Recent Trends in the Use of Bioceramics for Treatment of Osteomyelitis

Despite the inherent ability of bone for self-repair, this spontaneous healing capability in some bone disorders is not sufficient. Diseases as osteomyelitis, osteosarcoma, and osteoporosis, usually demand medical and/or surgical interventions to enhance tissue regeneration, control infection or to handle the clinical condition. Osteomyelitis (OM) is a bone infection disease, where Staphylococcus (S.) aureus is the main causative microorganism. OM is characterized by elevated rates of relapse and mortality. Coupling local osseous delivery of antibacterial agents with bioactive agents capable of bone regeneration was intensely studied for the treatment of OM, proving their effectiveness. Bioceramics are widely investigated due to their osteoconductive and osteointegration nature. Among these are calcium phosphates (CP), which are distinguished by a similar structure to that of bones and diverse resorption rates. CP is applied in the bone regeneration field, either solely or as composites with different polymers, as scaffolds, pastes, cement, and hydrogels. In this review we overview OM disease with its pathogenesis and treatment, especially focusing on different CP-bioceramics used for bone repair.


INTRODUCTION
Bone is a type of connective tissue in the higher vertebrates, characterized by its mineral architecture [1,2]. Being hard, it is responsible for locomotion, support and soft tissue conservation as well as storage of calcium and phosphate, and holding the bone marrow [2]. Bone frequently undergoes remodeling which is an active process, where old bone is resorbed by osteoclasts and new bone is formed by osteoblasts [3,2]. This regular bone remodeling which occurs through harmony between bone cells namely; osteoclasts, osteoblasts, osteocytes and lining cells, is essential for fissures healing, compliance of skeleton for mechanical benefits and calcium equilibrium in the body [4,2]. Any disturbance of the normal bone remodeling process leads to bone diseases such as osteoporosis and osteopetrosis, also known as marble bone disease or "stone bone" [2].
The basic framework of bones is comprised of outer cortical bones and inner trabecular bone tissues, as illustrated in Fig. 1 [3,5,6]. Cortical bones are compact, surround the bone marrow and the trabecular plates and are developed from the Haversian system. The latter is comprised of concentric lamellae encompassing blood vessels harbored in a medial canal. The spongy trabecular tissues form a grid with a honeycomblike structure composed of trabecular plates and rods, which are dispersed throughout the marrow cavities [1, 3, 5]. Bone diseases involve several skeletalrelevant disorders that can cause mobility difficulties to deaths along with time [7]. The most common bone disorders are osteoporosis, osteoarthritis, osteosarcoma, metastatic bone cancer, osteomyelitis, and bone degenerative disorders [7,8]. In bone defects management, surgery and bone tissue engineering are the most applied techniques with the local or targeted delivery of drugs, growth or bioactive factors [7, 8].

Osteomyelitis (OM)
OM is a grievous infectious disease of the bones, characterized by progressive destruction of the bone, associated with high recurrence rate, morbidity and high treatment cost [9,10]. The infection can include one part of the osseous tissues or extend to other sites as the bone marrow, cortex, periosteum or the neighboring soft tissues [9]. Several microbes can induce OM; the most predominant microorganism is (S. aureus) accounting for 90% of the cases [11,12]. Other microbes include Pseudomonas aeruginosa, Candida species, Mycobacterium tuberculosis, Brucella species, and others [9, 13,14].
S. aureus has a powerful adaptive competency and discharges virulence factors that alter the host immune response [15,10]. This bacterium is characterized by biofilm formation which is the leading reason for developing bacterial resistance [16]. It releases adhesive components on its surface that advocate attachment to bone extracellular matrix proteins as fibronectin, fibrinogen, collagen, bone sialoprotein, elastin, and others [9, 17,12]. Additionally, it can invade viable cortical bone cells resulting in biofilm deposition inside osseous lacunae [12].

OM Classification
OM can be classified in several ways based on; the chronicity (whether acute or chronic), the etiology of the disease (either due to hematogenous migration of the causative organism, contiguous spread following injury or trauma or secondary to vascular or neurologic insufficiency) according to Waldvogel classification, and the anatomic factors combined with the physiological classes (Cierny-Mader classification) [13]. The latter classification helps to stratify the basic elements for treatment according to the magnitude of bone necrosis, the patient's condition and the influence of OM on body functions [18, 19].

Pathogenesis of OM
Bones are normally resistant to infection due to the unique physiological and anatomic features. When an infection reaches the bone, a series of inflammatory processes occur due to inflammatory factors and leukocytes migration leading eventually to necrosis of bone tissues. The ischemia accompanying the inflammatory processes leads to compressed and destroyed vascularity, that in turn ends with necrotic bone tissues called sequestra [13]. These sectors of bones deprived of vascular supply can carry bacteria and pus despite antibacterial treatment [17]. Due to the active hyperemia on the infarction boundary, inflammatory cells and their cytokines provoke bone resorption by osteoclasts and fibrous tissue growth with new bone deposition on the damaged periosteum [20,21]. This new osseous tissue surrounding the necrotic infected sequestrum is termed involucrum. The pathogenesis of OM is divided into three stages, as illustrated in Fig. 2

Treatment of OM
In most cases, the treatment of chronic OM necessitates the combination of surgical intervention and systemic antibiotic administration [14]. Sufficient debridement of necrotic tissues and sinus tracts by surgery is a keystone for efficient treatment. Surgery has vital roles other than the removal of necrotic osseous tissues, these include the abolition of dead spaces left after the debridement of necrosis, osseous stabilization and covering of soft tissues [14]. As in oncology, conservative and provincial abscission is accompanied by high relapse rates [9,14]. Hence, the removal of the entire necrotic infected tissues and biofilm and assuring sufficient blood supply preliminary to medical therapy is mandatory [14]. This is followed by systemic administration of antibiotics for an extended time course; most probably 4 to 6 weeks of intravenous administration is the standard for OM treatment. However, some clinicians suggest longer courses for eight weeks followed by oral antibiotic therapy for three months in cases of high relapse and recurrence rates [14].
A major pitfall is that the systemic delivery of antibiotics hinders their efficacy owing to firstpass metabolism and their distribution to different body organs, hence, only a small fraction can hit the infection site [22]. Moreover, in the case of OM, the demolition of the local vascular supply makes it more strenuous for the antibacterial agents to reach their target site [22]. The systemic toxicity associated with high levels of antibiotics in the body as hepatotoxicity and nephrotoxicity, along with the emerging crisis of bacterial resistance; limit increasing the dosage of the given antibiotic to compensate for the low drug levels at the infection spot [23,24]. So, it is believed that the local delivery of antibiotics is beneficial for delivering sufficient concentration of the antibiotic at the bone tissues with low blood concentration levels [25]. Different systems for antibacterial delivery for the treatment of OM are illustrated in Fig. 3.

Local osseous delivery
Local drug delivery was primarily introduced to restrain bone infection in the sixties through "closed irrigation" of antibiotic solutions [26]. Nowadays, bone fillers loaded with actives are extensively employed to deliver drugs intraosseously. The optimum bone filler should be able to deliver its loaded antibiotic locally with a controlled release profile and to aid bone growth to restore the osseous cavities left after the debridement of necrotic tissues.
Ideally, the perfect bone scaffold for tissue repair should provide a suitable network with adequate porosity permitting vascularization and new bone cell penetration and growth. Besides, it should be able to arbitrate osteoconduction, osteoinduction, osteointegration, and osteogenesis processes. Osteoconduction is the stimulation of new bone deposition by providing the optimum conditions and the skeleton for osteogenic and neoplastic cells adhesion and bone penetration. Osteoinduction is the stimulation of stem cell differentiation into osteoblasts that are capable of bone formation similar to bone morphogenetic proteins (BMP). Osteointegration is the attachment between native bone cells and the bone filler, with new cells gradually substituting the device as they grow. Finally, osteogenesis is the process of new bone synthesis [27,28]. Another important aspect of the ideal bone scaffold is to degrade at a rate that matches the process of new bone growth to allow host cells to replace it, and to resorb with neither toxic byproducts nor inflammatory response [29].

Non-degradable fillers
Poly (methyl methacrylate) (PMMA) bone cement/beads bearing various antibiotics are used for local treatment of OM. They can release their loaded antibiotics slowly and act as bone fillers [26,30]. They are commonly used as they are the only approved pre-installed devices by the US Food and Drug Administration (FDA) [25]. However, PMMA is not clinically desirable as its polymerization reaction is exothermic producing heat [30,25], and their remnant unreacted monomers are toxic [31,30]. Thermolabile antibiotics are not suitable to be loaded on PMMA due to the high exothermic reaction temperature [32]. Besides, their loaded carriers fail to attain the sustained release required with only a small fraction of the loaded antibiotic being released, this may be due to the nonbiodegradability of the polymer which prohibits their release from the matrix core. After the initial burst release, the sub-inhibitory concentration of the antibacterial agent is released, causing the carrier itself to act as a surface where bacteria can thrive forming colonies and new biofilm further contributing to the development of bacterial resistance [31,25]. Moreover, PMMA is deprived of the osteoconduction features so their attachment to bone cells is inadequate [31,25]. Being nonbiodegradable, a second surgery is required for their elimination as their presence can hinder the new bone formation and regeneration process and may allow bacterial colonization on their surfaces [32, 22].

Biodegradable fillers
Biodegradable systems for local delivery of antibiotics are the focus of many researchers nowadays. They eliminate the need for a second surgery for their removal. Besides, being biodegradable they prevent the possibility of bacterial growth as in the case of bio surfaces Synthetic polymers can be facilely controlled in terms of their physicochemical, mechanical and bio-resorption rates [7]. For example, polylactide (PLA) is used in bone devices and implants due to its huge mechanical strength [38,7]. Poly-lactide-co-glycolic (PLGA) is a polyester that is approved for bone repair applications by the FDA [7]. It has optimal biodegradation behavior with minimum inflammatory reaction stimulation [39]. Polycaprolactone (PCL) is extensively used in bone regeneration due to its attractive mechanical properties and manufacturability [37]. Polyurethane (PU) comprises a family of synthetic elastomers composed of soft segments of polyester chains and hard segments including mainly polyurethane blocks. They have good mechanical properties driving their use in biomedical devices. However, they suffer low biocompatibility due to their released toxic degradation products, which can be alleviated through creating chemical linkages that are broken in the biological conditions [40].
However, the use of polymeric scaffolds/cement alone is restricted due to their low mechanical strength and the inflammatory reaction produced by the acidic environment resulting from the degradation of some of the synthetic polymers. Hence, composites of ceramics with biodegradable polymers offer suitable osteoconductive and osteoinductive systems for efficient treatment of OM [7, 41].

Bioceramics
Ceramics are inorganic compounds with ionic and covalent bonds combination. Bioceramics are those proposed to be intermingled with viable tissues [42]. Bioceramics; as calcium phosphates, calcium sulfates, and bioactive glasses, has the merits of biocompatibility and the ease of recognition and acceptance by the body [ CP bioceramic powders fabrication techniques are based on dry or wet chemical synthesis. Dry ones depend on reactions of the solid-state as redox reactions and thermal decomposition, while wet methods involve wet precipitation, solgel synthesis, hydrothermal synthesis, or spray drying [58,45]. Wet precipitation technique is favorable as it produces a homogenous product, the processing parameters as temperature and pH can be controlled, and including additives during the synthesis is possible [58,45]. Various CPbased systems are illustrated in Fig. 4.

Fig. 4. Various CP-based systems
Several studies based on composites that can be loaded with drugs and/or growth factors for local osseous delivery and bone repair and regeneration applications were described in the literature.

Calcium phosphate cements (CPCs)
CPCs are settable forms of calcium phosphates; they can be used as bone fillers and tailored into customized shapes according to the defect [59, 60]. They are CP powder, that upon admixing with the proper liquid they turn into a paste [59]. These pastes can be injected into the defect site and harden after implantation offering biocompatibility and osteoconduction to the bone defect  Stigter et al. applied a carbonated hydroxyapatite coating on titanium alloys through the biomimetic precipitation method. They incorporated different antibiotics into the coatings and assessed their release and antibacterial efficiency through in vitro studies, assuming that the biomimetic precipitation technique was favorable for drug incorporation within the coatings rather than the plasmaspraying technique, as the latter includes high temperature during processing. They concluded that the release of drugs incorporated into the coatings depends on the porosity and permeability of the coating and the chemical structure and binding of the drug with the coating

Hydrogels containing CP
Hydrogels are gels formed from networks of 3D hydrophilic crosslinked polymers. Being highly hydrophilic, they can absorb enormous amounts of water imparting excellent mechanical strength and outstanding cell growth support.

Ceramic-composite scaffolds
Owing to the composite nature of bones, it is relevant to develop composite scaffolds to achieve superior bioactivity and biomimicry in bone applications [48]. The bioactivity properties of scaffolds can be improved by the incorporation of materials capable of interaction with or attachment to viable tissues. This can subsequently enhance the osteoconductive function by inducing bone cell growth, augmenting osteointegration and fixation of the scaffold within osseous tissues, and boosting vascularization [48]. An ideal scaffold should be biocompatible, biodegradable, of matching mechanical strength to the bones, its resorption rate should match the rate of new cell growth, to be replaced by host native cells. Also, a scaffold with an optimal microarchitecture would allow the exchange of oxygen and nutrients and help cell migration through its interconnected porous structure [74, 75,48]. Fabrication techniques for scaffolds include solvent casting/particulate leaching, gas foaming, emulsification freezedrying, phase separation, electrospinning, and 3D printing techniques [48]. A brief on previous studies in the literature on ceramic composite scaffolds for localized treatment of OM is summarized in Table 1. Based on the aforementioned overview of the different systems for the treatment of osteomyelitis, the biodegradable bone fillers are optimum for the local intraosseous delivery and bone repair purposes. Among these are CP, which we thought to be the favorable bioceramics due to their structural similarity to native bones and optimum resorption rate. However, these should be coupled with polymers to enhance their mechanical strength for load-bearing applications.

Conclusion
OM is a difficult-to-treat disease, where surgical debridement of necrotic tissue with long term antibiotic therapy is demanded. Local osseous delivery of antibiotics coupled with bone regenerative therapy is always advantageous. Biodegradable bone substitutes are favorable over their counterparts, due to their osteoconductive and osteointegration nature. CP is widely applied as bone fillers as cement, pastes, hydrogels, coating or scaffolds, either alone or with polymers as composites. CP composites are distinguished with appropriate mechanical strength and resorption time. Loading of antibacterial agents with CP composites provide a proper solution for controlling local infection in bone tissues while replacing damaged osseous tissues with new ones.

TCP)
Vancomycin  Vancomycin-containing PLA/ ß-TCP composites were able to control antibiotic release and stimulate bone formation.  The in vitro experiments showed an antibiotic release in the inhibitory doses and biocompatibility based on cell culture studies of adhesion, proliferation, and mineralization.

[76]
PCL Calcium phosphate ceramic Vancomycin  Osteoconductive degradable composite loaded with vancomycin were successfully prepared.  The results delineate the system for local antibiotic therapy of osteomyelitis and other bone infections. [77] PLGA ß-TCP Gatifloxacin  The composites of gatifloxacin-loaded PLGA and ß-TCP were proven to be effective for the local treatment of osteomyelitis. [78]

PLGA HAp
Quaternized chitosan (HACC)  PLGA/HAp/HACC composite scaffold was fabricated using a 3D printing method.  The developed scaffold proved to have optimum antibacterial activity in vitro and inhibited adhesion of bacteria and biofilm formation on scaffolds implanted subcutaneously in rats.  They promoted cell proliferation, adhesion, and differentiation of human bonemarrow-derived mesenchymal cells while in-vivo biocompatibility test they showed great neovascularization and integration in rats' tissues.

[79]
 PLGA/HAp/HACC composite scaffolds were investigated in-vivo to assess their capability of regeneration of infected bones in rabbits with induced bone infections.  The antibacterial and bone repair efficacy was determined through radiographic, microbiological and histopathological evaluations.  The composite scaffolds showed optimum in vivo results which impose the system to be a model for local treatment of bone infections.  MGHA nanocomposite/ PVA scaffold was developed with rapid prototyping and coated externally with gelatin-glutaraldehyde.  Each drug was loaded in different sites in the scaffold yielding different kinetics of release and effective combined therapy: LFH was loaded into the bioceramic part, VAN loaded into PVA while RF loaded into the outer coating.  The multidrug loaded scaffolds achieved the destruction of bacterial biofilm that was detected by confocal laser scanning microscopy.  They achieved optimum proliferation, differentiation, and mineralization of MC3T3-E1 cells.  The 3D multidrug loaded scaffold offered a promising tool for local treatment of bone infections.

[84]
Oligolactide HAp Gentamicin  Oligolactide-HAp porous scaffolds were fabricated using the stereolithographic method and coated with gentamicin.  The composite scaffold had a well-structured interconnected porous framework.  The released gentamicin levels over 2 weeks were higher than the minimum inhibitory concentration of S. aureus and E.coli.  The findings suggest the potential use of scaffolds for the prevention of osseous infections.  CS scaffolds with or without BG was fabricated with the freeze-drying technique, the selected scaffolds were loaded with 5%, 10% or 20% CIP.  The selected composite scaffold composed of CS and BG in ratio 1:2 loaded with 5% CIP exhibited satisfactory release rate of Si and good biocompatibility on Saos-2 cells with promoted cell proliferation and differentiation.  PU-n-HAP/CIP composite scaffolds were developed.  CIP released from the scaffolds in a sustained manner for at least 2 weeks.  The antibacterial activity was determined by measuring the zone of inhibition, the drug-loaded scaffolds showed good activity against S. aureus and E. coli.  The scaffolds capability for promoting proliferation and osteogenic differentiation was tested using rat-bone-marrow-derived mesenchymal stem cells (BMSCs), the scaffolds showed positive results on BMSCs.  The composite scaffolds are a promising model as a pro-osteogenic space keeper in the treatment of OM.

[30]
Polymer Ceramic Active moiety Main findings Reference

Polyurethane (PU)
Nano-HAp Levofloxacin hydrochloride-loaded mesoporous silica microspheres (LFH@MSNs)  An antibacterial bone graft was developed through the immobilization of LFH@MSNs on the n-HA/PU bioactive composite scaffold.  The LFH was released on a sustained basis for 42 days from the scaffolds.  The in vitro MTT cytotoxicity test on L929 showed that n-HA/PU composite had no negative effect on cell proliferation in contrast to LFH@MSN/n-HA/PU scaffolds that affected the proliferation due to the released LFH, however, this effect may be diminished in vivo due to the dynamic circulation.  The scaffolds showed optimum antibacterial activity against Gram-positive (G +ve) and Gram-negative (G -ve) bacteria.  LFH@MSN/n-HA/PU porous scaffold is a promising model for the treatment of bone infections bone regeneration capabilities.  3% Ag/n-HA/PU and 10% Ag/n-HA/PU exhibited initial burst release followed by slower release profiles for 39 and 42 days respectively.  10% Ag/n-HA/PU exhibited a fast resorption rate that did not match the rate of new bone growth, so it is not suitable for bone regeneration with a possible toxic effect on viable tissues.  The in vivo study on New Zealand rabbits with induced OM showed that 3% Ag/n-HA/PU exhibited good bone repair with no evidence of infection or toxic effects. [92]

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All data generated or analyzed during this study are included in this published article in the main manuscript.

Competing interests
No competing interests were declared by the authors.

Funding Statement
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Acknowledgment
The authors would like to acknowledge all colleagues in the Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University for their continuous support.