Biological behavior of bioactive glasses and their composites

Abstract

Bioactive glasses (BGs) as third generation biomaterials have the ability to form an interfacial bonding more rapidly than other bioceramics between implant and host tissues in defect treatment. Therefore, BGs have shown great applications in the field of bone tissue engineering, dental materials, skin and other tissue regeneration. This review is based on inorganic and organic BG composites being used in bone tissue engineering and summarizes current developments in improving the biological behavior of BGs and their composites. A main focus was given to highlight the role of BGs and their composites in osteogenic differentiation and angiogenesis, followed by their cytotoxicity, protein adsorption ability and antibacterial properties. BGs were found to enhance the cell proliferation and cell attachment without any toxic effects with a significant increase in metabolic activity and possess osteogenic properties. Organic and inorganic dopants have been used to improve their cytocompatibility, osteoconductivity and promote stem cell differentiation towards the osteogenic lineage. BGs have also been used as graft materials because of their significant role in angiogenesis, as they stimulate relevant cells (i.e. fibroblasts, osteoblasts and endothelial cells) to release angiogenic growth factors. They show good protein adsorption because they act as templates for the adsorption of proteins which in turn depends upon surface properties. Antibacterial effects were also observed in BGs as a result of the high aqueous pH value in body fluids due to the presence of alkaline ions. There has been significant research work performed on silica-based bioactive glasses but not much literature can be found on phosphate- and borate-based bioactive glasses, which have good solubility and degradation, respectively.

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1 Introduction

Over the last two decades there has been a scarcity of tissues and organs for transplantation applications; tissue engineering has offered a promising role in regeneration of damaged tissues and organs.^1,2 Allografts and autografts pose potential risk of contamination, are limited in supply and have low morbidity from the donor’s side with a low clinical success rate. These shortcomings motivated scientists to look for alternatives with ideal porosity for tissue ingrowth, nutrient and waste transport and angiogenic properties with desirable mechanical properties.³ Bioceramics such as calcium phosphates (CPs),⁴ hydroxyapatite (HA)⁵ and bioactive glass (BG)⁶ are promising biomaterials used in bone and tissue engineering.⁷

Among them, BGs are third generation biomaterials discovered by Larry L. Hench in 1969.⁸ This review article focuses on research articles based on the biological behavior of bioactive glasses published within the duration of the last fifteen years. However, to provide some supporting information, a very few articles of older date have also been added based on the findings of various individuals about bioactive glasses and their biological behavior. All the research papers, articles, reviews and books for this review article were located using different databases: ISI Knowledge; NCBI (Pubmed); and Web of Science. Different papers were obtained using the terms which provided us articles related to our main topic. Terms such as ‘introduction to bioactive glasses’, ‘types and applications of bioactive glasses’, ‘properties of bioactive glasses’, ‘biological properties of biomaterials’, ‘bioactive behavior of bioactive glasses and their composites’, ‘mechanical properties’, ‘antibacterial properties’, ‘toxicity due to bioactive glasses’, ‘effect of bioactive glasses on cells’ and ‘tissue formation through bioactive glasses’ were used to extract important information. More than 250 research papers and review articles were found to reveal the biological properties of bioactive glasses. A total of 178 studies are cited in this review article.

2 Biological behavior of bioactive glasses and their composites

BGs have good biocompatibility, resist corrosion and compression, bond to soft and hard tissues, foster cell growth, activate osteogenic genes and stimulate angiogenesis.^8,9 BGs have extensive applications including bone grafts for bone regeneration,^10,11 bioactive coatings in orthopedic¹² and dental implants,¹³ for drug delivery,^14,15 wound healing¹⁶ and soft tissue repair.^17,18 BGs form a crystalline HA or amorphous CP phase on a glass surface and strengthen bonding with the surrounding tissue in vivo. The ease of controlling the structure and chemistry of glasses by changing either the environmental or thermal processing or composition allows the design of ideal scaffolds for bone ingrowth.³ BGs can be fabricated using a variety of methods including sol–gel,¹⁹ melt quench,²⁰ polymer foam replication,²¹ thermal bonding,²² solid free foam fabrication²¹ and freeze casting solid free foam fabrication.^3,22 Significant work has been done to develop silicate, borate/borosilicate, phosphate (Table 1) and metallic BGs with incorporated trace elements which have been used for different applications.²³ Among the different BGs, silicate BGs are the most famous, of which 45S5 and 13-93 BGs are the most widely used and researched.²⁴ Their structure consist of an SiO₂ based network in three dimensions where silicon is fourfold-coordinated to oxygen.²⁵ These BGs consist of a low SiO₂ content, a high Na₂O and CaO content, and a high CaO/P₂O₅ ratio. Silicate BGs are popular for supporting the proliferation and differentiated function of osteoblastic cells.²⁶

Table 1 Different types of commercial bioactive glasses with their compositions and applications

Common name	Composition	Applications
45S5 bioactive glass^34	45SiO2–24.5CaO–24.5Na2O–6P2O5	Bone^6 and soft tissue engineering,^17 orthopaedics,^35 antibacterial agents^36
13-93 bioactive glass^21	53SiO2–20CaO–6Na2O–12K2O–5MgO–4P2O5	Bone repair^37
13-93B1 bioactive glass^38	34.4SiO2–19.5CaO–5.8Na2O–11.7K2O–4.90MgO–3.8P2O5–19.9B2O3	Bone repair^38
13-93B3 bioactive glass^24	18.5CaO–5.5Na2O–11.11K2O–4.6MgO–3.7P2O5–56.6B2O3	Bone tissue engineering^24
58S bioactive glass^39	58.2SiO2–32.6CaO–9.2P2O5	Bone tissue engineering and tissue regeneration^40
S53P4 bioactive glass	53SiO2–20CaO–23Na2O–4P2O5	Bone graft substitutes, craniofacial reconstructions such as mastoid obliteration and orbital floor reconstructions^41

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Borate BGs are another type of bioglass which are well known for their faster degradation rates and completion towards HA-like material.²⁷ They are very reactive and have lower chemical durability, due to which their degradation rate is faster.^{24,26,28–31}

Phosphate BGs have an active role in tissue engineering as they can be tailored to induce a specific biological response. Biological functions can be greatly enhanced by modifying the glass using different dopants. They have unique dissolution and degradation properties in aqueous based fluids which depend upon the composition of glass. The building block of phosphate glasses, phosphorus with a +5 charge, is a tetrahedral unit. It shares three of its oxygen atoms while the lone pair of unshared electrons form a terminal double bond with P⁵⁺ ions. The presence of oxygen at the terminal of phosphate ions limits its connectivity with silicate based counterparts, thus reducing its rigidity.³² Phosphate glasses have a higher affinity with bone as compared to silicate or borate based bioglasses.^23,33

When BGs come in contact with body fluid they chemically react and produce HA.⁷ BGs can be resorbed in tissue from few days to several months as their bioactivity can vary from surface to bulk degradation.²³ In the case of surface degradation, which occurs layer by layer, the inside material does not erode until after the degradation of the exterior material. Meanwhile, erosion occurs throughout the whole material equally in case of bulk degradation.⁴² Gene expression of bone cells also depends upon the topography, surface chemistry, shear stress and dissolution ions released from BG. In situ regeneration of tissue by silicate BGs involves up-regulation of seven gene families that control different processes involved in bone regeneration. BG increases the release of vascular endothelial growth factor (VEGF) gene expression and promotes angiogenic effects.⁷ This review focuses on the in vivo and in vitro biological behavior of BG which includes osteogenic and angiogenic properties followed by cytotoxicity, protein adsorption and antimicrobial studies.

Partial crystallization of BGs through heating above the crystallization temperature forms glass ceramics. The crystallization and densification process shrinks the glass microstructure, reduces the porosity and increases mechanical strength. The brittleness and low fracture toughness are overcome by designing BG scaffolds from the macro to the nanoscale with ideal properties.⁷ The inclusion of organic biodegradable polymers and inorganic metal oxides enhances the mechanical properties of these glasses but success is uncertain.³ Properties peculiar to BGs are imparted by incorporation of inorganic oxides. Metal oxides such as CaO, K₂O, Na₂O and MgO adjust the bioactivity rate of BGs, while antibacterial materials such as ZnO, CuO, AgO and TiO₂ protect against microbes. Zinc (Zn) and magnesium (Mg) stimulate osteoblast proliferation, differentiation and bone mineralization.^43,44 Strontium (Sr) reduces bone resorption and enhances bone healing.^44–46

Enhanced new bone growth of glasses is the result of released ionic dissolution products especially the biologically active soluble silica (SiO₂) and calcium (Ca²⁺) ions. Ca²⁺ ions released from mesoporous BG show enhanced hemostatic activity, increased coagulation rate and reduced clot detection time as compared to nanoporous BG.⁷ These ionic products of BG ceramics regulate osteoblast proliferation, gene expression and differentiation, thus enhancing osteogenesis; as is evident from the increase in alkaline phosphatase (ALP), osteocalcin and osteopontin (which are differentiation markers).^7,47

2.1. Osteogenic differentiation

Ionic dissolution products released due to degradation of the BG matrix show increased osteogenic differentiation and thus promote bone regeneration.^48–50 They have the ability to enhance revascularization, osteoblast adhesion, enzyme activity and differentiation of mesenchymal as well as osteoprogenitor cells.^{18,45,51–57} Controlling the initial composition and processing parameters of BGs enhances these properties;^3,23,58 sodium (Na¹⁺), magnesium (Mg²⁺) and borate (BO₃³⁻) based BGs have been observed to be actively involved in bone remodeling and/or associated angiogenesis and also in biological regulation of osteogenesis.^59–63 Sharmin et al.⁶⁴ studied boron doped phosphate-based BGs with different concentrations of boron trioxide (B₂O₃) (Table 2) and observed that this had favorable effects on cell morphology, proliferation and metabolic activity.

Table 2 Different concentrations of B₂O₃ contents used in study along with their glass codes⁶⁴

Glass code	P2O5 content (mol%)	CaO content (mol%)	Na2O content (mol%)	MgO content (mol%)	B2O3 content (mol%)
P45B0	45	16	15	24	—
P45B1	45	16	14	24	1
P45B5	45	16	10	24	5
P45B10	45	16	5	24	10
P50B0	50	16	10	24	—
P50B1	50	16	9	24	1
P50B5	50	16	5	24	5
P50B10	50	16	—	24	10

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It is apparent that BG with 0–5% boron trioxide (B₂O₃) enhanced the ALP activity, however, there was no pronounced effect in the case of higher (10%) boron content when compared with the TCP control. As B₂O₃ increased, the dissolution rate, density and thermal expansion coefficient value decreased (Fig. 1).

Fig. 1 SEM images of MG63 cells cultured on P₄₅Ca₁₆Mg₂₄Na_(15−x)B_x (left) and P₅₀Ca₁₆Mg₂₄Na_(10−x)B_x (right) after 3 days and 14 days of culture. Micrometer scale bar = 50 μm.⁶⁴

Liang et al.⁶⁵ studied borate glass scaffolds having a porosity of 25–40% and found that scaffolds supported the attachment and differentiation of human bone marrow derived mesenchymal stem cells (hbMSC) and human mesenchymal stem cell derived osteoblasts (MSB-Obs) and generated high metabolic activity (Fig. 2).

Fig. 2 Weight loss of borate glass scaffolds as a function of reaction time in a 0.25 M K₂HPO₄ solution at 37 °C and a starting pH value of 9.0. Conversion of the glass to hydroxyapatite is accompanied by weight loss. Data are shown for scaffolds prepared from 90–150, 150–212, and 212–355 lm microspheres. The theoretical weight loss of the glass is shown by the horizontal dotted line.⁶⁵

Pawlik et al.⁶⁶ prepared sol–gel derived BG–TiO₂ composites with high calcium oxide (CaO) or SiO₂ contents and tested their effects on cell viability, ALP activity and total collagen production. Increase of BG content resulted in better integration of material microstructure and unification. Composites showed good biocompatibility in both undifferentiated and osteogenic media. BG promoted bone marrow derived stem cell (BMSC) proliferation and differentiation. Composites with high CaO introduced osteoinductive properties which led to a significant increase of collagen production and mineralization of the extracellular matrix (ECM). Cu, Zn and Sr are the trace elements required by the body⁶⁷ that have angiogenic and osteogenic properties and promote stem cell differentiation towards the osteogenic lineage.⁶⁸ Thus to achieve healthy bone growth, BGs have been modified by doping with these elements.⁶⁹ Biodegradable but mechanically robust non-crystalline BGs had been synthesized by Erol et al.⁷⁰ using boron containing silicate glass by the replication technique. Subsequently, these scaffolds were coated with sodium alginate and cross linked with copper (Cu²⁺). The coated scaffolds exhibited more compressive strength and improved bioactive behavior compared to the non-coated scaffolds. These newly synthesized scaffolds exhibited a controlled release of Cu ions which may promote the angiogenic potential of the scaffolds for bone regeneration.⁷⁰ Blaker et al.⁷¹ synthesized highly porous poly(DL-lactic acid) (PDLLA) and BG-filled PDLLA composite foams and carried out cell culture studies using the human osteosarcoma cell line (MG-63). The osteoblast cell infiltration studies revealed that, in case of BG–PDLLA composites, the cells were able to migrate through the porous network and colonize the deeper regions within the foam, showing that the composition of the foams and the pore structures are able to support osteoblast attachment, spreading, and viability. Blaker et al.⁷¹ observed rapid formation of HA on these composites (Fig. 3) and the attachment of MG-63 cells within the porous network of the composite foams that confirmed the high in vitro bioactivity and biocompatibility of these materials and their potential to be used as scaffolds in bone tissue engineering and repair.

Fig. 3 SEM micrographs showing the microstructure of (a) a pure PDLLA foam (orthogonal to the pore direction); (b) a PDLLA/bioglass-filled composite foam (5 wt% bioglass; orthogonal to the pore direction); (c) a PDLLA/bioglass-filled composite foam (40 wt% bioglass; orthogonal to the pore direction); and (d) a PDLLA/bioglass-filled composite foam (40 wt% bioglass; parallel to the pore direction).⁷¹

BG also had an effect on expression of bone sialoprotein. Tsigkou et al.⁷² prepared PDLLA/BG composites and studied their cell adhesion, viability and differentiation. They observed that incorporation of 5% of 45S5 BG within the PDLLA matrix significantly enhanced ALP activity and osteocalcin protein synthesis compared to tissue culture polystyrene controls, PDLLA alone and 40% 45S5 BG (Fig. 4). Enhanced alkaline phosphatase enzymatic activity and osteocalcin protein synthesis confirmed the differentiation and maturation of fetal osteoblasts.

Fig. 4 SEM images of cells on the surfaces following 7 days in culture, demonstrating the differences in cell phenotype on the materials. (a) Cells on control, (b) cells on PDLLA, (c) cells on P/BG5, (d) cells on P/BG40.⁷²

In another study, the incorporation of mesoporous BG into the polyamide (m-BPC composites) was found to enhance the attachment of cells as compared to PA scaffolds. It was observed that 79% of the bone defect area was filled with newly formed bone tissue, twelve weeks after the implantation of m-BPC. The m-BPC scaffolds greatly enhanced repair of the bone defect in the rabbit femur model and also revealed high efficiency of bone regeneration. Thus the m-BPC scaffolds showed not only good biocompatibility, but also rapid and more effective osteogenesis than PA scaffolds.⁷³ Gao et al.⁷⁴ observed that BG coated electrospun poly(vinyl alcohol) PVA fibers had a greater capacity to support proliferation of osteogenic MC3T3-E1 cells, ALP activity, and mineralization than the uncoated PVA scaffold (Fig. 5). In vitro cell culture studies showed that these BG-coated PVA scaffolds could be considered as candidate materials for bone tissue engineering applications.

Fig. 5 SEM images of MC3TC-E1 cell morphology on PVA (a–c) and BG-coated PVA fibrous scaffolds (d–f), after incubation for 1 day (a and d); 3 days (b and e); and 7 days (c and f).⁷⁴

Oudadesse et al.⁷⁵ synthesized BG/chitosan based nanocomposite films and tested them for chemical reactivity and bioactivity. Chitosan showed excellent apatite formation and formed a biologically active HA layer. Chitosan acted as a capping agent and delayed the release of silicon from the glassy network. Cytotoxicity assessment of these films was done by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) tests, proliferation was measured through DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity. These films were non-toxic and also increased cell viability and proliferation of osteoblasts. BG ceramic nanoparticles (nBGC) have been found to increase the biomineralization, cell attachment and cell proliferation of chitosan gelatin scaffolds. The same results have been observed in other studies for chitosan–BG composites.^76–78 Poly(glycerol sebacate)–BG elastomeric composites⁷⁹ and bioglass–collagen–phosphatidylserine (BG–COL–PS) improved the cytocompatibility, osteoconductivity and enhanced bone formation.^80,81 Electrical stimuli significantly help in bone healing at the injured area, and therefore biocompatible conductive polymers have been used to boost electrical signals among the cells. Shahini et al.⁸² used poly(3,4-ethylenedioxythiophene) and poly(4-styrene sulfonate) (PEDOT:PSS) conductive polymers in BG/gelatin composites which increased the viability of human mesenchymal stem cells (MSC). Moreover, the processing conditions and nature of acid of BG also affect cell proliferation. BGs were prepared by Lei et al.⁸³ by employing a sol–gel technique using different acids i.e. hydrochloride acid, lactic acid, citric acid and acetic acid as hydrolysis catalysts. The effect of acids on morphology, textures, apatite forming bioactivity and the cellular response of BGs were investigated. It was observed that these features could be manipulated by changing the acid species. The HCl and CH₃COOH derived BGs exhibited high surface areas and efficient apatite forming rates. A nanoscale surface morphology, relatively low surface areas and moderate apatite forming rates were observed while using lactic acid and citric acid. An enhanced effect on the cell proliferation of hMSC was observed for lactic acid- and citric acid derived BGs.⁸³ Growth factors such as VEGF, also increase the bone regeneration ability of BG. In a rat critical-sized defect model the capacity of VEGF-releasing polymeric scaffolds with a BG coating was studied in vivo and in vitro. The mitogenic stimulation of endothelial cell was found to be increased in the presence of BG modified with VEGF. These in vivo coated VEGF-releasing scaffolds exhibited significant improvements in the blood vessel density and bone mineral density, but only a slight increase in bone volume fraction (Fig. 6).⁸⁴

Fig. 6 (a) MicroCT image reconstruction of coated VEGF-releasing scaffolds at 12 weeks. The image on the left shows the distribution of new mineralized tissue, while the coronary section on the right shows almost complete bridging of the defect by newly formed mineralized tissue. (b) Quantitative analysis of bone volume fraction (BVF) in coated control (open bars) and coated VEGF-releasing scaffolds (filled bars). (c) Bone mineral density (BMD) in control (open bars) and coated VEGF-releasing scaffolds (filled bars). Area between dashed lines represents BMD of PLG scaffold alone (120 720 mg cm⁻³) in this identical model.⁸⁴

13-93 BG (53SiO₂–20CaO–6Na₂O–12K₂O–5MgO–4P₂O₅) scaffolds, either having a trabecular or columnar microstructure, support the soft tissue growth.⁸⁵ The cellular response of 13-93 BG scaffolds was evaluated by Liu et al.⁸⁶ using murine MLO-A5 cells. Cell cytotoxicity was determined by MTT assay, further ALP activity confirmed osteogenic differentiation. These results confirmed that 13-93 BG scaffolds were a good potential biomaterial to repair large bone defects.

2.2. Angiogenesis

Biomaterials with angiogenic potential are highly desirable in tissue engineering. For tissue development, cells entrapped within graft materials require oxygen and a continuous supply of nutrients. These requirements are fulfilled by blood vessels formed as a result of neovascularization. Therefore, engineered biomaterials should have the ability to neovascularize, support cell viability and finally tissue development.⁴³ In past, ceramics and synthetic polymer composites have been used to attain osteoconductive and osteoinductive approaches. VEGF, which is the most common regulator of angiogenesis and belongs to the family of placental growth factors, has been loaded on these biomaterials.⁸⁷ BGs play a significant role in angiogenesis without addition of growth factors and therefore, have recently been used as graft materials. Increased osteogenic differentiation due to the ionic dissolution products of BGs highlighted their osteoconductive as well as osteoinductive potentials.⁸⁴ BGs act as stimuli to allow fibroblasts to release angiogenic growth factors and the growth of blood vessels.⁸⁸ An increase in angiogenic indicators has been achieved through both direct and indirect contact of relevant cells with 45S5 bioglass.^43,89 Vargas et al.⁹⁰ reported the angiogenic properties of nano-sized BG particles (n-BG) when added to bovine type I collagen/n-BG composites. Quail chorio-allantoic membrane (CAM) was used as a model study to identify angiogenesis. Type I collagen which is the major constituent of extracellular matrix supported the exposure of endothelial cells at the injured area. An increase in vascularization and number of branch points of blood vessels was observed with 10 wt% n-BG containing collagen membranes, while 20 wt% BG resulted in the inhibition of angiogenesis due to an excessive inflammatory response (Fig. 7).

Fig. 7 Stereomicroscopic views of CAM tissue response at 24 h post-implantation. (a) Collagen film (b) 10 wt% n-BG containing collagen film (c) 20 wt% n-BG containing collagen film.⁹⁰

In another study, micron and nano-sized BG (n-BG) filled poly(D,L-lactide) (PDLLA) composites were investigated for cell culture studies. In vivo vascularization was observed through secretion of VEGF by human fibroblasts. The composites having 20 wt% BG released 5% higher VEGF and showed potential for the regeneration of hard–soft tissue defects and increased bone formation raised from enhanced vascularization of the construct.⁹¹ The angiogenic properties of micron-sized (m-BG) and nano-sized (n-BG) BG filled PDLLA composites were studied by Gerhardt et al.⁹¹ In vivo vascularization of the scaffolds was studied in a rat model and quantified using stereological analyses. The prepared scaffolds had porosities, permeability and compressive strength values in the range of trabecular bone. On composite films containing 20 wt% m-BG or n-BG, human fibroblasts produced 5 times higher VEGF than on pure PDLLA films. After eight weeks of implantation, m-BG and n-BG scaffolds were well-infiltrated with newly formed tissue and demonstrated higher vascularization and percentage blood vessels to tissue than PDLLA scaffolds. Although not statistically significant, total blood vessel volume and percentage of blood vessels was higher in n-BG as compared to m-BH. Leu et al.⁹² observed that the pro-angiogenic potential of collagen/BG substrate was related to the soluble dissolution products of BG and the subsequent production of cell-secreted angiogenic factors by stimulated cells (Fig. 8). BG loaded collagen sponges with 1.2 mg BG concentration showed higher cell proliferation as compared to 0.12, 0.6 and 6 mg. BG acts as a stimulus to allow fibroblasts to release angiogenic growth factors, growth of blood vessels, tubule formation within the co-culture of endothelial and fibroblast cells, and release of angiogenic factors.^92,93

Fig. 8 Tubule formation assay with BG-conditioned media. (A) The extent of tubule formation was dependent upon the mass of BG in the conditioned media. (B) Quantification of tubule formation. * P < 0.05 vs. collagen sponge without BG (0 mg BG).⁹²

Day et al.¹⁷ studied the effect of BG coated scaffolds and observed that 45S5 BG incorporated into polyglycolic acid (PGA) surfaces increased proliferation of a fibroblast cell line (208F) along with enhanced neovascularization. Bioglass significantly increased the number of blood vessels. The PGA mesh supported this well-vascularized developing tissue. After 60–90 days, PGA mesh dissolved and the developed tissue supported itself. Li et al.⁹⁴ found that a BG coated polyethylene terephthalate (PET) artificial ligament surface had a positive effect on the induction of artificial ligament osteointegration within the bone tunnel and safely promoted angiogenesis. Calcium and silicon ions have been found to increase the angiogenesis of endothelial cells.⁹⁵ Thereafter, in another study, bioglass/alginate (BG–ALG) composites had been found to support proliferation and osteogenic differentiation of MSC MC3T3-E1. Zeng et al. observed that calcium and silicon ions released from BG–ALG composites also supported the angiogenesis of endothelial cells.⁹⁶ These results indicated potential for the regeneration of hard-soft tissue defects and increased bone formation arising from enhanced vascularization.

The hypoxia pathway is key to initiate various processes such as progenitor cell recruitment, differentiation and angiogenesis. It is a condition in which oxygen has a low partial pressure due to which blood vessel formation is increased as a result of the hypoxia-inducible factor-1 (HIF-1α) pathway. As the formation of blood vessels has an important role during wound healing, treatment materials or wound dressings have been modified using certain dopants such as Cu which mediate the release of VEGF growth factors and cytokines and induces cell proliferation and angiogenesis.⁹⁷ Copper, strontium, zinc, cobalt, silicon and boron are therapeutic agents that have osteogenic as well as angiogenesic potential.⁹⁸ Cobalt⁹⁹ and nickel stimulate the hypoxia-inducible factor-1 (HIF-1α) and improve blood vessel formation.¹⁰⁰ Lin et al.⁹⁷ investigated the angiogenic potential of bioactive borate-based glass microfibers in rats and compared it with 45S5 silica glass microfibers and sham implants. Copper-supplemented bioactive borate glass promoted angiogenesis as compared to the control. The sample also showed a higher density of blood micro vessels which might be the result of borate and Cu ions released from BG.⁹⁷ Yang et al.¹⁰¹ also studied the effect of borate-based 13-93B3 bioactive glasses on cell migration, proliferation and wound healing. Cell migration and proliferation of the keratinocytes and fibroblast cells is the key indicator of new tissue formation during wound healing. These results indicate that, in the case of borate based BGs, presoaking procedures potentially stimulated higher cell proliferation. Boron has the ability to induce angiogenesis and perform functions in osteogenesis. Durand et al.¹⁰² observed that boron doped 45S5 BGs stimulate the release of interleukin 6 (IL6) and the basic fibroblast growth factor (bFGF). Boron stimulates cell migration and proliferation as well as tubule formation. Wu et al.¹⁰³ investigated the angiogenic capacity of copper doped BG towards angiogenesis and obtained positive results. Cu stimulates the proliferation of endothelial cells and upregulates VEGF thus promoting neovascularization. Zhao et al.¹⁰⁴ also observed similar results. He observed that ionic dissolution products of Cu doped borate BGs and promoted cell migration, tubule formation and secretion of vascular endothelial growth factor (VEGF), and stimulated the expression of angiogenic-related genes of the fibroblasts.

2.3. Cytotoxicity

Cytotoxicity testing is the estimation of how much a material is toxic to cells in terms of cell viability, adhesion, morphology and proliferation. Biocompatibility, which is one of the most important requirements for clinically used biomaterials, is measured in terms of cytotoxicity. Rismanchain et al.¹⁰⁵ studied the cytotoxicity of nano and micro sized BG and found that both had no significant difference in cell viability when seeded with human HGF1-PI53 gingival fibroblast cells. Both glasses had the same cytotoxic effect up to 2 mg mL⁻¹ concentration for 48 h, but at higher concentration micro-sized BG was more toxic than the other sample. Cytotoxicity was also found to be time dependent. At the start, the particle-free medium showed more cell viability, but after 72 h the control and the tested samples showed no significant difference, which may be due to the adaption of cells in the new environment.

Daguano et al.^106,107 studied the effect of crystallinity of BG and glass ceramic samples on biocompatibility using the neutral red uptake method with NCTC clones L929. No significant influence of the partial crystallization on cytotoxicity was observed. Bakry et al.¹⁰⁸ compared cytotoxicity of 45S5 BG having 50% phosphoric acid with commercially available dentines. The synthesized BG did not affect the cell viability of rat pulpal cells when compared to the controls. Dental pulp–BG paste loaded with 30% phosphoric acid did not differ in cell viability when compared to commercial desensitizing agent and intermediate restorative material.^108,109 Mallick et al.⁵⁷ checked the cytotoxicity of HA and BG scaffolds fabricated by camphene-based foam reticulation (ARM) and camphene freeze casting (CFC). These scaffolds facilitated cell adhesion and proliferation and were proved to be non-cytotoxic when cultured with MG63 cells. The viability of cells was determined by resazurin assay which showed that the scaffolds possess metabolic activity. In vivo testing has also shown that BGs are biocompatible and have no cytotoxicity. Amaral et al.⁶³ synthesized BG–PLA composites and tested them in a rat calvarial tissue for biocompatibility, systemic toxicity and tumorigenicity. These composites were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). Bioglass did not cause organ damage, long-term systemic inflammatory reactions or tumor formation, making it biocompatible. Doostmohammadi et al.¹¹⁰ observed that bioglass at the nanoscale range also had no cytotoxic effect on hMSC and had a tendency towards agglomeration. MTT assays showed the highest cell viability in BG- and HA-containing cultures.^58,110 In a study, cytotoxicity tests of ZK30 alloy-BG composites carried out on rat bone marrow stromal cells showed that they stimulated cell proliferation and were cytocompatible.¹¹¹ Nair et al.¹¹² studied the effect of sol–gel derived BG coatings on HA (BGHA) and observed no cytotoxic effects. BG coated HA provided an adherent surface for the attachment of cells. Huang et al.¹¹³ synthesized BG–high density polyethylene (BG-HDPE) composites. As compared to unfilled HDPE, these composites favored the growth of cells without any cytotoxic effect with a significant increase in metabolic activity. Na¹⁺, Ca¹⁺, P³⁺ ions leached from BG provided a favorable environment for the growth of cells. Eldesoqi et al.¹¹⁴ developed PLA composites having different BG concentrations. Glutamic oxaloacetic transaminase (GOT) and creatinine were analyzed as organ damage markers while malondialdehyde (MDA) and sodium oxide dismutase (SOD) were markers of tumor progression. These composites caused neither damage to organs nor caused any inflammation. Huang et al.¹¹³ studied the biocompatibility of BG–high density polyethylene (HDPE) composites on primary human osteoblast-like cells. Composites with varying BG concentrations were synthesized to determine the effect of varying concentrations of BG on cell viability. It was observed that cellular metabolic activity significantly increased in the case of composites. Bioglass created a favorable microenvironment for cell proliferation and growth and stimulated cellular activity (Fig. 9).¹¹³

Fig. 9 Viability of HOB cells following 24 h exposure to 24 h eluted media from test and control materials; statistically significant differences from TCP using Student’s t-test indicated by * p(0.001).¹¹³

Zhou et al.¹¹⁵ observed that PLLA–BG composites enhanced cell adhesion and proliferation and showed no adverse effect to cells (Fig. 10).

Fig. 10 Morphologies of cultured fibroblasts: (a) PLLA/BG group, 12 h; (b) PLLA/BG group, 3 d; (c) negative control group, 3 d; (d) positive control group, 3 d.¹¹⁵

Oliveria et al.¹¹⁶ observed the same non-toxic properties of BG–PVA hybrids which supported cell attachment and proliferation. 45S5 bioglass was found to be biocompatible with osteoblasts and favored cell adhesion after 72 hours.¹¹⁷ The cytotoxicity of silver doped S70C30 BG was studied by Lohbauer et al.¹¹⁸ and the effect of soluble Ag, Ca and SiO₂ ions on human epidermal keratinocytes was evaluated. The viability of these cells showed that silver ions caused no toxic effect to cells. The alkaline ions released from BG raised the pH slightly, but imposed no negative effect on cells.

Although many studies show the non-toxic behavior of bioactive glasses however there is some evidence which show that presence of alkaline ions may pose a harmful effect to cells. Many compositions of bioactive glass contain significant amount of alkali oxides.^119–123 Although these alkalis have several advantages, their increased coefficient of thermal expansion, increased crystallization ability as a result of degradation of sintering ability,¹²⁴ and cytotoxicity due to presence of alkali content make them unfit for use as coatings. Kansal et al.¹²⁵ synthesized alkali-containing alkaline-earth phosphosilicate glasses and used varying sodium concentrations: Na-0, Na-2, Na-6, and Na-10. Cellular responses in vitro using a mouse-derived pre-osteoblastic MC3T3-E1 cell line were observed. The results showed the cytotoxic effects of bioglass due to increasing alkali concentration in the cell culture medium. In vitro osteoblast proliferation studies showed that the cytotoxic effects of bioglasses were due to the sudden release of sodium ions. Alkali-free BG (Na-0) showed the highest cell proliferation as compared to alkali containing BG.¹²⁵ Wallace et al.¹²⁶ also observed the same results due to higher concentration of Na₂O leached from soda-lime phosphosilicate bioglass. Bioactive glass exchanges sodium ions in place of protons from the physiological environment resulting in an increase in pH. Like alkali containing bioglasses, 45S5 bioglasses may in some cases have cytotoxic effects due to acidic pH. Although 45S5 glass has a favorable healing capacity, the high dissolution of alkali content caused fast resorption of bioactive glass which might have a negative effect on angiogenesis and natural bone remodeling. According to Bakry et al.¹⁰⁸ 45S5 bioglass used for dentine hypersensitivity treatment had some cytotoxicity due to its initial acidic pH. However, the cytotoxic effect gradually reduced due to high calcium and phosphate contents of the powder.¹⁰⁸ In order to overcome the cytotoxicity of alkali containing bioglasses, scientists introduced alkali-free bioactive glasses so that the toxic effects could be reduced or totally eliminated. Goel et al.¹²⁴ designed an alkali-free series of bioactive glass along the diopside (CaMgSi₂O₆)–fluorapatite (Ca₅(PO₄)₃F)–tricalcium phosphate (3CaO·P₂O₅) join. He introduced rat bone marrow derived stem cells to check the viability of the cells. Cells slowly proliferated for up to 7 days but the growth continued with prolonged culture period reaching the cell growth rate of the control. This suggested that bioactive glass provided suitable substrate conditions for the adherence and proliferation of cells.¹²⁴

2.4. Protein adsorption

Biomaterials provide space for adsorption of proteins which help with cell growth, differentiation and tissue formation. They mediate protein adsorption on their surface and stimulate adhesion of cells and integration of implants into the tissue. The quantity of adsorbed protein and its orientation on the surface of biomaterial is important for cell attachment because it exposes the active sites which help to bind specific receptors on the cell membrane. BGs act as a template for adsorption of proteins. The capability of a protein such as bovine serum albumin (BSA) to be adsorbed on a BG surface depends on the amount of Ca²⁺ and PO₄³⁻, as these are the main binding sites.¹²⁷ However, the crystallization of BG enhances the negative zeta potential and decreases the protein adsorption on its surface.¹²⁸ EI-Ghannam et al.¹²⁹ studied the effect of crystallization of BG on protein adsorption behavior by sintering it at different temperatures (Fig. 11).

Fig. 11 Comparison of the amount of serum protein adsorbed onto thermally treated and control untreated BG. Control untreated BG adsorbed a statistically significant higher amount of serum protein than BG samples containing 5% crystallization (p < 0.02).¹²⁹

Protein adsorption on BG decreased with increase in sintering temperature while non-sintered BG showed a higher amount of protein bonding. Thus bonding between BG and bone might be reduced due to inhibition of protein adsorption caused by sintering or the adsorbed protein being unable to perform its function due to conformational changes on the surface. However, Bahniuk et al.,¹³⁰ observed that protein adsorption on BG depends upon surface properties in spite of material crystallinity. Amorphous BG adsorbed 3–4 times as much protein as the crystalline BG. However, small differences were observed in the amount and variety of the adsorbed proteome in case of melt-cast BG. Sol–gel BGs produced with high stabilization temperature and decreased medium pH had been found to adsorb more fibrinogen protein due to their specific surface chemistry. BGs sustained proteins within their nanoporous network which helped with sustained protein delivery.^131,132 Gruian et al.¹³³ reported that BG caused structural and adsorption changes in the proteins methemoglobin (MetHb) and 5-methyl-aminomethyl-uridine forming enzyme (MnmE). Unfolding of proteins results in a decrease of α-helix due to interactions between the internal hydrophobic protein domains and the hydrophobic BG. However, BG functionalized with glutaraldehyde (GA) was found to preserve the α-helix of adsorbed protein. In another study, the adsorption of horse methemoglobin protein on BG in high NaCl concentration was observed through FTIR and electron paramagnetic resonance (EPR). The applied chemical treatment to bioglass influenced the adsorption of methemoglobin.^134–136 Vulpoi et al.¹³⁶ studied the protein adsorption of silver doped BG. X-Band cw-EPR (continuous wave electron paramagnetic resonance) spectra of methemoglobin protein adsorbed to silver doped BG showed an increased immobilization of protein to the surface. This was due to interaction of Ag with the thiol group of the protein. The amount of adsorbed protein increased with increase in the concentration of silver. Cell proliferation was also enhanced due to the compact protein structure.

Protein adsorption also depends on the types of precursors used for the synthesis of BG and their methodology. BG aluminosilicate samples prepared with silicic acid favored the adsorption of fibrinogen rather than that of a sample prepared with triethylorthosilicate (TEOS).¹³⁷ Misra et al. used the flame spray method to synthesize 45S5 bioglass with a particle size of 29 nm. Poly(3-hydroxybutyrate) loaded BG films were obtained through a solvent casting technique. α-Tocopherol (vitamin E) was incorporated into the poly(3-hydroxybutyrate) (P(3HB))/BG and found to improve hydrophilicity, which resulted in an increase in total protein adsorption, confirmed through XPS.¹³⁸

In another study, nano sized BG (n-BG) induced a nanostructured topography in poly(3-hydroxybutyrate) (P(3HB))/BG composite systems which resulted in enhanced protein adsorption as compared to micro sized BG and unfilled polymer (Fig. 12). Amount of adsorbed protein increased with increasing concentration of BG in composites.¹³⁹

Fig. 12 Total protein adsorption study on P(3HB)/BG composites containing m-BG and n-BG particles in different concentrations (wt%) using foetal bovine serum. The data (n 1/4 3; error bars 1/4 SD) were compared using Student’s t-test and differences were considered significant when * p < 0.05, ** p < 0.01 and *** p < 0.001.¹³⁹

2.5. Antibacterial properties

BGs possess an antibacterial effect against common bacteria as a result of the high aqueous pH value of the medium.^140–145 The pH of the medium increases as a result of the dissolution of alkaline ions such as Na, P, Si and Ca ions from BG.¹⁴⁶ BGs show a variation in sensitivity against both Gram-negative and Gram-positive bacteria, which is mainly linked to the composition of BG, in particular, the content of calcium ions (Table 3).^140–145

Table 3 Antibacterial properties of bioactive glass

Glass	Bacterial species	Possible reason
S53P4	Enterococcus faecalis (E. faecalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), Actinomyces naeslundii (A. naeslundii), Streptococcus mutans (S. mutans), and Streptococcus sanguis (S. sanguis)	High pH, Si-conc.^147 rise in pH and osmotic pressure, Na-conc.^148
45S5	E. faecalis	High pH ^149
	Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus)^150
	S. sanguis, S. mutans, Actinomyces viscosus (A. viscosus)	High pH^151
	A. actinomycetemcomitans, P. gingivalis, Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia)
76SiO2–22CaO–2P2O^150	E. coli, P. aeruginosa, S. aureus
CaPSiO2	Staphylococcus epidermidis (S. epidermis)	High Ca-conc.^152
58S, 63S, and 72S	E. coli, P. aeruginosa, Salmonella typhi (S. typhi), and S. aureus	Ca-conc.^153

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Microbial contamination at the targeted site is reduced as a result of leaching of ions from antimicrobial agents.¹⁵⁴ The needle like BG debris consisting of Na and Ca ions inactivates bacteria by damaging their cell wall.³⁶ These agents also attack bacterial DNA and RNA and thus cause damage to them.¹⁵⁴ Several antibacterial agents such as silver Table 4 (ref. 154 and 155) and antimicrobial drugs such as ibuprofen¹⁰³ have also been added to BGs to provide antimicrobial properties.

Table 4 Antimicrobial properties of dopant modified bioactive glasses

Glass	Doping material	Concentration	Bacterial species
Bioactive glass (Na2O–CaO–P2O5–CuO)^156	Cu	1, 5 and 10 mol%	S. epidermis
Mesoporous bioactive glass^103	Cu	5%	E. coli
45S5 bioactive glass^150	Ag	0.05 to 0.20 mg mL^−1	E. coli, P. aeruginosa, and S. aureus
45S5 bioactive glass^157	Ag	0.05 to 40.0 mg mL^−1	E. coli
Bioactive glass (SiO2–P2O5–CaO–Ag2O)^154	Ag	0.05 to 20 mg mL^−1	E. coli
58S bioactive glass^158	Au	0.1% wt and 1% wt	E. coli, S. aureus
Mesoporous nano-bioactive glass^159	TiO2	1–3 mol%	E. coli, S. aureus
Mesoporous bioactive glasses (MBGs)^160	Ga2O3	1, 2 & 3 mol%	E. coli, S. aureus

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The antibacterial effect of a BG could also be greatly enhanced by lowering the particle size of the BG.¹⁴⁹ Micro- and nano-scale BG incorporated poly(3-hydroxybutyrate) P(3HB) foams prepared by Misra et al.¹⁶¹ showed bactericidal properties against S. aureus (Fig. 13). This study showed more a pronounced antibacterial effect of nanoscale BG than macroscale BG.

Fig. 13 Effects of 58S, 63S, and 72S BG nanopowders on the growth of S. aureus (a) and E. coli (b) at a concentration of 100 mg mL⁻¹ broth. 4, good growth (positive control); 3, moderate growth; 2, sparse growth; 1, very sparse growth; and 0, no growth.¹⁵³

Waltimo et al.¹⁴⁹ also observed similar results. The mean number of viable cells was reduced and were significant in presence of n-BG. So, BG and P(3HB) both acted as antibacterial agents and reduced the number of viable cells. Mortazavi et al.¹⁵³ studied the antibacterial effect of three different percentage compositions (58S, 63S and 72S) of BG nanoparticles. It was found that sol–gel derived BG showed bactericidal and bacteriostatic effects on E. coli and S. aureus at optimum concentrations. They had no cytotoxicity as the cells showed good viability and proliferation. 72S BG with 72.88% silica showed no antibacterial activity while 63S with 57.72% silica was found to be bactericidal and bacteriostatic against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) only. 58S BG exhibited antibacterial effect against all the tested microbes such as E. coli, Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhi (S. typhi) and S. aureus (Fig. 14).¹⁵³ 45S5 bioglass exhibited a strong antibacterial effect against the bacteria, and the sensitivity of Gram-negative and Gram-positive bacteria to BG was different.^162,163 BG S53P4 showed a marked bactericidal activity against methicillin resistant S. aureus, Staphylococcus epidermidis (S. epidermidis), P. aeruginosa and Acinetobacter baumannii (A. baumannii) isolates, which are commonly involved in osteomyelitis.¹⁶⁴

Fig. 14 SEM micrographs of S. aureus attachment after 48 h on (a and c) P(3HB) foams and (b and d) P(3HB)/n-BG composite foams. A decrease in S. aureus cells on the surface of the P(3HB)/10 wt% n-BG foams is evident (d) in comparison to P(3HB) foams (c), respectively.¹⁶¹

BGs have been doped with different antibacterial agents in order to provide an antimicrobial effect. Kawashita et al.¹⁶⁵ prepared silica glass by a sol–gel method and doped it with aluminum and Ag ions. Aluminum ions accompanied silver ions in the three dimensional silicon tetraoxide (SiO₄) network. These microspheres, having a diameter of 1 μm, released silver ions slowly into the water and had a good antibacterial effect on S. aureus and P. aeruginosa with a minimum inhibitory concentration of 400. Hu et al.¹⁶⁶ studied the antibacterial effect of silver-loaded nanoporous BG, synthesized by a sol–gel method, and applied this as an antibacterial hemostatic dressing. BG with 0.02 wt% silver showed antibacterial properties against E. coli and the inhibition effect reached up to 99% in 12 h. In another study, a silver doped bioglass system with a silver concentration of 0.02–0.20 mg inhibited the growth of E. coli.¹⁵⁴ Blaker et al.,¹⁶⁷ coated commercial surgical sutures with silver doped BG in order to impart antibacterial properties to the affected site. Silver oxide was proved to be bactericidal and provided BG with a bacteriostatic effect. Bioactive glass coatings on different biomaterials have also been used to induce bactericidal effects. Pratten et al.¹⁶⁸ observed that silver-containing BG coatings are effective in reducing bacterial contamination as compared to coatings without silver. These antibacterial coatings limited the attachment of bacteria to the coated surfaces. Verné et al.¹⁶⁹ showed that these silver containing BGs also show antibacterial effects against S. aureus. Bioactive glass composites without silver also exhibited antibacterial activity against S. aureus, E. coli and P. aeruginosa depending upon the concentration without any cytotoxic effects.^170–172 Instead of using silver ions, which are toxic to cells beyond the optimum levels, MgO was used as antibacterial agent by Fooladi et al.¹⁷³

Another study showed that copper (Cu) also acted as an antibacterial agent. The antibacterial effect of Cu was studied by incorporating it in bioactive scaffolds. Cu underwent redox reactions and switched between the redox states from Cu⁺ to Cu²⁺ by donating or accepting electrons. These redox reactions also resulted in the formation of hydroxyl groups which could cause damage to bacteria directly or by oxidizing the lipids and proteins thus promoting their antibacterial effect.¹⁰³ Mouriño et al.¹⁷⁴ studied the antibacterial activity of gallium crosslinked alginate–BG composites against S. aureus and found these composites to be bactericidal.

Bioglasses have been used as local drug delivery systems although these still require extensive research. Pronounced antibacterial effects have been generated by using BGs as drug carriers.¹⁷⁵ Tetracycline hydrochloride (TCH) incorporated BG/collagen showed antibacterial effects against Staphylococci strains by inhibiting S. aureus cell growth.¹⁷⁶ Domingues et al. used bioactive glass as a drug delivery system for tetracycline hydrochloride and the tetracycline–β-cyclodextrin complex. Drug-loaded BG samples showed a bacteriostatic effect against A. actinomycetemcomitans.¹⁷⁷

3 Conclusions and future scope

BGs are biocompatible and are excellent materials for bone and tissue regeneration due to their osteogenic and angiogenic properties; they support cell adhesion, proliferation and osteogenic differentiation. Studies show that BGs are non-toxic and enhance cementoblast viability, metabolic and mitochondrial activity. Although there are few reports on the cytotoxicity of alkaline glasses, however, this can be overcome by modifying the composition of the glass. Controlling the initial composition and processing parameters of BGs enhances their abilities of revascularization, differentiation of mesenchymal stem cells (MSC), enzyme activity and osteoblast adhesion. BGs act as stimuli to allow fibroblasts to release angiogenic growth factors, and help blood vessel formation and revascularization. BG/polymer composites have also been studied by many research groups to tailor their physical and biological properties. These polymers not only promote cell proliferation and cell adhesion but also affect the mechanical properties of bioactive glasses which are brittle in nature. Different metal oxide dopants such as B₂O₃, CuO, CoO have been found to help in bone remodeling, angiogenesis, collagen production and mineralization. Bioactive materials as implants should not cause any infection at the site of surgery. To control bacterial infection, Ag, Cu, Au, Mg, Ti and Ga have been used, which impart antibacterial properties in bioactive glasses and their composites.

However, before going into clinical trials there is a need to investigate in vivo degradation, bone formation and metabolism. Understanding the mechanism of the in vivo biological behavior of bioactive glass shall help to make them promising biomaterials in future. There are a few reports on both in vitro and in vivo studies, but these studies are limited to short periods. Long-term degradation and biological responses in realistic biological system should be investigated to reveal the effect of degradation products on biological systems. Many polymeric composites of bioactive glass may lead to formation of by-products which may interact with biological systems; therefore, these issues need to be addressed. Studies have shown that nano and micro BGs have different properties, but their behavior in combination with polymers also needs to be addressed in terms of how they affect cell adhesion, proliferation and osteogenic differentiation, and other metabolic activities. Inorganic metal oxides have been added in bioactive glasses but their effects in vivo and in vitro need to be studied before their clinical use. A detailed study of the behavior of these metal oxides is very important for future applications of these composites. Furthermore, there is a need to understand the mechanism of gene expression and up-regulation of markers involved in bone and tissue regeneration. One important concern for clinical trials and commercialization of BGs and their composites is their sterilization procedure. Especially in the case of BG polymer composites, the maintenance of properties of polymers after sterilization is very important, as in case of composites these sterilization conditions affect the degradation rate and degradation products.

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