cells into
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CS/GP hydrogels for improving the physicochemical and biological properties suited for

BTE applications. To date, no single combination of CS/GP has shown both biological and
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mechanical properties adequate for BTE. Designing CS/GP hydrogel combinations should

foresee certain clinically important circumstances that should be seriously pursued for treating
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bone defects (Fig. 6).

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Figure 6: Future perspectives in the development of CS/GP hydrogel systems to combat

various clinical scenarios in bone repair.

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The importance of CS/GP hydrogels manifests its ability to deliver drugs,

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bioactive molecules, and cells to the site of bone loss. In these situations, sustained,
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controlled a n d sequential delivery of bioactive components till the growth of new bone

tissue is essential. Predilections based on induction of stem cell homing by CS/GP

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hydrogels are yet another area to be strengthened with wide investigations. A novel drug

delivering CS/GP hydrogel should be able to support hierarchical release of drugs/progenitor

cells at the site of defect. An ideal hydrogel containing a delivery system for bioactive cues

should support the initial burst release of chemokines to recruit sufficient number of stem

cells from bone marrow and followed by the sustained delivery of osteogenic and growth

factors that stimulate the differentiation of recruited stem cells to osteoblasts (Fig. 6).

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Cell-laden hydrogels often fail to prolong the survival of encapsulated cells upon its

injection into the site of bone loss. Cell survival is limited by (a) loss of cell adhesion in the

hydrogel matrix and (b) free radicals generated after bone injury, which deteriorates the

function and viability of injected cells. Cell microenvironment controls the function and fate

of cells. Encapsulating cells in a hydrogel defined microenvironment suitable for cell

attachment will improve the cell survival post transplantation. Hence, ECM containing CS

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hydrogel (Fig. 6) are widely investigated with superior proliferation and differentiation

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promoting properties, which can maintain cell viability and functions.

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The success of CS/GP hydrogels is challenged by various limitations halting its

endeavor into the clinical side. Understanding mechanism of fracture healing where
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hypoxia, a neglected factor is to be considered in designing hydrogels that can negate

hypoxia with antioxidant-rich biomolecules/plant polyphenols. Preventing anoikis of

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transplanted cells by extending the research on including various ECM mimics would

account for the success of CS/GP based cell delivery system to bone. Controlled delivery of
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drugs, antibiotics, and other functional molecules to attain therapeutic effects requires intense
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investigations on the modification of hydrogel properties to augment bone loss. Regardless

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of the considerable amount of investigations on utilizing injectable CS/GP hydrogels for

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bone tissue repair in experimental animals, translation of hydrogel-based therapies to

humans remains to be elusive. Certain pathological conditions decrease the efficacy of the
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injected hydrogel (Fig. 6). In obesity, the inherent ability of mesenchymal stem cells to

differentiate into adipocyte and osteoblasts is altered favoring towards adipocyte lineage,

which decreases the osteo-differentiation of injected cells and host cells recruited to the site.

Therefore, the implant survival is a question to be answered for developing patient-specific

grafts. Hence, incorporating factors that reverses this pathology would preferentially support

osteo-differentiation and hence would promote bone healing. In diabetic individuals,

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persistent hyperglycemia alters the parathyroid hormone regulation and inhibits osteoblast

activity, which induces apoptosis of bone lining cells, reduces the deposition of collagen

(reduced ECM deposition) during callus formation and increases osteoclasts activity. These

pathologies ultimately decrease the efficacy of various implant strategies. Systemic

administration of aminoguanidine significantly promoted osseointegration of implants in

diabetic animals [191]. Alternatively, recombinant rat insulin-like growth factor 1 release

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from PLGA microspheres improved osseointegration of titanium calvarial implant in diabetic

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rats [192]. Adiponectin may also increase bone density through enhancement in osteoblast

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activity and inhibit osteoclastogenesis in both obese and diabetic individuals due to its anti-

inflammatory properties. These bioactive additives can be delivered locally through the
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hydrogel to eliminate its loss upon systemic administration. Such restorative strategies should

be integrated into the CS/GP based hydrogels for improving bone formation post injection.
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It is anticipated that cracking insights into the synthesis of CS/GP based hydrogels

that can substantiate a significant improvement in mechanical strength, osteoconduction

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and delivery o f bioactive cues will provide new strategies to improve bone regeneration.
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Acknowledge ment
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We thank Dr. Swaminathan Sethuraman and Dr. Uma Maheswari Krishnan for their
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technical help in the manuscript preparation. We also acknowledge Department of Science

and Technology, Inspire Faculty Program, Government of India for the research grant to
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S. Vimalraj (grant no. DST/INSPIRE/04/2017/002913).

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