Journal Pre-proof

Chitosan-based drug delivery systems: From synthesis strategy to

osteomyelitis treatment – A review

Fenghua Tao, Sijia Ma, Hai Tao, Lin Jin, Yue Luo, Jian Zheng, Wei
Xiang, Hongbing Deng

PII: S0144-8617(20)31236-4
DOI: https://doi.org/10.1016/j.carbpol.2020.117063
Reference: CARP 117063

To appear in: Carbohydrate Polymers

Received Date: 8 July 2020

Revised Date: 22 August 2020
Accepted Date: 3 September 2020

Please cite this article as: Tao F, Ma S, Tao H, Jin L, Luo Y, Zheng J, Xiang W, Deng H,
Chitosan-based drug delivery systems: From synthesis strategy to osteomyelitis treatment –
A review, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.117063

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Chitosan-based drug delivery systems: from synthesis
strategy to osteomyelitis treatment – A review
Fenghua Tao a, b,1, Sijia Ma a,1, Hai Tao a, Lin Jin a, Yue Luo a, Jian Zheng a, Wei Xiang
a, b, *
and Hongbing Deng b, *
a
Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan University,
Wuhan 430060, China
b
Hubei International Scientific and Technological Cooperation Base of Sustainable
Resource and Energy, Hubei Key Laboratory of Biomass Resource Chemistry and

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Environmental Biotechnology, School of Resource and Environmental Science,
Wuhan University, Wuhan 430079, China

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*
Corresponding authors.
Tel.: +86 27 88041911-83330; Fax: +86 27 88402292 (W. Xiang)
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Tel.: +86 27 68778501; Fax: +86 27 68778501 (H. Deng)
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E-mail addresses: xiangwei_13@whu.edu.cn (W. Xiang)
hbdeng@whu.edu.cn (H. Deng)
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1
Contributed equally to this work.

Highlights
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 Chitosan is a polycation polysaccharide with excellent biological properties

 Chitosan can be a carrier for osteomyelitis-targeting drug delivery
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 Chitosan modifying can prevent implant-related infection and promote

osseointegration
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 Chitosan derivatives can improve the therapeutic effects of osteomyelitis

Abstract

Osteomyelitis is a complex disease in orthopedics mainly caused by bacterial pathogens

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invading bone or bone marrow. The treatment of osteomyelitis is highly difficult and it

is a major challenge in orthopedic surgery. The long-term systemic use of antibiotics

may lead to antibiotic resistance and has limited effects on eradicating local biofilms.

Localized antibiotic delivery after surgical debridement can overcome the problem of

antibiotic resistance and reduce systemic toxicity. Chitosan, a special cationic

polysaccharide, is a product extracted from the deacetylation of chitin. It has numerous

advantages, such as nontoxicity, biocompatibility, and biodegradability. Recently,

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chitosan has attracted significant attention in bacterial inhibition and drug delivery.

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Because chitosan contains many functional bioactive groups conducive to chemical

reaction and modification, some chitosan-based biomaterials have been applied as the
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local antibiotic delivery systems in the treatment of osteomyelitis. This review aims to
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introduce recent advances in the biomedical applications of chitosan-based drug

delivery systems in osteomyelitis treatment and to highlight the perspectives for further
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studies.
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Keywords: Chitosan; drug delivery system; antibiotic; implants-related infection;

osteomyelitis
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Introduction

Osteomyelitis is a complex inflammatory disease always caused by a bacterial

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infection (Kavanagh et al., 2018). The prolonged osteomyelitis often recrudesce and

can lead to progressive bone destruction, and it has become a major challenge in

orthopedics (Roderick, Sen, & Ramanan, 2018). The pathogenesis of osteomyelitis is

complex and varied, including endogenous bacterial seedings such as hematogenous

osteomyelitis and exogenous bacterial infection caused by open injury and surgical

implant contamination (Seebach & Kubatzky, 2019; Singh, Bierrum, Wormald, &

Eastwood, 2020; Thakolkaran & Shetty, 2019). With the popularity of bone fracture

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internal fixation, joint replacement, and bone substitute biomaterials, the surgical

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implants-related infections have become the most important causes of osteomyelitis,

leading to considerable difficulties and challenges in clinical treatment (Metsemakers

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et al., 2018). If not controlled well, a local infection can develop into chronic
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osteomyelitis and exist for months or even years, resulting in a prolonged hospital stay

for patients (Urish & Cassat, 2020). Furthermore, sinus tract and skin squamous
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epithelium transformation can be detected around the long-lasting infection lesions in

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osteomyelitis, requiring multiple debridement operations and systematic antibiotic

treatment (Jiang et al., 2020; Q. Li et al., 2015). In severe cases, osteomyelitis can lead
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to limb necrosis, dysfunction, and even sepsis, which requires amputation or ICU rescue

and eventually leads to permanent disability (M. Shi et al., 2020).

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The standard treatment for osteomyelitis involves the extensive debridement of

the infected bone, the obliteration of dead space, and a long-term systemic

administration of antibiotics (Masters et al., 2019; Pincher, Fenton, Jeyapalan, Barlow,

& Sharma, 2019). For implant-related osteomyelitis, particularly the periprosthetic

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infection, the treatment first requires the removal of the infected prosthesis.

Subsequently, the bone and joint repair surgery should be performed after prolonged

local and systemic antibiotic therapy (Masters et al., 2019). The challenge of surgical

treatment is to perform a thorough debridement and preserve the normal bone tissue in

order to avoid bone nonunion and limb dysfunction. Meanwhile, the systemic antibiotic

administration requires a long-term and a large dose because the localized bacterial

plaque can inhibit the antibiotic diffusion from blood to the infection site (Nandi et al.,

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2016). The limited availability of antibiotics at the target site is often insufficient to

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eradicate the local bacteria (Mahmoudian & Ganji, 2017). Additionally, the use of long-

term and high-dose systemic antibiotics may lead to antibiotic resistance and cause side
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effects affecting organs (Bhattacharya, Kundu, Nandi, & Basu, 2013; Nandi et al.,
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2016). Recently, to overcome these restrictions, local antibiotic delivery system (Figure

1) has been established as an effective approach to cure osteomyelitis (Nandi et al.,

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2016). Local drug delivery systems are superior to systemic administration because
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they can control the long-term and high-dose release of antibiotics directly to the target

tissue with few hazards of toxicity to organs (Boles et al., 2018). Filling polymethyl
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methacrylate (PMMA) bone cement and beads loaded with antibiotics in the local

infection sites has been considered as the standard practice in osteomyelitis treatment
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for decades. The usage of antibiotics-loaded PMMA in osteomyelitis is advantageous

for delivering a high concentration of drugs to local infection sites and reducing the

organic toxicity (Masters et al., 2019). However, many problems have been found in

the application of PMMA-based drug delivery systems. For example, the rapid thermal

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diffusivity, the non-biodegradability and the poor drug release kinetics can affect the

therapeutic effects of antibiotic-loaded PMMA bone cement on osteomyelitis (Inzana,

Schwarz, Kates, & Awad, 2016). Therefore, it is necessary to develop more satisfactory

drug delivery systems that should be biocompatible and biodegradable and also have a

desired loading efficiency and an appropriate release rate to eradicate osteomyelitis

(Zeng, Hoque, & Varghese, 2019). Natural biomaterial-based drug delivery systems

can provide reliable solutions to these problems.

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Figure 1. Schematic diagram illustrating the process of local drug delivery system for

the treatment of osteomyelitis.

Chitosan (CS) is a kind of polysaccharide polymer extracted from the

deacetylation of chitin, which is the second most abundant biological resources in

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nature next to cellulose. It mainly exists in the shells of crustaceans, insects, and fungal

cells walls (Bakshi, Selvakumar, Kadirvelu, & Kumar, 2020). CS is a cationic

biopolymer with active functional groups, which is advantageous for modification and

chemical reactions (Negm, Hefni, Abd-Elaal, Badr, & Abou Kana, 2020). In recent

years, multiple special properties of CS, such as biodegradability, biocompatibility, low

immunogenicity, non-toxicity, and intrinsic antibacterial activity, have been reported

(Tao et al., 2020). These special characteristics have attracted increasing attention in

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tissue engineering. CS-based biomaterials have been widely used in biomedical

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applications such as biological substitute materials, wound dressing, drug carriers, and

antibacterials (Ding, Deng, Du, Shi, & Wang, 2014). Owing to the advantageous
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bioactivities, significant progresses have been made in the field of CS-based drug
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delivery systems. The application of CS-assisted antibacterial delivery has attracted

immense attention in curing bone infections (Kimna, Deger, Tamburaci, &

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Tihminlioglu, 2019). This review aims to summarize and discuss recent developments
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and applications of CS-based drug loading and delivery systems in the treatment of

osteomyelitis.
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1. Biological properties of CS

As a naturally derived amino polysaccharide, CS has abundant bioactive hydroxyl

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and amino groups, which determine the changeable solubility, chemical reactivity, and

bioactivity of CS (Qin & Li, 2020). The molecular structure of CS is similar to collagen

and can be applied to mimic the extracellular matrix (ECM). Due to the excellent

biological properties, it has been widely used in tissue engineering (Chen et al., 2018).

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CS has been proven to be biodegradable, biocompatible, nontoxic, nonallergenic and

bioadhesive (Tao et al., 2020). Furthermore, it can be degraded into small molecular by

enzymes and readily absorbed in vivo (C. H. Fang, Lin, Sun, & Lin, 2019).

Consequently, CS is a promising biomaterial with broad application prospects in the

field of biomedicine.

CS-based biomaterials consistently demonstrate excellent biocompatibility to cell

and bioactive substances (H. Li, Koenig, Sloan, & Leipzig, 2014). The molecular

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structure of CS determines high thermal and chemical stability (Karakecili, Topuz,

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Korpayev, & Erdek, 2019). CS can be fabricated into various scaffolds for tissue

engineering by modifying its structure and functions (Abinaya, Prasith, Ashwin, Viji
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Chandran, & Selvamurugan, 2019). Biomaterials with CS as the main component are
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conducive to enhancing cell bioactivities, such as adhesion, proliferation, and

differentiation, which are essential for tissue regeneration and repair (Riaz Rajoka,
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Zhao, Mehwish, Wu, & Mahmood, 2019). In bone tissue engineering, CS-based
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biomaterial scaffolds exhibit excellent biocompatibility to seed cells, such as

osteoblasts, osteocytes, and stem cells (Tao et al., 2020). For example, a bioprintable
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CS-hydroxyapatite (HAp) hydrogel shows good affinity to osteoblast and can maintain

cell viability and proliferation, as well as promote biomineralization and osteogenic

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differentiation in an early stage (Demirtas, Irmak, & Gumusderelioglu, 2017). CS-

based membranes are good candidates for guiding bone regeneration. The

glycerylphytate crosslinked CS membranes are beneficial for mesenchymal stem cells

(MSC) growth, and can promote calcium deposition and alkaline phosphatase (ALP)

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activity to facilitate bone regeneration (Mora-Boza et al., 2020). The CS bioactive glass

nanoparticles hybrid scaffolds exhibit adequate degradation properties and are

conducive to the deposition of HAp to promote osseointegration (Oudadesse et al.,

2020). CS scaffolds incorporated with HAp and pyrophosphatase can significantly

promote osteoblast proliferation and calcium phosphate deposition. These hybrid CS

scaffolds can enhance the callus formation to facilitate bone fracture healing (Jahan,

Manickam, Tabrizian, & Murshed, 2020). CS nanoparticles can be used to deliver

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BMP-2 plasmid to MSC and facilitate endogenous BMP-2 protein secretion, which

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significantly enhances MSC-mediated osteogenesis and accelerates bone formation in

critical-sized defects (Raftery et al., 2018). The CS derivatives can also improve bone
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biomaterials physicochemical properties and bioactivity. Lin et al. reported that the
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quaternary ammonium chitosan (QTS) can improve the antibacterial efficacy, washout

performance and osteogenic activity of calcium silicate-based hybrid cements. These

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CS derivative-based hybrid cements can be promising candidates for promoting bone

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defects repair (Lin, Chen, Wu, & Ding, 2020). The carboxymethyl chitosan (CMCS)

doped synchronous self-assembly/mineralization collagen scaffolds exhibit good

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antibacterial effects and can synergistically promote bone marrow mesenchymal

stromal cells (BMSCs) proliferation and osteogenic differentiation (H. Liu et al., 2020).
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The CS derivatives involved multifunctional scaffolds can meet the clinical challenges

of promoting bone regeneration. These properties significantly expand the applications

of CS-based biomaterials in bone tissue engineering.

As a polycation polymer, CS exhibits good affinity for negatively charged

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bioactive molecules, such as antigen (Weber et al., 2010), antibody (Polexe & Delair,

2013), enzyme (Marquardt et al., 2016), cytokines (Anouz, Repanas, Schwarz, & Groth,

2018; Jimi, Jaguparov, Nurkesh, Sultankulov, & Saparov, 2020), DNA (Ahmadi, De

Llano, & Barisic, 2018), and polyanion polymers (Francesko et al., 2018). A colloidal

vaccine delivery system loaded with an antigenic protein is synthesized by the

combination of CS and dextran sulfate. This delivery system can activate the

humoral and cellular immune response to facilitate a high production of antibodies

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(Weber et al., 2010). The antibody functionalized colloids can be prepared by charge

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neutralization between polycation CS and polyanion hyaluronic acid. These nano-

structured particles can immobilize the antibodies on the surface and show a broad
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application prospect in target drug delivery (Polexe & Delair, 2013). CS-based scaffolds
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can retain a biological function and control the release of loaded growth factors. The

BMP-2 and TGF-β1 loaded visible light-cured glycol CS hydrogels can increase the
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bone volume and mineral density in the bone defect sites via prolonged releasing the
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bioactive molecules, and demonstrate the potential for clinical applications in

orthopedic practice (Yoon et al., 2018). An antibacterial freestanding nano-

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biocomposite film is synthesized by the layer-by-layer (LBL) assembling method. The

polycationic CS-silver nanoparticles and the polyanion hyaluronic acid are sequentially
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deposited to form a stable 3D scaffold, which can inhibit bacteria growth and prevent

biofilms formation (Francesko et al., 2018). These specific biological properties

determine that CS can be widely used in the biomedical field.

2. CS-based drug delivery systems

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 The presence of free amine groups in the backbone chain confers to CS the unique

polycationic character that ensures the encapsulation of negatively charged proteins and

peptides (Sultankulov, Berillo, Sultankulova, Tokay, & Saparov, 2019). Simultaneously,

the inherent biodegradability, bioadhesion, plasticity, and modifiability of CS allow its

use as a material component to prepare drug delivery systems. These CS-based

scaffolds can be designed and fabricated into various structures, shapes, and functions

to meet different requirements of disease treatments (J. Li et al., 2018). For example,

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novel CS polymeric micelles fabricated by combining the benefits of PEGylation with

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acylation can efficiently encapsulate and protect hydrophobic drugs. The new

amphiphilic CS drug carriers can act as efficient delivery systems to load hydrophobic
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drugs in tissue engineering (Almeida et al., 2020). A kind of -helical antimicrobial
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peptide (AMP) can be covalently immobilized on the CS coating. This composite

scaffold exhibits good bactericidal, hemostatic and osteoconductive properties, and can
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be used in several infection scenarios, such as wound infection and bone implants-
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related infection (Monteiro et al., 2020). The kartogenin-loaded CS hydrogels modified

by N-(β-maleimidopropyloxy) succinimide ester demonstrate the enhanced shear

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modulus. This kind of CS-based drug delivery system is injectable and thermosensitive.

Moreover, it can control a sustained release of kartogenin to enhanced chondrogenic

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differentiation (Dehghan-Baniani et al., 2020). To introduce more beneficial insulin

delivery system for diabetes treatment, the quaternized CS-based thermosensitive

hydrogel is prepared for insulin delivery through the nasal cavity. This drug delivery

system demonstrates a uniform porous structure, desirable swelling rate, and adequate

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encapsulation. These properties contribute to the prolonged release of insulin to

improve the therapeutic effects on diabetes (Bahmanpour, Ghaffari, Milan,

Moztarzadeh, & Mozafari, 2020). In cancer treatment, the CS-based pH-sensitive anti-

tumor drug delivery system can enhance the biological activity of the drug and

selectivity against target tumor cells. The pH-sensitive polymer can improve

encapsulation efficiency and release profile of doxorubicin by atomic interaction (J. Li

et al., 2020). CS-based drug delivery systems are widely utilized in the target disease

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therapy of biomedicine.

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In bone tissue engineering, CS-based drug carriers often possess good mechanical

properties and controlled drug release efficiency. These scaffolds demonstrate great
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potential for promoting bone regeneration and treating bone diseases (Venkatesan, Anil,
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Kim, & Shim, 2017). An injectable biological scaffold with good mechanical properties

is prepared by introducing electrospun SiO2 nanofibers into CS hydrogels, followed by

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homogenous dispersion and lyophilization. The composite nanofibrous scaffolds can

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mimic ECM without toxic chemical crosslinking. These composite scaffolds are found

beneficial for BMSCs adhesion and stretching. Further, the bioactive BMP-2 can be
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efficiently immobilized on the scaffolds to exert the promotive function of osteogenesis

(L. Wang et al., 2020). Suitable mechanical properties of biomaterials are essential to
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support osteogenesis and osteointegration in bone tissue engineering. Because CS lacks

mechanical strength, constructing composite materials or undergoing chemical

modification is required to enhance mechanical property to facilitate biomineralization

(Deepthi, Venkatesan, Kim, Bumgardner, & Jayakumar, 2016; Saravanan, Leena, &

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Selvamurugan, 2016). Mechanical properties of CS-based scaffolds are always

enhanced by adding HAp, bioglasses, ceramics, and some inorganic salts. The novel

ultralong HAp microtube-CS composite scaffolds can demonstrate high drug loading

efficiency, sustained drug release capacity and high antibacterial activity. The addition

of HAp provides the composite drug delivery system with excellent mechanical

properties for bone regeneration (Y. G. Zhang, Zhu, Chen, & Sun, 2017). The

poly(vinyl alcohol)-bioglass/CS-collagen porous scaffolds exhibit significantly

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improved mechanical properties, calcium deposition, biological function and controlled

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drug release. These essential characteristics make the composite scaffolds promising

candidates in bone tissue engineering (Pon-On et al., 2014). Porous CS scaffolds

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combined with ceramics show significantly improved osteoinductive capacity by
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enhancing the mechanical properties and biocompatibility. Moreover, they can control

the release of a bioactive platelet-derived growth factor to improve tissue regeneration

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efficacy (J. Y. Lee et al., 2002). A biodegradable bone graft dual-delivery system,
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fabricated by embedding CS microspheres in calcium sulfate, can efficiently load and

release antibiotic and growth factors at the same time. This dual-delivery system is an
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efficient alternative in bone tissue engineering due to the prevention of bone infection

and the facilitation of bone regeneration simultaneously (Doty, Leedy, Courtney,

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Haggard, & Bumgardner, 2014). These CS-based composite drug delivery systems can

meet the requirements of mechanical and biological properties of bone substitution

biomaterials. Increasing attention has been paid to the application value of CS-based

drug delivery systems in treating osteomyelitis and bone defect repair.

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 In recent years, three-dimensional (3D) printing technique has gained popularity

in bone tissue engineering. Biomaterial scaffolds prepared by 3D printing always have

unique inherent characteristics and enormous advantages(Pandey, Singh, Singh, Jha, &

Prakash, 2020). The antibiotic delivery systems fabricated by 3D printing method have

been widely used and are promising options for osteomyelitis treatment(Mills,

Jammalamadaka, Tappa, & Weisman, 2018; Shnol & LaPorta, 2018). A kind of

rifampicin-loaded PCL drug delivery system can be fabricated by 3D printing under

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low temperatures. This computer-aided design and manufacturing porous scaffold can

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maintain the antibacterial activity and control the release of rifampicin (J. H. Lee et al.,

2020). The rifampin and sitafloxacin loaded 3D-printed calcium phosphate scaffolds
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can control the antibiotics release in osteomyelitis animal model. The antibiotic loaded
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scaffolds can significantly reduce local bacterial colonization for a long time and show

good osteoconductive property in osteomyelitis treatment (Trombetta et al., 2019). A

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kind of 3D-printed calcium phosphate bioceramics can also be fabricated as dual

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antibiotics delivery systems for the treatment of implant-associated bone infection

(Inzana, Trombetta, Schwarz, Kates, & Awad, 2015). These novel 3D printing scaffolds
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are promising patient-customized treatment options for osteomyelitis because of their

good antibacterial activity and osteoinductivity. Recently, the CS-based biomaterials

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prepared by 3D printing technology have been widely applied in bone tissue

engineering (S. Li et al., 2019). The fused filament fabricated CS reinforced poly-lactic-

acid (PLA) biomaterials are prepared by 3D printing. These scaffolds have shape

memory effects and good wettability for cell adhesion and proliferation. The shape

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recovery characteristics and biological activities endow the scaffolds excellent self-

healing properties for bone defects repair (Pandey et al., 2020). The CS-based

biomaterial scaffolds can be fabricated as good drug delivery systems by 3D printing

method. The 3D-printed lactoferrin-loaded carboxymethyl cellulose-glycol CS

scaffolds have stable hydrogel network. This kind of injectable gels show sustained

drug release and good biocompatibility for osteoblasts and BMSCs growth

(Janarthanan et al., 2020). The 3D-printed BMP-2 loaded poly(L-lactic-co-glycolic acid)

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(PLGA)/HAp/CS scaffolds show sustained growth factor release and can control the

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early burst release of BMP-2. This kind of composite scaffolds can significantly

facilitate new bone formation in bone defect site (Deng et al., 2019). The crosslinker
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modified CS scaffolds can improve the osteoinductivity and osteointegration abilities
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in bone tissue engineering (Mora-Boza et al., 2020). These CS-based biomaterials are

promising implants for promoting bone regeneration and defects repair. Besides, the
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3D-printed CS scaffolds with multifunctional-therapeutic properties exhibit many

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advantages in challenging the infected bone defects. The 3D-printed

quaternized CS/PLGA/HA scaffolds exhibit enhanced antibacterial and

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osteoconductive properties. The dual-functional 3D-printed CS biomaterials are

promising implants in orthopedic surgery (Y. Yang et al., 2018). Thus, the preparation
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of patient-customized CS drug delivery systems via 3D printing method has

tremendous application value in the treatment of osteomyelitis.

3. Local drug delivery systems for osteomyelitis treatment

The treatment of osteomyelitis is a long and complex process. The standard

14
treatment includes extensive surgical removal of the infected bone and adjacent soft

tissue, removal of dead bone, elimination of dead space and long-term systemic use of

antibiotics (Masters et al., 2019). However, local recurrence of osteomyelitis may still

occur. After thorough debridement, local filling of a drug delivery system is capable of

maintaining the sustained release of antibiotics for a long time. It is beneficial for

keeping high concentrations of antibiotics locally to remove the residual bacteria and

prevent the recurrence of osteomyelitis (Ford & Cassat, 2017). The development of the

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local drug delivery systems can significantly improve the therapeutic effects of

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osteomyelitis (Nandi et al., 2016). High concentrations of antibiotics can be achieved

at the infection sites with reduced systemic toxicity via local antibiotic delivery
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systems. For a long time, PMMA bone cements beads or coated rods antibiotic delivery
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systems are treated as the standard practice for preventing

periprosthetic joint infections and treating local infection in osteomyelitis (Tan et al.,
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2012). PMMA bone cements do not show significant tissue toxicity in vivo, and some
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broad-spectrum antibiotics such as gentamicin and vancomycin exhibit good

biocompatibility with the PMMA drug delivery systems (Wentao et al., 2017).
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However, some disadvantages have been found in the treatment of osteomyelitis with

PMMA bone cements-based drug delivery systems. More precisely, 1) the temperature
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of drug-loaded PMMA implants will increase during the preparing process and

therefore, the loaded drugs are required to have thermal stability; 2) PMMA implants

are non-biodegradable and often require the second surgery to remove them from the

local sites; 3) PMMA scaffolds demonstrate poor drug release kinetics because of the

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low drug release efficiency and the rapid burst release in the early stage; 4) The surface

of PMMA scaffolds may become the seeding place for bacteria colonization and

biofilms formation after antibiotics complete elution; 5) PMMA bone cements beads

are prone to breakage because of a lack of stability, and certain complications may

occur with PMMA coated rods or nails, such as cements delamination or debonding

(Cho et al., 2018; Masters et al., 2019). To overcome these limitations, the development

of novel carriers for drug delivery is critical for the treatment of osteomyelitis.

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The desired local drug delivery systems should have certain essential properties,

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such as biocompatibility, biodegradability, suitable drug elution kinetics,

biomineralization capacity and osteoinductivity (Sarigol-Calamak & Hascicek, 2018).

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Currently, synthetic biodegradable polymers, natural polymers, ceramics, bioglasses,
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and their blending composites are widely used in preparing novel antibiotic delivery

systems for osteomyelitis treatment. The structure, shape, and composition are designed
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based on the requirements of eliminating bacteria and promoting bone regeneration

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(Inzana et al., 2016). For example, poly (ε-caprolactone) (PCL) and poly (D,l-lactic

acid) (PLA) are used to prepare antibiotics-loaded emulsion microspheres. The

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scaffolds exhibit appropriate antibiotic release profiles and show enhanced bone

affinity when functionalized by bisphosphonate drugs (Rotman et al., 2020). Novel

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silica-polymer local antibiotic delivery systems can preserve the sustained

ciprofloxacin release for a few weeks without significant toxic effects toward

osteoblasts (Skwira et al., 2019). The mesoporous bioactive glass and PLGA composite

scaffolds demonstrate controlled degradability, improved hydrophilicity, sustained

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antibiotics release efficiency, and reliable osteoinductivity. These novel inorganic-

organic composites have good application prospects in the treatment of bone infections

(Cheng, Qu, Zhang, & Zhang, 2018). Ceramic beads with high antibiotic loading

capacities can be incorporated into the porous HAp matrix scaffolds via freeze gelation.

This kind of scaffolds has large open pores and high surface area to enhance the

maximum loadable amount and prolong the tunable release of antibiotics. It is feasible

to increase the drug loading capacity and improve the therapeutic effects of

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osteomyelitis(Hess et al., 2016). Because of high antibiotic adsorbability,

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HAp/collagen composite scaffolds can be used as antibiotics delivery systems to

improve the therapeutic effects of osteomyelitis (Egawa et al., 2020). The silk fibroin
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coated Titania nanotubes can control the antibiotics freely release, inhibit the biofilms
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formation, and further facilitate osteoblast adhesion and development. It is a promising

alternative for osteomyelitis treatment (Fathi, Akbari, & Taheriazam, 2019). These
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novel drug delivery systems show more advantages than PMMA-based scaffolds,
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including controlled drug loading and release rate, suitable biodegradability, excellent

biocompatibility, excellent mechanical properties, plasticity, modifiability,

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osteoinductivity, and osseointegration characteristics. Because of these special

physicochemical and biological properties, CS-based biomaterials are not only used to
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mimic the natural ECM, but also considered as excellent drug carriers in biomedicine.

The inherent antibacterial and osteoinductive properties of CS have attracted immense

attention in bone tissue engineering. CS has become one of the most appealing

biopolymers in preparing local drug delivery systems for osteomyelitis treatment.

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4. CS biomaterials in osteomyelitis treatment

As a natural derived polycation polymer, CS is considered as an important

ingredient for fabricating biomaterials in tissue engineering. The biodegradability and

biocompatibility of CS enable the diverse bioactive functions, especially as promising

drug carriers for disease therapy (S. Wei, Ching, & Chuah, 2020). CS-based drug

delivery systems have large drug loading capacity, a controlled drug release profile, and

improved drug bioavailability due to their polycation properties, abundant functional

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groups, and special behaviors for water absorption and swelling (Kundu et al., 2010).

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At present, CS-based local drug delivery systems have gained immense attentions in

the treatment of osteomyelitis. Here, a systematic overview of alternative CS-based

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biomaterials in the management of osteomyelitis is provided.
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4.1 CS-based drug carriers in treating osteomyelitis

CS-based biomaterials have a high encapsulation efficiency for antibiotics, and the
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local implantations are proven as more effective than systemic administration (Cevher
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et al., 2006; Orhan et al., 2006). Novel three-dimensional fibrous scaffolds combined

the vancomycin loaded CS with ZIF8 nanocrystals are fabricated by the wet-spinning
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method. They are metal-organic frameworks with superior properties, such as high

porosity, high thermal/chemical stability, and acceptable toxicity. In the composite

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scaffolds, CS fibers provide a large surface area for osteoblast adhesion and growth.

Furthermore, the antibacterial activity of the composite can be enhanced by the

controlled release of vancomycin to improve the therapeutic effects of osteomyelitis in

a pH-responsive manner (Karakecili et al., 2019). The CS sponges with customizable

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sizes and porous structure can be fabricated by lyophilization. The controllable pore

sizes and thicknesses of the sponges are conducive to loading high concentrations of

antibiotics by passive absorption and delivering to the infected tissues by local diffusion.

CS-based drug delivery systems show great potential for managing infected traumatic

injuries (Boles et al., 2018; Wells et al., 2018). The CS-polycaprolactone blend sponges

can also be prepared as drug delivery systems in osteomyelitis treatment. The composite

sponges can carry and control the release of ciprofloxacin hydrochloride and ibuprofen

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simultaneously. This dual delivery system demonstrates antibacterial and anti-

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inflammatory activities concurrently to support managing chronic osteomyelitis after

surgical debridement (Pawar & Srivastava, 2019).In addition, CS can also be used as
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an adhesive material to immobilize antibiotics in the scaffolds. The CS-based
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antibacterial implants can provide optimum drug elution kinetics in managing

osteomyelitis (J. Wei et al., 2019).

lP

Silver (Ag) is a traditional antibacterial drug that has been widely applied in the
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clinics for a long time. Ag nanoparticle-loaded CS scaffolds show good mechanical

strength and swelling properties, making the scaffolds with sufficient protein adsorption
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and mineralization capacity. The CS-based nanocomposites can be fabricated into

highly efficient antibacterial scaffolds for the treatment of osteomyelitis (Hasan,

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Waibhaw, Saxena, & Pandey, 2018). A new type of vancomycin delivery system is

fabricated by embedding hydroxy propyl methyl cellulose (HPMC)microparticles into

CS thermosensitive hydrogels to treat local bone infection. The CS-based injectable

hydrogels achieve sol-gel transition under the physiological temperature and play an

19
important role in extending drug release. The first burst release is mainly caused by the

diffusion of the absorbed antibiotics on the scaffold surface, while the longer-term

release is affected by the drug diffusion from pores within the scaffolds and penetration

through the barriers of HPMC microparticles and hydrogel matrix (Mahmoudian &

Ganji, 2017). Injectable CS wrapped vancomycin liposomes nanosuspension can be

prepared by electrostatic deposition. This antibiotic delivery system possesses a

sustained drug release in vitro and a long retention time in vivo. The coating of

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polycationic CS can improve the stability and targeting of liposomes. Meanwhile, CS

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can make the liposomes positively charged to interact with the polyanionic cellular

membrane to promote the absorption and transportation of hydrophilic macromolecules.

-p
The positively charged properties and nanoparticle size are conducive to reducing the
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immune response and prolonging the circulation time in vivo, which can enhance the

therapeutic effects of antibiotics in osteomyelitis treatment (Z. Yang, Liu, Gao, Chen,
lP

& Huang, 2015). The promising therapeutic effects of the ciprofloxacin-loaded

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CS/bioactive glass scaffolds on osteomyelitis treatment have been found. In this

composite scaffold, CS plays an important role in supporting cell adhesion and

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proliferation, while the bioactive glass upregulates the ALP activity to facilitate cell

osteogenic differentiation. The CS/bioactive glass composite scaffolds exhibit suitable

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burst release of antibiotic followed by a sustained release profile, because CS can

provide the exceptional hydrophilic nature and fast biodegradation rate. Furthermore,

the addition of bioactive glass can fill up the micropores of the scaffolds to reduce the

hydration of the polymer matrix to delay the antibiotic release (Mostafa, El-Sayed,

20
Mahmoud, & Gamal-Eldeen, 2017). CS coating can also enhance the chronic

osteomyelitis treatment effects of calcium sulfate-based drug delivery systems. The

deacetylation degree of CS has an impact on cellular affinity and the elution rate of

antibiotics. Thus, the CS/calcium sulfate composite beads can achieve a high local

concentration of antibiotics and promote osteogenic cell adhesion, proliferation, and

bone mineralization (Beenken et al., 2014). In an animal experiment, ciprofloxacin

embedded CS films show high local concentration of drug and low side effects.

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Accordingly, they can be used as local antibiotic delivery systems for the prophylaxis

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of osteomyelitis in open fractures (Paiva Costa et al., 2016). A novel injectable

CS/borate bioactive glass cements can be prepared as local vancomycin carriers for
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treating osteomyelitis. Because CS possesses high viscosity, excellent biocompatibility,
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suitable degradation rate, and complexation property, the composite drug carriers

exhibit optimal elution characteristics of antibiotics and enhance compressive strength

lP

to eradicate osteomyelitis and support bone regeneration in the weight-bearing sites (H.
na

Ding et al., 2014). Similarly, the gentamicin or teicoplanin-loaded CS/borate bioactive

glass drug delivery systems also demonstrate a good therapeutic effect on bacteria-
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induced osteomyelitis and support the ingrowth of new bone in vivo (Jia, Zhang, Luo,

et al., 2010; Jia, Zhang, Zhang, et al., 2010; Xie et al., 2013; X. Zhang et al., 2010).
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Furthermore, the local release profile of the antibiotics in CS/bioactive glass composite

scaffold is correlated with the sizes of scaffolds and the concentration of the loaded

drug (Soundrapandian, Datta, Kundu, Basu, & Sa, 2010). Recently, a novel CS-calcium

phosphate composite scaffolds loaded with moxifloxacin hydrochloride are fabricated

21
by in situ precipitation method. These composite scaffolds are highly porous and

display a complete antibiotic release over three days. The CS-calcium phosphate

scaffolds not only show promotive effects on osteoblast proliferation and differentiation,

but also exhibit inhibitory effects on bacterial growth, inflammation and intra-

medullary fibrosis in osteomyelitis models (Radwan, Nasr, Ishak, Abdeltawab, & Awad,

2020). These results indicate that the CS-based bioactive scaffolds can be used as

antibiotic carrier materials to eradicate osteomyelitis and facilitate local bone

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regeneration.

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A disadvantage of the CS-based drug delivery system is the uncontrollable initial

burst of antibiotics, especially in porous scaffolds. To overcome the drawback of fast

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initial burst in porous CS/β-tricalcium phosphate ( β -TCP) composites, synthetic
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polymer PCL is utilized as a coating to retard the release of vancomycin. This composite

drug delivery system contributes to the enhancement of mechanical strength and the
lP

improvement of the drug release profile. It can be progressively substituted by the

na

newly formed bone (T. Fang, Wen, Zhou, Shao, & Dong, 2012). CS crosslinked

bioactive ceramics are fabricated as biocompatible antibiotic carriers. More than 75%
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of the entrapped vancomycin can be sustained and steadily released for 12 days to

maintain a high local concentration of vancomycin for inhibiting biofilms formation

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(Thanyaphoo & Kaewsrichan, 2012). In some cases, high concentrations of some

antibiotics at the bone infection sites can delay bone regeneration by disturbing the

osteoblast proliferation and differentiation. Novel complex antibiotic carriers are

composed of porous nano-HAp/CS/konjac glucomannan and loaded with cationic

22
liposomal vancomycin. The biodegradable scaffolds can control the release rate of

vancomycin by adjusting the composition ratio of CS and konjac glucomannan to avoid

the side effects for osteoblast growth and development (T. Ma et al., 2011). BMP-2 and

berberine can be loaded in the oppositely charged CS microspheres (CSM) and O-

carboxymethyl chitosan microspheres (CMCSM), respectively (Figure 2 A). These

scaffolds are scattered and compact microspheres with different diameters and a few

attached small flakes on the surface of CMS and CMCSM (Figure 2 B-E). There are

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many nanopores and micropores formed in their assembled structure (Figure 2 F-I) and

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can provide suitable microenvironment for osteoblast-like cell adhesion, spreading and

proliferation (Figure 2 J-L). These drug-loaded composite scaffolds possess dual

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effects of high antibacterial activity due to berberine/CMCS and
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significantly enhance osteoinductivity with the assistance of BMP-

2/CSM (Cai et al., 2018). CS coatings have good affinity for water absorption and
lP

can swell to form a diffusional barrier to retard drug release into the dissolution medium.
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β-TCP scaffolds bilayer coating of CS with antibiotics by the foaming method exhibit

a highly interconnected porous microstructure. The swelling properties of CS

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contribute to the prolonged drug release profile for combating osteomyelitis (Kundu et

al., 2010). CS has been found of good antibacterial activity because the positively
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charged CS can react with polyanion molecules on the cell surface to change the

permeability of bacteria. Additionally, the active amino groups of CS can interfere with

DNA to disrupt the synthesis of downstream RNA and protein (Adhikari & Yadav, 2018;

P. Shi et al., 2010). The gentamicin-impregnated CS/nano-HAp/ethyl cellulose

23
microspheres granules show a controllable drug release profile with a prolonged elution

time for 49 days in vitro and 45 days in vivo, meeting the requirement of four to six

weeks curative duration for chronic osteomyelitis treatment (P. Shi et al., 2010).

These results indicate that CS-based drug delivery systems can be prepared via

chemical conjugation and physical interaction. They are assembled to achieve diverse

shapes and structures, such as sponges, hydrogels, microparticles, fibrous scaffolds,

coating films, porous materials, and composites (Table 1). These drug delivery systems

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possess thermal and chemical stability to maintain normal antibacterial efficiency.

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Furthermore, the CS-based antibiotic carriers demonstrate optimized release kinetics in

osteomyelitis treatment by maintaining and continuously releasing high concentrations

of antibiotics locally.
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Table 1. CS-based drug delivery systems for osteomyelitis treatment
(MIC: minimum inhibitory concentration; DD: degree of deacetylation)

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Composite Method Mw/ DD Crosslinking Morphology Drugs Burst release Cumulative release Application Reference

Chitosan-calcium phosphate In situ 100-300 kDa / Glutaraldehyde CaP crystals homogenous Moxifloxacin 77.3% and 90.7 % on the Almost complete release Promising platforms in (Radwan et al.,

composite scaffolds precipitation 85% distribute within chitosan hydrochloride first day by the third day prevention of post-operative 2020)

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method matrix. osteomyelitis

CS-ZIF8 nanocrystals Wet-spinning -/85% Glyoxal 3-D fiber mesh with high Vancomycin 25% at pH 7.4 and 42% at 30% at pH 7.4 and 45% at Reduce infection and promote (Karakecili et al.,

and interconnected pH 5.4 in the first 24 h pH 5.4 for 144 h osteoblast proliferation 2019)

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porosity

CS-PCL sponges Lyophilization 190-310 - Highly porous Ciprofloxacin 30% for the first 3 h 78% for 12 d Anti-inflammatory and against (Pawar &

kDa/>75% interconnected surface hydrochloride infection Srivastava, 2019)

CS crosslinked mesoporous In situ foaming -/- Vanillin Micropores on walls and Levofloxacin - Sustained release for 42 d Candidate for osteomyelitis (J. Wei et al.,
a
silica microspheres modified method and microspheres on surface hydrochloride treatment 2019)

nano-HAp/polyurethane soaking
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CS, carboxymethyl cellulose Lyophilization 50-190 kDa/75- - Highly crystalline and Silver - - Overcome bone related (Hasan et al.,

and silver nanoparticle 85% porous structure nanoparticles infections like osteomyelitis. 2018)

modified cellulose
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nanowhiskers
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HPMC microparticles and Spray drying -/95% - Spherical structure with Vancomycin 42% for the first 8 h 94% for 72 h Treat osteomyelitis (Mahmoudian &

CS/glycerophosphate method different particle size Ganji, 2017)

thermosensitive hydrogel

CS with bioactive glass Lyophilization 190-480 - Interconnected porous Ciprofloxacin 50% for the first 9 h About 70% for 14 d As a localized osteomyelitis (Mostafa et al.,

kDa/65-82% structure with glass treatment 2017)

25
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particles dispersed

CS coated vancomycin Modified reverse 50 kDa/- - Spherical shapes with Vancomycin 40% for the first 4 h About 70% for 70 h Treat osteomyelitis, (Z. Yang et al.,

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hydrochloride liposomes phase evaporation different particle size endocarditis and pneumonia 2015)

and electrostatic

deposition

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CS-coated calcium sulfate Mixture and drying 210 kDa/78% - Pellets Daptomycin 1000 times MIC after 1 d 100 times MIC after 10 d Treat chronic osteomyelitis (Beenken et al.,

implants in a desiccator after surgical debridement 2014)

CS-bonded borate bioactive Mixture -/- - Heterogeneous Vancomycin 44% for the first 2 d 86% for 36 d Eradicate osteomyelitis and (H. Ding et al.,

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glass particles microstructure with some promote new bone regeneration 2014)

pores

HAp and CS nanoparticles Ultrasound- 190-310 kDa/- - Round particles with Clindamycin 15% in the first 24 h About 90% for 21 d Treat osteomyelitis and (Uskokovic &

lP
assisted sequential sharp edges promote bone regeneration Desai, 2014)

precipitation

PCL coated CS/β-tricalcium Soaking 200 kDa/91% Highly porous structure Vancomycin 7.12% in the first 1 d About 72% for 42 d Treat MRSA-related (T. Fang et al.,

phosphate with a coating layer osteomyelitis 2012)

a
CS crosslinked Si-HAp porous Wet chemical -/- CS Bone-like apatite Vancomycin 12%-15% in the first 4 h 20%-30% for over 12 d Aid long-term healing of (Thanyaphoo &

scaffold method deposited on the surface osteomyelitis Kaewsrichan,

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2012)

Nano-HAp/CS/konjac Mixture -/- - Scaffold with small Cationic - About 50% for 72 h Treat biofilms infection caused (T. Ma et al., 2011)

glucomannan scaffolds cracks and pores liposomal osteomyelitis

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vancomycin
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CS-bonded borate bioactive Mixture -/98% - Smooth surface with Teicoplanin 47% in the first day 83% for 30 d Treat MRSA induced chronic (X. Zhang et al.,

glass pellets drying cracks and air osteomyelitis 2010)

bubbles remnants

CS/nano-HAp/ethyl cellulose Mixture and 40 kDa/95% - Round granules with Gentamicin 38% at the first day Sustained 49 days in vitro Treat chronic osteomyelitis (P. Shi et al., 2010)

microspheres granules freeze-drying coarse surface and 45 days in vivo

26
 of
CS microspheres Spray drying 190-310 kDa/ - Spherical with porous Vancomycin - 58.68% for 170 h Eradicate implant-related (Cevher et al.,

method 75-85% surface and roughness hydrochloride osteomyelitis 2006)

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27
 of
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Figure 2. (A) Scheme of the fabrication of the oppositely charged microspheres
lP

(chitosan microspheres CSM, O-carboxymethyl chitosan microspheres CMCSM,

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berberine Bbr). The SEM images of the morphology (B-C) and particle size

distribution (D-E) of the CSM and CMCSM. (F) SEM image and (G) fluorescence
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image of the composite scaffold. (H) LSCM fluorescence image of the

assembled scaffold containing CSM (red) and CMCSM (green) and the 3D
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reconstruction images (I). (J) SEM observations of cell adhesion; (K)

Fluorescence staining of cell morphology (red: F-actin, green:

mitochondria, blue: nucleus); (L) Fluorescence images of living cells

growing in CSM and CMCSM after culturing for 1 day and 4 days. Reprinted

28
with permission from ref.(Cai et al., 2018). Copyright 2018 American

Chemical Society.

4.2 CS-modified implants against osteomyelitis

Implants-related infection is an important cause of osteomyelitis. The risk of

implants-related infection is 2-5% in orthopedic surgeries (Chouirfa, Bouloussa,

Migonney, & Falentin-Daudre, 2019). The colonization of bacteria on implants can

induce tissue inflammation, promote biofilms formation, disrupt the osseointegration

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process, and accelerate metal corrosion, eventually leading to implants failure (Arciola,

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Campoccia, & Montanaro, 2018). The treatment of implants-related infection

represents an orthopedic surgical challenge. The dual functions of anti-bacteria and

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osseointegration are required for the design of ideal orthopedic implants (Zhao et al.,
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2020). Surface modifications and coatings of implants can reduce bacteria adhesion,

inhibit biofilms formation and improve the affinity of osteogenic cells to facilitate
lP

osseointegration (Chouirfa et al., 2019). Titanium (Ti) and its alloys are the most
na

commonly used orthopedic implant materials, which have excellent mechanical

properties and biocompatibility (Jing et al., 2020). However, implants-related infections

ur

remain the main cause leading to Ti implants failure. Owing to the special biological

properties of CS, the CS-based biomaterials can be applied to modify and functionalize
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the orthopedic implants to prevent the implants-related infections (Pawar, Topkar, &

Srivastava, 2018). Recently, the CS modified implants are widely used in the treatment

of osteomyelitis (Table 2).

TiZr alloys with a nanostructured surface can be modified by the gentamicin-

29
loaded CS delivery system. This composite implant exhibits more hydrophilic and

higher molecular polarity than the simple TiZr. The thickness of CS film controls the

drug release with predictable kinetics, which enables the implants to have desired and

targeted treatment effects for osteomyelitis. The CS-modified implants can suppress

inflammation in the short-term, demonstrate a better antibacterial effect in the middle-

term, and promote fracture repair in the longer-term (Stoian, Demetrescu, & Ionita,

2020). The CS modification can also improve biocompatibility and antibacterial

of
properties of Ti implants. The CS/alginate multilayer films modified TiO2 nanotube

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arrays can be coated on the surface of the Ti substrate. The surface modification can

accelerate osteoblast growth, enhance osseointegration, and reduce bone infection by

-p
inhibiting bacteria adhesion and controlling antibacterial drug release from TiO2
re
nanotube arrays (P. Liu et al., 2017). TiO2-SiO2/CS-Lysine modified Ti alloys can be

good carriers for the local delivery of gentamicin in osteomyelitis treatment. The
lP

composite implants show high gentamicin loading efficiencies because of the abundant
na

nano pores and active OH- and NH2 groups, which provide the implants excellent

affinity to gentamicin molecules. Furthermore, these ceramic/biopolymer coated

ur

implants demonstrate not only the first burst release and sustained release of gentamicin,

but also appreciable corrosion resistance in vitro (Mohan Raj, Priya, & Raj, 2018).
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Vancomycin-loaded CS/gelatin coatings can be immobilized on Ti implants by

electrophoretic deposition without the cytotoxic chemical crosslinking. The addition of

CS and gelatin can facilitate the ingrowth of biomineralization and vascularization.

These composites demonstrate dual functions of inhibiting biofilms formation and

30
enhancing cellular interaction at the bio-interface, making them promising candidates

for eradicating osteomyelitis and promoting bone regeneration (D & N, 2018; David &

Nallaiyan, 2018). Vancomycin loaded CS/HAp composites can be immobilized on the

surface of Ti6Al4V implants by the electrochemical method. Vancomycin and CS are

tightly conjugated by the hydrogen bonds and filled in the pores of HAp coatings to

form a dense and smooth film (Figure 3 A-B). The first burst release of vancomycin is

reduced to 55% and sustained for more than 30 days after modification due to the polar

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water molecular mediated destruction of hydrogen bonding (Figure 3 C). The enhanced

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antibiotic elution kinetics contributes to maintaining the stable antibacterial function

for chronic osteomyelitis treatment in animal models (Figure 3 D). Additionally, the
-p
vancomycin loaded CS/HAp composite coating can also promote osteointegration on
re
the Ti6Al4V implants by facilitating osteoblast-like cell proliferation, differentiation,

and mineralization (Figure 3 E-H) (C. C. Yang, Lin, Liao, & Yen, 2013). The Ti6Al4V
lP

alloys surface-modified by LBL deposition of hyaluronic acid/CS polyelectrolyte

na

multilayers demonstrate excellent antibacterial properties. The CS-based coating can

significantly reduce the contact angle of the implant surface and improve hydrophilic
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behavior similar to the smooth surface. It is beneficial for inhibiting bacteria adhesion

and proliferation to prevent implants-related infection (Valverde et al., 2019). The

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physical and chemical properties of CS can be changed by functional groups

substitution, and the derivates of CS can also exert promotive effects on the

improvement of antibacterial and osteogenic properties of implants. The

hydroxypropyltrimethyl ammonium chloride chitosan (HACC) based multilayer

31
coating on the porous Ti is fabricated by the LBL covalent-immobilized method. The

CS derivative mediated surface modification can inhibit bacteria colonization and

biofilms formation, and prevent bone destruction in vivo. The slow release of HACC

after degradation by collagenase can eliminate the planktonic bacteria, while the

remaining HACC contributes to the suppression of the colonized bacteria. The CS

derivative HACC assisted surface modification is a novel potential strategy to prevent

implants-related infections in orthopedics (Ao et al., 2019).

of
Therefore, the antibacterial activities of implants can be significantly improved by

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modifying or coating with CS-based antibiotic delivery systems. This therapeutic

strategy is conducive to maintaining prolonged antibiotic release to inhibit bacteria

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colonization on implants surfaces and providing an affiliative interface for osteogenic
re
cell growth and osseointegration. Thereby, CS-based implants modification is a

promising strategy to prevent the occurrence of implants-related osteomyelitis and

lP

promote the peri-implant osseointegration.

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33
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Table 2. CS modified implants for osteomyelitis treatment

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Composite Method Morphology Drug Application Reference

CS covered nanaotubular TiZr Dip-coating Nanoporous surface Gentamicin Anti-inflammatory and reduce infection (Stoian et al., 2020)

CS coated porous Ti Direct metal printing and Coating with Ag Ag Against infection in vitro, but not reduce (Croes et al., 2018)

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electrophoretic deposition nanoparticles infection in vivo

TiO2-SiO2 and CS-Lysine coated Electrophoretic deposition Hierarchically porous Gentamicin Inhibit infection and against corrosion (Mohan Raj et al., 2018)

Ti alloy structures

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CS deposit on HAp coated Electrolytic deposition Dense and smooth film Vancomycin Prophylaxis and therapy of osteomyelitis (C. C. Yang et al., 2013)

Ti6Al4V implant

Ti coated with TiO2-SrHAP and Co-precipitation and A smooth uniform well- Vancomycin Control osteomyelitis and favor bone (D & N, 2018)

lP
CS/Gelatin electrophoretic deposition oriented morphology mineralization

CS/alginate multilayer films LBL self-assembly A uniform smooth surface Gentamicin Improve osteoblast growth and enhance (P. Liu et al., 2017)

coated TiO2 nanotube arrays on Ti antibacterial properties

Hyaluronic acid/CS (Mw: 261 LBL deposition A smooth surface Triclosan Enhance the antibacterial properties of (Valverde et al., 2019)
a
kDa, DD: 79.4%) polyelectrolyte implant

multilayers coated Ti6Al4V

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alloys

GHK-Cu loaded mesoporous Electrophoretic deposition Particles homogeneously Cu2+ Show excellent antibacterial properties (Ning et al., 2019)

silica nanoparticles -CS (DD: distributed

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≥75%) coated Ti
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(DD: Degree of deacetylation)

34
 of
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lP
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Figure 3. SEM observation of the vancomycin-CS/HA composite coated specimen for

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cross section (A) and surface morphology (B). (C) The curve of drug release from the

vancomycin-CS/HA composite coated specimen. (D) The X-ray image for Ti6Al4V

alloy pin implanted in the tibia of rabbit. (E-H) SEM images of osteoblast-like cells on

the vancomycin-CS/HA composite coated specimen. Reprinted with permission from

35
ref.(C. C. Yang et al., 2013). Copyright 2013 Elsevier.

4.3 CS derivatives in osteomyelitis treatment

CS, a kind of polysaccharide polymers, also has some characteristics that limit its

application in tissue engineering. For example, CS always needs to be dissolved in

acidic solvents for preparing biomaterial scaffolds. The residual solvents can disrupt

the homeostasis of the microenvironment in vivo and have a negative impact on cell

growth and development (Tao et al., 2020). The unmodified CS molecules are easily

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hydrolytically cleaved and metabolized by lysozyme and N-acetyl-β-D-

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glucosaminidase in vitro and in vivo (Lim et al., 2008). These characteristics will restrict

the application prospects of CS in tissue engineering. Therefore, the chemical

-p
modification and functionalization of the CS molecular chain enable the scaffolds with
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special biological properties to improve the therapeutic effects of osteomyelitis and

promote bone regeneration. The chemical modification and functionalization of CS can

lP

be realized by substituting and reacting with the active hydroxyl and amino groups in
na

the molecular chain. These structural and functional changes can expand the application

of CS in different fields (J. Ma, Zhong, Peng, Xu, & Sun, 2020). Recently, various
ur

studies have found that CS derivatives demonstrate excellent drug delivery efficiency

for the treatment of osteomyelitis (Table 3).

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Carboxymethyl chitosan (CMCS), as a water-soluble derivative of CS, possesses

better hydrophilicity than CS and accordingly, it has attracted increasing attention in

osteomyelitis treatment. CMCS-based scaffolds exhibit excellent antibacterial

properties and promotive effects on bone regeneration (Z. Shi, Neoh, Kang, Poh, &

36
Wang, 2009). A polysaccharide-based hydrogel is fabricated by introducing tetracycline

hydrochloride (TH) loaded calcium carbonate microspheres (CM) and HAp into CMCS

and oxidized alginate (OAlg). The composite scaffold is crosslinked by the Schiff-base

reaction between CMCS and OAlg to enhance bioactivity and mechanical strength

(Figure 4 A). This drug delivery vehicle is injectable (Figure 4 B) and the drug-loaded

CMs show a coarse surface to delay the burst release for TH (Figure 4 C-D). The freeze-

dried composite hydrogels exhibit porous structures and the porous walls can uniformly

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disperse in PBS (Figure 4 E-J). These special structural features can contribute to

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maintaining the good mechanical property and the high antibacterial activity in bone

tissue engineering (Ren et al., 2018). Owing to the hydrolyzation and ionization of the
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acidic carboxyl groups and basic amino groups, CMCS can be treated as an amphoteric
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polysaccharide at different pH conditions. For example, it can be positively charged in

an acidic condition and negatively charged in a neutral or alkaline environment (Tu et

lP

al., 2019). The active amino groups and carboxyl groups of CMCS can bind
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to the charged surface of bacteria to destroy the membranes homeostasis

and disrupt the substances transmission of an intra- or extra-cellular

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environment. This characteristic contributes to the improvement of the

inherent antibacterial properties of the polysaccharide polymer

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derivative (Maya et al., 2012; Tu et al., 2019). A kind of

multifunctional scaffolds with the porous structure is fabricated by

the addition of copper (Cu) nanoparticles into anionic CMCS and

alginate. These composite scaffolds are gradually crosslinked by the

37
sustainable release of the antibacterial Cu2+ ions and show multiple

functions of inhibiting bacteria and promoting osteoblast adhesion and

osteogenic differentiation (Lu et al., 2018). CMCS can also be applied

to modify and functionalize the surface of implants to facilitate

antibacterial activity and bone regeneration simultaneously. The CMCS-

functionalized Ti substrates exhibit excellent antibacterial

properties and enhance osteoblast mineralization by conjugating

of
vascular endothelial growth factor (VEGF) on the CMCS surface (Hu et al.,

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2010). The CMCS modified Ti substrates show an inhibitory effect on

bacterial colonization and contribute to the promotion of osteoblast

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growth and mineralization after being functionalized by BMP-2 (Zheng,
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Neoh, Shi, & Kang, 2013). A kind of newly developed CMCS and bone-

forming peptide-conjugated biocomposites demonstrate dual functions of

lP

significantly bacterial adhesion inhibition and osteointegration

acceleration (Xu et al., 2016). Additionally, CMCS-Zn2+ coated implants

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show antibacterial properties in vivo and are effectively used in

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preventing pin tract infection (Martin et al., 2018). Thus, CMCS-based

biomaterials possess good antibacterial activity, excellent biocompatibility, and

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osteoinductivity, and can be treated as potential transplantable scaffolds for

osteomyelitis treatment.

The antibacterial activity of CS is always restricted by its poor solubility in water. To

solve this problem, a new quaternary ammonium CS derivative has been prepared. The

38
antibacterial activity and water solubility can be improved by increasing the chain

length of the alkyl substituent (Z. Shi, Neoh, Kang, & Wang, 2006). PMMA bone

cements modified by adding quaternary ammonium CS derivative nanoparticles show

high antibacterial effectiveness and have no significant negative effects on the

mechanical properties of bone cements. These composite bone cements have potential

applications in combating joint implants-related infection (Z. Shi et al., 2006). CS

derivative HACC exhibits excellent biocompatibility in biomedical applications and

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can be fabricated as desirable drug delivery systems. A kind of BMP-2-loaded HACC

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scaffold exhibits good antibacterial efficiency and controlled release of a growth factor.

This HACC-based biomaterial can be a multifunctional scaffold with sequential

-p
antibacterial and osteoinductive properties for treating osteomyelitis (D. Wang et al.,
re
2019). Furthermore, HACC-loaded PMMA scaffolds demonstrate strong antibacterial

activity and good biocompatibility with osteogenic cell simultaneously. This composite
lP

scaffold can effectively inhibit biofilms formation on the implant surface and suppress
na

virulence-associated gene expression of antibiotic-resistant staphylococcus. Thus, it is

utilized in combating implants-related infections and osteomyelitis (Tan et al., 2012).

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Additionally, another water-soluble CS derivate N-trimethyl chitosan (TMC) can be

fabricated into a high-efficiency antibiotic delivery system. The vancomycin-loaded

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TMC nanoparticles exhibit great biodegradable, cytocompatible, and antibacterial

properties. In this drug delivery system, the positively charged feature and the large

surface area contribute to the high protein adsorption and promoting vancomycin

transport across the cytomembrane. The cumulation of active regulatory proteins on the

39
scaffolds can facilitate osteoblast adhesion, infiltration and proliferation. These

vancomycin-loaded TMC nanoparticles show great potential for promoting bone

healing and can be promising candidates for chronic osteomyelitis treatment (Y. Zhang

et al., 2017).

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Table 3. CS derivates drug delivery systems for osteomyelitis treatment

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Composite Method Mw / DD Crosslinking Morphology Drug Application Reference

Quaternised CS-loaded PMMA Mixture 200 kDa / 91.83% - Well-connected highly porous Gentamici Combat implant infections and (Tan et al., 2012)

structures n osteomyelitis

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N-trimethyl CS nanoparticles- Mixture and drying in -/- - Spherical nanoparticle on Vancomyc Treat chronic osteomyelitis and (Y. Zhang et al.,

poly(trimethylene carbonate) vacuum surface in promote bone healing 2017)

Hydroxypropyltrimethyl ammonium chloride Mixture 150 kDa / 95–98% - Granules embedded in CS BMP-2 Treat infected bone defects (D. Wang et al.,

re
CS network 2019)

Copper-containing CMCS/alginate scaffolds Freeze-drying - 2% CaCl2 An interconnected porous Cu2+ Promote new bone formation (Lu et al., 2018)

structure and avoid bacterial infection

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CMCS decorated polyetheretherketone ternary Polydopamine tag strategy 9.5-20.9 kDa / ≥86% - Homogeneous dispersion of Bone- Have high disinfection efficacy (Xu et al., 2016)

biocomposite carbon fibers and nano-HA forming and better osteointegration

particles on the surface peptide

CMCS-modified Ti Anchoring by dopamine -/ ≥75% - - BMP-2 Inhibit bacterial colonization (Zheng et al.,
a
and enhance osteoblast 2013)

functions
rn
CMCS/oxidized alginate hydrogel blended with Freeze-drying -/- Chemical cross-linking via Interconnecting porous three- Tetracycli Show effective antibiotic (Ren et al.,

calcium carbonate microspheres/ HAp Schiff-base reaction dimensional structure ne activity 2018)

nanoparticles hydrochlo
u

ride
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Hydroxypropyltrimethyl ammonium chloride Mixture 200 kDa / 91.83% - Particles distributed and well- - Inhibit biofilms formation (Tan et al., 2012)

CS loaded PMMA connected pore structure

Quaternary ammonium CS derivative Mixture -/ 84% Tripolyphosphate Nanoparticles distributed on - Combat bacterial infections in (Z. Shi et al.,

nanoparticles in PMMA the surface joint replacements 2006)

41
 of
ro
-p
re
lP
na
ur

Figure 4. (A) Schematic summary of the TH loaded CMCS-OAlg-HAp-CMs hydrogel

scaffolds (carboxymethyl chitosan CMCS, and oxidized alginate OAlg, hydroxyapatite HAp,
Jo

calcium carbonate microspheres CMs, tetracycline hydrochloride TH). (B) An image shows the

gel scaffold injected from a syringe needle. (C-D) SEM images of the TH loaded CMs under

different magnification. (E-H) SEM images of 6% HAp/gel scaffolds combined with different

concentration of CMs after immersed in PBS for 1 h. (I-J) High magnification images of (G).

42
Reprinted with permission from ref. (Ren et al., 2018). Copyright 2018 Elsevier.

However, many factors affect the antibacterial properties of biomaterials and the

elution profile of antibiotics, such as physicochemical properties, porosity,

biodegradability, drug loading capacity, and the differences in microenvironments.

Specific capacities for inhibiting bacteria of many CS-based biomaterials have been

proven. However, not all CS-assisted drug delivery systems show significant

therapeutic effects on combating osteomyelitis. For example, the CS-based coatings on

of
Ti implants incorporated with different concentrations of Ag nanoparticles demonstrate

ro
enhanced antibacterial activity in vitro but poor antibacterial effects on osteomyelitis in

vivo. Furthermore, this composite CS/Ag coating shows negative regulatory effects on
-p
bone remodeling by enhancing osteoclast activity and disturbing innate immune
re
response, resulting in the abnormal regeneration and the persistent infection of the bone

(Croes et al., 2018). A novel drug carrier fabricated by impregnating CS films with
lP

ciprofloxacin presents a high local concentration of antibiotic and low systemic side
na

effects. However, this CS-based drug delivery system is not effective in eradicating

infection in vivo and does not exhibit significant efficacy in the prophylaxis of
ur

osteomyelitis secondary to open fracture (Paiva Costa et al., 2016). The nanoparticulate

HAp/CS composite scaffolds are considered as potential drug delivery systems. These
Jo

scaffolds can mitigate the initial burst release and promote sustained release kinetics of

antibiotics in the treatment of osteomyelitis. However, the antibacterial efficacy of the

clindamycin-loaded HAp/CS scaffolds has been reported as markedly lower than that

of the antibiotic-loaded HAp. Furthermore, the addition of CS to HAp nanoparticles

43
shows negative effects on osteoblast proliferation, decreasing osteoblast spreading and

adhesion in biomaterials/cell interface, and delaying osteoblast osteogenic

differentiation in vitro (Uskokovic & Desai, 2014). These results indicate that the

therapeutic effects of CS-based biomaterials as drug delivery systems for osteomyelitis

is not invariable, and various factors affecting material properties and drug release

kinetics need to be considered. The loading capacity of the drug, the observation time

of the experiment and the differences in microenvironments in vivo and in vitro may

of
directly affect the release efficiency and antibacterial activity. Therefore, more rigorous

ro
experimental schemes and material designs are required to verify the therapeutic effects

of CS-based biomaterials on the treatment of osteomyelitis in vivo.

5. Conclusion and future perspectives

-p
re
Osteomyelitis represents a severe infectious disease that needs to be treated timely

and efficiently. Most CS-based local drug delivery systems exhibit dual functions of a
lP

controlled antibiotics release profile for eradicating osteomyelitis and promotion effects
na

on bone regeneration. CS, as a naturally derived polymer, possesses numerous

advantages such as biocompatibility, biodegradability, bioadhesion, nontoxicity and

ur

antibacterial activity. These biological properties significantly contribute to various

applications in tissue engineering. The extensive literature reveals that CS-based drug
Jo

carriers can mitigate the first burst release and facilitate the prolonged release of

antibiotics in the treatment of osteomyelitis. These CS-based drug delivery systems

show desired elution kinetics for antibiotics release and the versatile bioactive

characteristics for bone regeneration, attributing to the polycationic properties, the

44
abundant bioactive groups, and the deacetylation degree of CS. These critical biological

properties of CS are conducive to eliminating bacteria in local infected sites and coating

the orthopedic implants to prevent infection. However, it is necessary to pay more

attention to the negative regulatory factors on biomaterial physicochemical properties,

drug elution profile, and bone regeneration mechanism. In conclusion, CS-based

biomaterials are promising candidates for eradicating osteomyelitis and favoring bone

regeneration.

of
Declaration of Competing Interest

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There are no conflicts of interest to declare.

Acknowledgements
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This work was supported by the National Natural Science Foundation of China (No.
re
51873157), National Key R&D Program of China (No. 2016YFB0303303), and the

Fundamental Research Funds for the Central Universities (2042019kf0064).

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