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Selective Contribution of Bioactive Glasses to Molecular and Cellular Pathways Maryam Rahmati, and Masoud Mozafari ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01078 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Biomaterials Science & Engineering

Selective Contribution of Bioactive Glasses to Molecular and Cellular Pathways

Maryam Rahmati 1, Masoud Mozafari 2,3,4,*

1

Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, 0317 Oslo, Norway 2

Bioengineering Research Group, Nanotechnology and Advanced Materials

Department, Materials and Energy Research Center (MERC), P.O. Box 14155-4777, Tehran, Iran 3

Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran 4

Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

* Corresponding author: M. Mozafari, PhD 1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

E-mail address: [email protected] Tell: +98-912 6490679; fax: +98-263 6280033 (Ext. 477)

2 ACS Paragon Plus Environment

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Abstract Over the past few decades, biomedical scientists and surgeons have paid substantial attentions to bioactive glasses as promising long-lasting biomaterials that can chemically make connections with the neighboring hard and soft tissues. Several studies have examined the cellular and molecular responses to bioactive glasses as suitable biomaterials for tissue engineering and regenerative medicine. In this regard, different ions and additives have been currently used to induce specific characteristics for selective cellular and molecular responses. This review briefly describes foreign-body responses (FBRs) mechanisms and the role of adsorbed proteins, as the key players in starting interactions between cells and biomaterials. Then, it explains the physicochemical properties of the most common bioactive glasses, which significantly impact on their cellular and molecular responses. It is expected that with the development of novel strategies the physiochemical properties of bioactive glasses can be engineered to precisely control proteins adsorption and cellular functions after implantation.



Keywords: Bioactive Glass; Biomaterials; Tissue engineering; Cell response; Regenerative medicine


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1. Introduction All implanted biomaterials cause immunological system responses in the body after their implantation, which frequently lead to incomplete in vivo functionality and durability of biomaterials

1-2.

During implanting biomaterials in the body, the adsorbed

proteins, besides macrophages and dendritic cells (DCs), play key roles in initiating communications between cells and biomaterials 3. Through the rapid adsorption of proteins on the surface of biomaterials, foreign body responses (FBRs) mechanisms start happening. The functions of adsorbed proteins are highly dependent on the physicochemical and biological properties of both the biomaterials surface and proteins’ properties

4-5.

Therefore, in designing each biomaterial carefully considering the details

of immune system responses to tits surface is essential. Over the past few decades, surgeons and biomedical scientists have paid substantial attentions to bioactive glasses as promising long-lasting biomaterials that can chemically make connections with the neighboring hard and soft tissues

6-8.

A bioactive ceramic is defined as a material which

in biological conditions potentially causes a progressive response to the interface of the implanted biomaterial 9. A porous biologically active layer, known as hydroxycarbonate 5 ACS Paragon Plus Environment


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apatite (HCA) layer, starts forming around the bioactive material after its implantation in the biological conditions 9. According to their bioactivity mechanism of action, these biomaterials are categorized into two groups including ceramics that initiate bioactivity owing to their chemical properties, and ceramics that their bioactivity could be encouraged by surface modification strategies or by using biological active agents in the ceramic’s porous structure

10-11.

Bioactive glasses (such as Bioglass®) and glass-

ceramics are frequently used in tissue engineering. Glass-ceramics are polycrystalline materials that encompass at least one crystal phases inserted into a remaining glass

12.

Glass-ceramics are commonly fabricated by controlling heat treatment concurrently with using sintering and crystallization, as well as sol-gel approaches 10-11. The most common crystallinity between glass-ceramics are between 30 and 70%. Differences between glass-ceramics crystallinity lead to observe a wide range of bio-ceramics with dissimilar biological, and physicochemical properties. In addition, introducing novel glass-ceramic biomaterials with different kinds of additives leads to observe dissimilar biological responses 13-14.


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This review emphasis on research concerning using bioactive glasses for tissue engineering applications. In the presented paper, a brief explanation about FBRs mechanisms and the role of adsorbed proteins as key players in starting interactions between cells and biomaterials will be given. Then, the main body of paper goes on the physicochemical, and mechanical properties of the most important bioactive glasses including, silicate, phosphate, and borate based glasses, which significantly impact on their biocompatibility responses. The discussion will then highlight the recent studies in which by the implementation of some novel strategies and additives the physiochemical properties of bioactive glasses have been engineered to present some promising solutions for more precisely controlling proteins adsorption and cellular functions after their implantation.

2. Biological Responses to Biomaterials After implanting a biomaterial into the body, a tissue/material interface is instantly formed. The main biological responses are started by the nonspecific adsorption of blood and tissue fluid proteins onto the biomaterial’s surface 15. Several of studies have proved 7 ACS Paragon Plus Environment


that the physicochemical properties of biomaterials have direct impacts on the degree and extent of FBRs 16-17. In fact, after implanting biomaterials, the injury to tissue causes a series of acute and chronic inflammatory and wound healing reactions, which are mainly directed by proteins, macrophages and DCs 3, 17. The acute inflammation phase, which is mainly in charge for provisional matrix formation and wound site cleaning, could endure from hours to days and is mainly directed by the adsorption of proteins, neutrophils, and macrophages type 1

18.

Briefly, during this phase, vessels expand which cause flowing

extra blood into the damaged area. Subsequently, several of blood and tissue proteins, such as fibronectin (FN), fibrinogen (Fg), vitronectin, complement C3, and albumin (ALB), are released; and also leukocytes stick to the blood vessels’ endothelium 1, 3, 18. Following that monocytes are presented into the area, which differentiate into macrophages type 1 and type 2 (M1, M2). M1 is responsible for acute inflammatory phase by releasing proinflammatory factors, however M2 is in charge for chronic inflammatory stage by releasing anti-inflammatory factors

3, 5, 18-19.

It has been shown that the determined inflammatory

provocations, such as the persistent existence of biomaterial/tissue contact, lead to the M2 activities and chronic inflammatory responses. In addition, the tissue in chronic 8 ACS Paragon Plus Environment

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inflammation stage is histologically less even than acute inflammation, and is mainly described by monocytes, M2, lymphocytes release, and subsequently the blood vessels and connective tissue formations 19-20. Based on the degree of injury at the implanted site, the acute phase takes generally less than one week, however the chronic phase typically lasts about two weeks 15, 21. The granulation tissue is then substituted by an extracellular matrix (ECM), which acts as a physical scaffold and a key modulator of the biological responses. During the chronic inflammatory responses and the presence of macrophages, the tissue granulation, fibroblasts infiltration, and neovascularization are also recognized

19.

Granulation tissue could be considered as the precursor of fibrous

capsule formation, and is detached from the biomaterial’s surface through the cellular constituents. The inflammatory reactions at the surface of a biocompatible material should not take further than three weeks 18. The physiochemical properties of bioactive glasses and glass-ceramics have a direct influence on the protein adsorption and subsequently biological responses to them, which will be discussed in detail here.

3. Bioactive Glasses and Their Physicochemical Properties 9 ACS Paragon Plus Environment


A bioactive material has been demarcated as a material that in biological circumstances experiences particular surface interactions with cells and biomolecules, which finally cause forming an HCA-like layer 22. The ability of a material to create a HCAlike surface layer after exposing to a simulated body fluid (SBF) in vitro, is the common signal of its bioactivity in biological conditions

22.

However, it should be noted that there

is a far distance between in vitro and in vivo environments’ conditions which makes it difficult to decide about materials’ function in the body based on in vitro examinations 2324.

Bioactive glasses which have a great ability of forming HCA-like layer in both in vitro

and in vivo conditions have recently gained considerable attentions among scientists and surgeons 9, 24-25. These materials are fabricated from glass formers including silica (SiO2), boric acid (B2O3), and phosphoric oxide (P2O5), network modifiers, and intermediate oxides

26.

In the following sections, the physicochemical properties of main types of

bioactive glasses including silicate, borate, and phosphate bioactive glasses which could potentially affect their biological responses will be in detail given.


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3.1. Silicate Bioactive Glasses For the first time in 1969, Hench and his co-workers introduced silicate-based bioactive glass (with the formulation of 45 % SiO2, 24.5 % Na2O, 24.5 % CaO and 6 % P2O5 known as 45S5 bioactive glass), as a biomaterial which could potentially make connections to the bone tissue in biological conditions

27.

Silicate-based bioactive glass

is the key type of bioactive glass constructions, which has been widely suggested as a promising candidate in tissue engineering applications 28-29. In this system, Na, Ca and P components are mixed in various relative amounts to formulate a three dimensional silicate structure. Furthermore, in this network Si is 4 fold organized to O. The main structural properties which make 45S5 glass a bioactive material are its low concentration of SiO2 content, high concentration of Na2O and CaO, as well as high amount of CaO/P2O5 proportion 24. Additionally, the main mechanism of bonding 45S5 glass to bone is related to the carbonate substituted HCA layer which constantly makes connections with the bone and its surrounding soft tissue

10-11, 27.

As defined by Hench,

27

the HCA

layer is commonly supposed to create as a result of some reactions on the implanted bioactive glass surface. Just after the formation of an HCA layer, the biological 11 ACS Paragon Plus Environment


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mechanisms stimulate the adsorption of growth factors, and consequently cells responses to the surface of bioactive glasses

30.

At this time, osteoblasts provoke the

formation of ECM, which mineralizes to create a nano-crystalline mineral and collagen on the surface of glass, whereas its chemical biodegradation comes to an end by passing the time 31. The molecule and cell responses to 45S5 glass has been widely studied 32-34. During the degradation of bioglass, in addition to Na and Ca release, Si(OH)4, is also released

35.

However, 45S5 glass still is appreciated as the gold standard for bioactive

glass materials, it has some drawbacks, including the difficulty of processing 45S5 glass into porous 3-D scaffolds, the restricted ability of 45S5 glass to sinter by viscous flow upper its glass transition temperature (Tg), the constricted space between Tg and the beginning of crystallization, as well as low mechanical behavior

36.

In addition, it has a

slow rate of chemical degradation and subsequently transformation to the HCA-like layer, which causes some mismatches between the degradation rate of body tissues and the implanted glass 37. Moreover, the transforming mechanism of bioglass to a HCA-like layer is frequently imperfect, which causes remaining some amounts of SiO2 in the substrate and increasing concerns about SiO2 cytotoxicity. Some studies have demonstrated that 12 ACS Paragon Plus Environment

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as a consequence of chemical degradation, the concentration of some ions such as Na+ and Ca2+ increased and also some changes in the pH were detected

37-39.

It has been

suggested that these changes could have some biological effects which predicting them by in vitro studies is difficult. Additionally, the toxicity of the released ions from this material is still questionable. Catauro et al. 40 have more recently investigated the specific effects of Ca/P molar ratio and heat treatment preparation approach on the bioactivity and biocompatibility properties of silicate bioactive glasses. The authors synthesized two bioactive glasses with dissimilar Ca/P molar ratios by sol-gel method and then exposed them to heat-treatment at three temperatures including; 120 °C, 600 °C and 1000 °C. NIH 3T3 murine fibroblast cells responses to the samples were investigated which revealed that both Ca/P molar ratio and heat-treatment approach could have noteworthy effects on bioactive glass biocompatibility. The sample with the greater calcium concentration and treated at 600 °C was detected to be the most bioactive ceramic. In addition, the biocompatibility of the glasses modified at 600 °C and 1000 °C was significantly greater than that heated at 120 °C. Moreover, Lei et al. 41 have synthesized some bioactive glass microspheres (BGMs) by the implementation of sol–gel-polymer approach, and then 13 ACS Paragon Plus Environment


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examined the impacts of their morphology on their physicochemical, bioactivity, and biological properties. They reported that the BGMs had a regularly spherical morphology, which in comparison with irregular bioactive glass (IBG), they demonstrated more stable and slower silicon release kinetics with pH stability in SBF. More even and needle flakelike apatite was shaped on the BGMs surfaces (see fig. 1). The human mesenchymal stem cells (hMSCs) viability tests indicated better cell growth, and osteogenic differentiation on BGMs in comparison with IBG. The authors suggested the suitability of BGMs for bone tissue engineering and drug delivery applications.

Figure. 1. A) Surface morphology of irregular bioactive glass (IBG) after immersion in simulated body fluid (SBF) for a) 1 day, b) 7 days. B) Morphology of bioactive glass microspheres (BGMs) after immersion in SBF after c, d) 1 day, e, f) 7 days. C) Cells morphology after seeding on IBG for g) 4 hours, h) 7 days and BGM for i) 4 hours, and j) 7 days. Reproduced with permission from Chemistry.


41.

Copyright 2011 Royal Society of

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3.2. Borate Bioactive Glasses Borate glass is another main class of bioactive glasses which with a more complex network has been currently used in biomedical applications 42-43. It has been shown that borate glass structure could be made of trigonal planar BO3 and/or tetrahedral BO4 components, which the addition of metal oxides to its network cause converting the planar units into tetrahedral ones leading to increasing the network connectivity

26.

Borate

bioactive glass is naturally bioactive, possesses inferior chemical sturdiness and could to a high extent convert to HCA-like layer. Some studies have reported that the degradation rate of this bioactive glass could be well-adjusted by controlling its chemical composition. Its degradation rate could be highly different by partly substituting SiO2 in silicate 45S5 or 13–93 glass with B2O3 or completely substituting the SiO2 with B2O3. The compositional flexibility and more precise control over the degradation rate of this type of materials make it more suitable for implanting it in biological conditions

26, 39.

In addition, it has been

reported that the borate bioactive glass powders are further reactive and bioactive than the silicate 45S5 ones 44. Some studies have revealed that borate bioactive glass has the 15 ACS Paragon Plus Environment


ability to support in vitro cell growth and also in vivo tissue infiltration with the aim of treating bone infections. Cui et al. 45 have suggested using borate bioactive system as a controlled drug delivery system for antibiotic drugs such as vancomycin for osteomyelitis treatment. The synthesized borate-based cement released vancomycin over twenty-five days in phosphate-buffered saline, which during that the borate glass transformed to HCA-like layer. The in vivo study on rabbit tibial defects infected with methicillin-resistant Staphylococcus aureus (MRSA)-persuaded osteomyelitis, indicated that the vancomycinloaded cement delivery system changed to HCA-like layer and improved new bone development in the defects during eight weeks. The osteomyelitis infection was treated in 87% of the defects injected with the vancomycin-loaded borate glass cement. This research team in another study more deeply

46

studied the efficacy of this system for

controlled releasing of vancomycin in a rabbit tibial model. The authors reported that the cement had an injectability greater than 90% within the first three minutes after mixing, hardened about thirty minutes, and also had a compressive strength about 1862 MPa. The vancomycin was released from the system into phosphate-buffered saline for more than 36 days, and the amassed concentration of released vancomycin was 86%. In 16 ACS Paragon Plus Environment

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addition, based on the results obtained from radiography and microbiological tests, 2 months after injection the borate bioactive glass and calcium sulfate (CS) cements had an improved capacity to destroy bone infection in comparison with intravenous vancomycin injection; however there was no meaningful dissimilarity between the two injected cements. Besides, the histological tests indicated the biocompatibility of boratebased bioactive glasses (see fig. 2).

Figure. 2. A) Radiographic images of rats before and after implantation. Illustrative preoperative radiographs presenting tibial osteomyelitis in the 4 various groups (1a, 2a, 3a, 4a), categorized by bone damage



(arrows), periosteal new bone development (arrowhead) and sequestral bone formation (asterisk). (1b) The images of Group 1 after surgery indicated worsening of osteomyelitis combined with periosteal new bone formation (arrowhead), damage of bone (arrows) and sequestral bone formation (asterisk). (2b) The images of Group 2 after surgery indicated that the osteomyelitis was partially well-ordered. (3b, 4b) The images of Group 3 and 4 after surgery indicated that the osteomyelitis was treated. B) Illustrative images of H&E stained segments of rabbit tibiae at two months after implantation. (a) Group 1 (without scaffold): Characteristic symbols of osteomyelitis, comprising damage of bone (DB), intramedullary abscess (IA), with practically no new bone development; (b) Group 2 (intravenous injection of drug): no distinctive marks of osteomyelitis and tiny new bone development; (c) Group 3 (drug-encapsulated CS cement): the CS cement was commonly degraded with inadequate new bone development (white arrow) neighboring it, and some macrophages near the degraded material (CS); (d) Group 4 (vancomycin-loaded borate BG cement): a substantial quantity of new bone (white arrow) was shaped which was in direct interaction with the degraded implant (BG). C) SEM images of the cement after surgery for eight weeks in rabbit tibiae infected with MRSA-induced osteomyelitis. The BG implant became porous (d), was attached to osteoid tissues (OT) at the interface (arrow) (e), and some cells (f) and fibrous tissue (F) connected to the substrate (c). Reproduced with permission from

46.

Copyright 2014 Ding, H.; Zhao, C.-J.; Cui, X.; Gu, Y.-F.; Jia, W.-T.;

Rahaman, M. N.; Wang, Y.; Huang, W.-H.; Zhang, C.-Q.

Yang et al.

47

have compared the physicochemical and biological properties of

using three bioactive glass nano-/micro-fibers, namely, silicate-based 45S5, boratebased 13-93B3 and 1605 which were doped with CuO and ZnO. Interestingly, faster ion releasing and glass changing to HCA-like layer were detected in borate-based fibers, which were related to their unique boron content and neighboring rheological form. Furthermore, 13-93B3 and 1605 fibers exhibited dissimilar glass alteration and


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biocompatibility properties, representing that the difference in ions’ concentration could also affect fiber's bioactivity and biocompatibility. Based on the suggested in vitro rheological module, they finally concluded that borate-based glass fibers could be promising candidates for wound healing applications. In another investigation the efficacy of using borate-based bioactive glass in comparison with β-TCP on radius defects regeneration was studied. The histological and morphological tests demonstrated that borate glass meaningfully improved the creation of new bone tissue. Also, its degradation rate was quicker than β-TCP. The ions released from borate glass improved bone marrow stem cells’ (BMSCs) viability, alkaline phosphatase’s (ALP) activity, and osteogenicrelated genes expression. Additionally, the key genes including Smad1/5 and Dlx5 in the bone morphogenetic protein (BMP) pathway and also the p-Smad1/5 proteins were considerably increased after borate glass implantation. The researchers concluded that borate glass could regenerate large segmental bone defects by stimulating the BMP/Smad pathway and also osteogenic differentiation in BMSCs

48.

Although, some

researchers have reported the poor cell responses to borate bioactive glasses. For instance, Lopes et al. 49 have compared the cytotoxicity of MG63 and hMSCs responses 19 ACS Paragon Plus Environment


to silicate and borate bioactive glasses. As it can be observed in fig. 3, they found appropriate cellular responses to silicate ceramics; however, the cellular responses to borate based systems were poor, which were likely owing to its high degradation rate leading to increasing of B and Mg ionic amounts in the cell culture medium. There is still doubt in the literature regarding the cytotoxicity of this material as a contradiction between related-studies could be seen which is likely because of the differences between experimental conditions, using different ion and additive dosages, as well as different preparation approaches.


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Figure. 3. Confocal laser scanning microscopy remark, for silicate glass scaffolds encapsulated with human bone marrow stem cells (hBMSCs) and cultured for a) one hour, b, c) 24 hours, d) 7 days, e) 14 days and f) 21 days. For borate glass composite g) one hour, h, i) 24 hours, j) 7 days, k) 14 days and l) 21 days. Reproduced with permission from 49 . Copyright 2011 Springer Nature.

3.3. Phosphate Bioactive Glasses Phosphate-based glasses have currently attracted a high attention in tissue engineering and regenerative medicine fields

owing to their great solubility,



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biodegradability, biocompatibility, and also chemical resemblance with the inorganic phase of natural bone tissue

9, 50.

Phosphate bioactive glasses encompass P2O5 as the

network former oxide as well as CaO and Na2O as their modifiers. It has been shown that this type of bioactive glasses in comparison with silicate glasses has a lower chemical stability, which restricts their application in biomedical fields

51.

Some studies have

suggested that by manipulating their solubility and composition these glasses could be used in biomedical applications as resorbable materials

24.

It has been reported that by

reducing the concentration of CaO in the structure the solubility of these bioactive glasses and subsequently their biocompatibility were increased

52-53.

In addition, decreasing the

CaO concentration in the phosphate-based bioactive glass compositions, which resulted in having lower dissolution rate, increased the osteoblast cells’ proliferation and also releasing bone sioloprotein (BSP), osteonectin (ON), and FN

54.

Although, in another

investigation, it has been demonstrated that phosphate-based bioactive glasses with different concentrations of Na2O decreased the cell adhesion and proliferation of human hBMSC and human osteoblast in vitro 55.


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4. Bone-like Apatite Formation on the Surface of Bioactive Glasses Bone-like apatite layers formed on the surface of bioactive glasses are analogous to the natural bone mineral, which could potentially make some interactions with collagen fibrils of injured bone tissue

27.

In the interactions between HCA layer and bone tissue

some molecular and cellular pathways are involved including protein adsorption, collagen fibrils integration, bone progenitor cells adhesion, cell differentiation, as well as the bone ECM secretion and its mineralization. The interactions between bioactive glass ions and osteoprogenitor cells provoke osteogenesis and subsequently new bone tissue formation 30-31.

The mechanism of HCA-like layer formation contains some phases including the

calcium ions of bioactive glass dissolve into the biological fluid whereas a rich layer of silica is formed on the glass surfaces. The dissolution of the calcium ions in the biological fluid causes starting HCA nucleation. Concurrently, the rich layer of silica liquefies substantial concentrations of silicate ions, which suggest suitable positions for HCA nucleation. Following that, the reactions of the calcium, phosphate, hydroxide, carbonate, and fluoride ions stimulate HCA layer growth

56.

It has been widely suggested that SBF

which has the ion concentrations similar to the human blood plasma could be potentially 23 ACS Paragon Plus Environment


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used for bio-mimicking the conditions of biological fluids environment and investigating the efficacy of bioactive materials in HCA-like layer formation

57.

The immersion in SBF

could be potentially used for anticipating the in vivo bone bioactivity prior to in vivo investigations, which to a great extent could decrease the number of used animals and also the time of trials 58.

5. Antimicrobial Properties of Bioactive Glasses It is a well-known fact that employing biomaterials in biological conditions is highly connected with the risk of adherence and colonization of bacteria on their surface, which could cause biomaterials’ failure and subsequently bacterial infections. A several of pathogens could be presented at biomaterials’ surface such as pseudomonasaeruginosa, Escherichia coli, staphylococcusaureus and staphylococcusepidermidis

59.

After

bioactive glass dissolution, owing to cation release the pH increases which could provide the conditions for eradicating microbes

60.

It has been reported that bioactive glasses,

could potentially show antibacterial properties and kill pathogens relate to enamel caries (Streptococcus mutans), root caries (Actinomyces naeslundii, S. mutans) and 24 ACS Paragon Plus Environment

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periodontitis (e.g. Actinobacillus ctinomycetemcomitas)

61.

A favorable bioactive glass

should possess antibacterial components such as metals, which help in hindering infections and decreasing the post-surgery sensitivity. It has been shown that metals could have a potential bioactivity against microorganisms and subsequently solve the low stability difficulty of bioactive glass materials 62.

6. Innovative Strategies for Selective Contribution of Bioactive Glasses Over the past few years, several papers have been published on modifying the physicochemical properties of bioactive glasses with the aim of improving the cellular and molecular interactions. The main current suggested approaches are through ion doping, salinization, and biological functionalization which will be discussed in the next sections.

6.1. Effects of Doping Different Ions on Cellular and Molecular Behavior of Bioactive Glasses A several of studies have recently suggested using various dopants to modify the physicochemical properties of bioactive glass materials which directly affect their cellular 25 ACS Paragon Plus Environment


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and molecular responses. Incorporating these ions in the structure could potentially cause alterations in the crystal network, surface area, thermal stability, morphology, solubility, and biochemical and biological properties. In addition, some studies have revealed that doping some ions could increase the mechanical strength properties of bioactive glass materials, which have direct effects on their cellular and molecular responses.

6.1.1. Fluorine-doped Bioactive Glasses Fluoride ions have been suggested as favorable doping agents for improving the physicochemical properties and subsequently biocompatibility behavior of bioactive glasses, by partially converting the HA phase of calcified tissue into fluorapatite (FA) 65.

63-

It has been shown that FA in comparison with HA had a greater physicochemical

stability, and subsequently a high resistance against dissolution by acids 66. Incorporating fluoride in bioactive glasses could be potentially used for dental caries treatment which are one of the most common bone tissue disorders. The main cause of dental caries is acidogenic bacteria, which can cause demineralization and complete tissue destruction 67.

A several of investigations have demonstrated the ability of fluorine ions to act as 26 ACS Paragon Plus Environment

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antimicrobial mediators, which improve the stability of apatitic structure against demineralization by acid

68.

It has been reported that the cytotoxicity of fluoride is totally

dependent on its concentration; as in low concentrations it encourages the osteoblast cells growth, whereas at high concentrations hinders the cells’ growth. Hence, this should be taken into consideration during its incorporating into bioactive glasses 69. Wang et al. 70

have currently investigated the effects of adding fluoride ions on biological and

mechanical properties of bioactive glasses. They fabricated 2 series of Na2O-CaO-SiO2P2O5 glass-ceramics which were doped with NH4HF2 (G-NH4HF2) or CaF2 (G-CaF2). Their results indicated that the mechanical properties of the sample containing G-NH4HF2 were inferior to that possessing G-CaF2, however its bioactivity was better. More interestingly, the thermal expansion coefficients of both samples were near to that of Ti6Al4V. Moreover, the hemolysis, in vitro cytotoxicity, systemic toxicity, and in vivo implantation tests all indicated the biocompatibility and good osseointegration of bioactive glasses. We have currently investigated the biological and scolicidal properties of fluoride-doped bioactive glasses as promising candidates for the prevention of post-surgery infections, particularly hydatidosis. As it can be seen in fig. 4, the samples exhibited a severe 27 ACS Paragon Plus Environment


scolicidal property, in the way that toxicity consequently started from 5 min and the substrates with 20, 10, 5 and 0% fluoride had 98 ± 2, 93 ± 5.8, 76.2 ± 6 and 5.8 ± 1.7% scolicidal activity, correspondingly, after 8 h exposing time. By increasing the fluoride doses, the scolicidal activity of glasses increased. In addition, the in vitro and in vivo studies indicated no noteworthy cell toxicity and also infiltration of inflammatory cells in comparison with control samples. Besides, they showed a remarkable antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa 71.


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Figure. 4. A) Microscopic images of Eosin-stained protoscoleces after exposing to FBG nano-powders in different times. B) Disk diffusion. Different samples of FBG were placed on Mueller–Hinton agar plate cultured with E. coli. C) Counting inflammatory cells seven days after implantation. (D, E) The H&E stained samples that received subcutaneous injection of normal saline (as negative control) and 20%-FBG seven days after implantation. (F) Counting inflammatory cells 28 days after implantation. (G, H) The H&E stained samples that received subcutaneous injection of normal saline (as negative control) and 20%-FBG 28 days after implantation Reproduced with permission from 71. Copyright 2015 Elsevier.



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6.1.2. Magnesium-doped Bioactive Glasses Magnesium (Mg) is an important metallic ion which plays some critical roles in the biological environment. For instance, Mg is a cofactor for several of enzymes, enhances the stability of nucleic acids, and also improves the ions metabolisms in body

26, 72.

In

addition, it could potentially encourage bone formation by increasing the activity of osteoblasts 73. Several of reports have been recently published on incorporating Mg into the microstructure of bioactive glasses. However, there is a contradiction between studies, as some of them suggested using this agent as modifiers, and others used it as an intermediate oxide, which partly entered in the silicate structure as MgO42− tetrahedral units 74-76. Additionally, a big paradox could be seen between studies that have been done on the effects of this agent on physicochemical and biological properties of bioactive glasses. It has been suggested that adding this metallic ion improved the surface area and porosity of bioactive glasses 77. However, in another investigation it has been found that incorporating Mg in glass composition had an insignificant effect on its physical features

78.

Additionally, some researchers have reported the effects of using this agent

on reducing degradation and subsequently retarding the apatite formation of bioactive 30 ACS Paragon Plus Environment

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glasses, whereas others found that it does not impact on the apatite formation

39, 78-79.

Moreover, some investigators have revealed that Mg could enhance the osteoblastic cells’ adhesion, proliferation, and differentiation responses to bioactive glass materials 80. Because there was poor data in the literature regarding the impacts of Mg concentrations on the bioactivity of bioactive glasses, Al-Noaman et al.

81

prepared seven bioactive

glasses containing different doses of Mg and then investigated their thermal properties, structure and bioactivity. Increasing the Mg concentration led to reducing the crystallization, TEC, Tg, softening temperature, and glass density, however, their oxygen density increased. In addition, however, Mg did not hinder the HCA-like layer formation, it delayed the apatite deposition time. Additionally, the authors found that the fibroblast cell responses to Mg-doped glasses were good, however, osteoblast cell responses to them were unfortunate 81,82.

6.1.3. Strontium-doped Bioactive Glasses Strontium (Sr) is one of the key metallic ions of the bone tissue which highly affects the bone metabolism

25, 83-84.

Strontium ranelate and strontium chloride are two 31

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commercial available agents which are commonly used for osteoporosis treatment. A several of studies have suggested a novel type of bioactive glass through replacing Ca2+ ions with Sr to more improve bone regeneration

25, 83-84.

It has been found that Sr-

substituted bioactive glasses could be useful in treating vertebral compression fractures 85.

In addition, the presence of Sr in bioactive glass materials makes it possible to more

prevent the growth of bacteria and microbial infections of bone tissue

25, 83.

Some

researchers have revealed that Sr-substituted bioactive glass demonstrated antimicrobial activity against sub-gingival bacteria, A. actinomycetemcomitans and P. gingivalis which was highly reliant on the concentration of Sr in the glasses 86. It has been shown that the biocompatibility of Sr-doped bioactive glasses was highly dependent on Sr concentration, as in low concentration it enhanced the bone formation, whereas, at high concentrations could cause bone abnormality 87. In addition, it has been exhibited that bioactive glasses which had smaller than 5 mol% SrO showed greater cell proliferation and alkaline phosphatase activity than glasses without Sr or with its high concentration

88.

Some

studies have reported that however the replacement of Sr with Ca caused a slight enlargement of glass structure, it did not cause substantial structural variations. In fact, 32 ACS Paragon Plus Environment

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doping Sr in bioactive glasses improved their density, whereas reduced their oxygen density

89.

Zhang et al.

90

have currently doped Sr in borate bioactive glass particles

containing a chitosan phase for improving their biocompatibility and biodegradability. Their results indicated that doping Sr to the system significantly improved the hBMSCs responses to the cement. In addition, histological evidence (see fig. 5) showed the osteogenic capacity of Sr containing samples. New bone tissue was detected at dissimilar spaces from the Sr-BBG implants during eight weeks, and the bone-implant contact (BIC) index was meaningfully greater for the cements containing Sr. Our researchers also found that the human osteosarcoma cells responses to the bioactive glasses containing different amounts of Sr were promising without any cytotoxicity effect 25.



Figure. 5. A) Three dimensional recreated images (column a, b and c) and sagittal images (column d) by micro-CT imaging of the region neighboring the scaffold, presentation freshly shaped bone at dissimilar spaces from the edge of the scaffold after surgery for four weeks and eight weeks in critical-sized rabbit femoral condyle defects. B) Histological test of freshly shaped bone and bone−implant contact (BIC) index for BBG and Sr-BBG scaffolds fixed for four weeks and eight weeks in critical-sized rabbit femoral condyle defects. e) Un-decalcified sections stained with Van Gieson’s picrofuchsin. The red color shows the new bone, and the cement seems black. B: bone; yellow arrow: BIC part; (f) BIC index for the BBG and Sr-BBG scaffolds defined by the histomorphometric measurements. Reproduced with permission from 90. Copyright 2015 American Chemical Society.


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6.1.4. Silver-doped Bioactive Glasses Silver (Ag) ions have gained great attentions among biomedical scientists owing to their potential antibacterial properties

65, 91-93.

A several of studies have revealed that

Ag-doped bioactive glasses could potentially prevent the growth of bacteria such as

Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus in biological conditions

94-96.

The Ag-doped bioactive glasses have also anti-inflammatory properties

which in combining with antibacterial properties make them favorable candidates for tissue regeneration and particularly wound healing applications. It has been demonstrated that the antibacterial and biological properties of Ag-doped bioactive glass were greater than the pure bioactive glass 97. Xiao et al. 98 have investigated the efficacy of Ag-containing borate bioactive glass in improving the biocompatibility and antibacterial properties of titanium implant. The authors reported that the coatings containing 1.0 wt% Ag was the most operative sample for the concurrent extermination of the infection and fracture fixation after implanting in a rabbit tibial model. In another study, Goh et al. 35 ACS Paragon Plus Environment

99


have compared the efficacy of using Ag and Cu in nano-sized bioactive glasses. The first release of Ag was significantly faster than Cu ions which indicated the better capability of Ag-doped bioactive glasses for showing fast antibacterial activities, however, cu-doped samples owing to a prolonged ions release, was more suitable for long-term antibacterial activities. We have currently investigated the effects of adding Ag and fluoride on the antibacterial activities of bioactive glasses against multidrug-resistant bacterial strains isolated from patients suffered from burns. Interestingly, however adding fluoride did not affect antibacterial activities, adding even one percent Ag had significant effects on killing bacteria without any toxicity against fibroblasts and also human osteoblast cells 91, 100-101. Catauro et al.

102

have also currently investigated the biological activities of Ag-doped

bioactive glasses. They fabricated bioactive glasses containing different amounts of Ag (in the range of 0.08-0.27%) by sol–gel technique. Scanning electron microscope/Energy dispersive X-Ray (SEM/EDX) tests demonstrated the ability of glass films in provoking the HCA nucleation on their surface after immersing in SBF. Moreover, the Colorimetric Cell Viability Kit I (WST-8) test indicated good 3T3 cells responses to the titanium implants coated with Ag, however, the bioactivity and biocompatibility of coatings reduced by 36 ACS Paragon Plus Environment

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increasing the concentration of Ag. In other hands, by increasing the amounts of Ag the antibacterial activity of films against the Staphylococcus aureus increased.

6.1.5. Copper-doped Bioactive Glasses Copper (Cu) is another well-known element which potentially possesses antibacterial properties 99, 103. Copper by connecting to the protein functional groups and microbial membrane has substantial effects against bacterial mediators such as E. coli, methicillin-resistant S. aureus and Clostridium difficile (C. difficile)

104-105.

Some studies

have revealed that based on the properties of Cu, its incorporation into bioactive glasses could promote the cell responses to these materials. However, it should be noted that increasing the concentration of copper leads to reducing apatite formation 106. It has been demonstrated that Cu ions generally act as network modifiers which disturbed the silicate structure of bioactive glasses; however, its influences on the structure is lower than Mg and Zn ions

107.

It has been reported that the impact of Cu ions on the physicochemical

properties of bioactive glass is highly dependent on the composition of bioactive glass. Bejarano and his coworkers have reported that doping copper in bioactive glass improved 37 ACS Paragon Plus Environment


the surface area and pore volume of 58S bioactive glass (60SiO2–36CaO–4P2O5), while a contrary outcome was found regarding Na-bioactive glass (60SiO2–25CaO–11Na2O– 4P2O5) 106. Wang et al. 103 have currently synthesized a controlled delivery system based on borate bioactive glass substrate containing Cu ions for encouraging angiogenesis and osteogenesis in a rodent calvarial defect model. Different concentrations of Cu (0–3.0 wt% CuO) were doped in the borate glass scaffolds with the pore size in the range of 200–400 µm by using a polymer foam replication method. The authors reported that after immersing the samples in SBF the scaffolds started releasing Cu ions at a rate which was reliant on the concentrations of Cu. Additionally, the in vitro cytotoxic tests on hBMSCs revealed that not only the Cu ions at the mentioned doses were not cytotoxic, but also by increasing the concentration of Cu in the scaffolds the ALP activity of the cells improved. Moreover, the results of implanting the scaffolds in rat models indicated that the bioactive glass substrates containing 3 wt% CuO had a meaningfully greater ability for provoking angiogenesis and osteogenesis in comparison with the un-doped samples (see fig. 6).


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Figure. 6. A) Field-emission scanning electron microscope images of hBMSC cells cultured on the 4 substrates (BG, BG–0.5%Cu, BG–1%Cu and BG–3%Cu) for 48 h: a) BG; b) BG–0.5%Cu; c) BG–1%Cu; (d) BG–3%Cu. B) Micro-CT assessment of bone repair in the rat calvarial defects treated with the BG–3Cu ceramics and the BG ceramics and in the unoccupied defect at eight weeks after surgery. e) Top, bottom and cross-sectional views of rebuilt images; f) and g) bone mineral density (BMD) and bone volume/total volume (BV/TV) in the defects treated with the ceramics and in the empty defects. C) h) Transmitted light images of van Gieson picrofuchsin stained segments of rat calvarial defects treated with BG–3Cu and BG scaffolds and the unoccupied defects at eight weeks after surgery. Red color is the new bone while the scaffold seems black. i) Percentage of new bone part in the defects treated with the scaffolds and in the empty defects. Reproduced with permission from 103 . Copyright 2014 Royal Society of Chemistry.

In addition, Zhao and his coworkers have also suggested using Cu-doped borate bioactive glass substrates as promising candidates for angiogenesis and wound healing applications. They synthesized some borate bioactive glass microfibers with the diameter in the range of 0.4–1.2 μm; and 6Na2O, 8K2O, 8MgO, 22CaO, 54B2O3, 2P2O5 composition; which different concentrations of Cu (0–3.0 wt% CuO) were doped in them. They found that after immersing in SBF the bioactive glass microfibers were degraded



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and simultaneously changed to HCA-like layer during seven days. Furthermore, the in

vitro cell culture exhibited that the released ions from microfibers were not toxic to human umbilical vein endothelial cells (HUVECs) and fibroblasts. In addition, they encouraged the HUVEC migration, tubule creation, as well as expression of VEGF and angiogenicrelated genes of the fibroblasts. The Cu-doped microfibers with the Cu concentration of 3.0 wt.% could also provoke angiogenesis of full-thickness skin defects in rodents, meaningfully better than the un-doped samples at 7 and 14 days after surgery 108.

6.1.6. Zinc-doped Bioactive Glasses Zinc (Zn) has also gained a considerable attention in the recent years for doping in bioactive glass materials owing to the fact that it is one of the key elements of body which can be found in the bone tissue. In addition, it commonly acts as a cofactor for many enzymes, and also as a stimulating factor for protein synthesis

109-111.

Some

researchers have exhibited that doping Zn in bioactive glass improved the chemical and mechanical sturdiness of the glass in biological environment

112-114.

In addition,

substituting Zn in bioactive glass assists in preserving the biological pH solution and 40 ACS Paragon Plus Environment

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consequently aids in the typical osteoblasts growth. However, using Zn in biological conditions also is highly dependent on its concentration as in high concentrations could cause cells death. Brauer et al. 115 have reported that although doping low concentrations of Zn in glass ceramics increases their mechanical properties, the amounts of Zn equal or more than 400 μM could cause cells’ death and fail even in basic biocompatibility investigations.

6.1.7. Cobalt-doped Bioactive Glasses Cobalt (Co) is a crucial element of the body which could potentially exhibit anticancer and antimicrobial properties. Co ions could also potentially provoke angiogenesis and osteogenesis in bone tissue by initiating hypoxic circumstances, the hypoxia-inducible factor-1 (HIF-1) in mesenchymal stem cells, and HIF-α target genes 25, 83, 116-117.

The biocompatibility of Co is also highly dependent on its concentration as its

high concentrations could be cytotoxic and genotoxic

118.

Hence, bioactive glass

substrates could be used for the controlled release of Co in biological conditions. Our researchers have more recently revealed that Co-doped bioactive glass promotes HCA41 ACS Paragon Plus Environment


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like layer formation in biological conditions 25, 83. Its activity in bioactive glass structure is highly reliant on its doses as it acts as a network modifier at one percent and as a network former at concentrations more than five percent

116.

Substituting Co in 45S5 bioactive

glasses and glass-ceramics improved its physicochemical and mechanical properties 116, 119.

In addition, it has been revealed that 1393 bioactive glass with 1 wt.% of Co could

promote the osteoblast-like cells and endothelial cells growth, whereas, doping 5 wt.% of it prevented good cellular responses in both cell types

119.

Hoppe et al.

116

have more

recently synthesized a Co-doped silicate bioactive glass scaffold by substituting CaO with CoO in “1393” glass (53 wt % SiO2, 6 wt % Na2O, 12 wt % K2O, 5 wt % MgO, 20 wt % CaO, and 4 wt % P2O5) for angiogenesis purposes. A comprehensive understanding into the physicochemical reactions happening at the scaffold−fluid border was investigated by using advanced micro-particle-induced X-ray emission/Rutherford backscattering spectrometry (PIXE-RBS) test. As it can be detected in fig. 7, the authors found that the activity of Co was highly dependent on its concentration as both a structure former and also a structure modifier. At 5 wt % Co doses, the Tg of the glass was decreased owing to the exchange of sturdier Si-O bonds with Co-O bonds in the glass structure. In addition, 42 ACS Paragon Plus Environment

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they detected a calcium phosphate layer formation in all scaffolds, which for Co-doped materials traces of Co were detected. Additionally, it has been reported that incorporating Co ions in 45S5 Bioglass could highly improve its density and mechanical properties 120.

Figure. 7. Essential scattering in the cross section of a series of scaffolds after responses to SBF for seven days. A) 1393 glass scaffolds (53 wt % SiO2, 6 wt % Na2O, 12 wt % K2O, 5 wt % MgO, 20 wt % CaO, and 4 wt % P2O5), B) 1393-1Co scaffolds, C) 1393-5Co scaffolds. Reproduced with permission from Copyright 2014 American Chemical Society.


116.

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Table 1. The effects of doping different ions on the biological responses of bioactive glass-based scaffolds based on the most recent studies.

Ions

Biological responses



Ref

The cell responses to fluoride is dose dependent. In low amounts it shows appropriate biocompatibility; however, adverse responses can be seen by increasing the concentration.

Fluoride



The fluoride incorporation of 1–1.5% has been frequently reported to be the ideal

121-125

concentration to meet the highest bioactivity and biocompatibility. 

Sodium-free fluoride bioactive glass scaffolds had higher biocompatibility in comparison with that of sodium-containing ones.

 Magnesium

The cell responses to magnesium is dose dependent. However, there has been always a contradiction between studies, as some of them have reported better cytocompatibility for scaffolds containing 5 wt% magnesium in comparison with that of samples having higher amounts this element in the structure.


126-128

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

There are also some few studies that reported better cytocompatibility for scaffolds containing 10 wt % of magnesium.



Most studies concluded that doping Sr at different concentrations could not provoke FBRs. However, it has been shown that at high amounts (15 mol%), it inhibited cell viability. 9, 83, 129-135

Strontium 

It has been reported that this ion is biocompatible in different sizes (micro and nano), our group has shown that 9-micron particles containing Sr had higher cytotoxicity than the 725-µm ones.

Silver



however, at higher concentrations could cause cell toxicity.

 Copper

Doping low concentrations of Ag (up to 1%) in the scaffolds had no cytotoxicity;

40, 136-137

The cell responses to fluoride is dose dependent. In low amounts (below 10 mg/L), it showed appropriate biocompatibility; however, adverse responses could be seen by

138-140

increasing the concentration. 141-143

Zinc 45 ACS Paragon Plus Environment

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

Page 46 of 88

Incorporating low concentrations of zinc (0.5 wt%) showed no cytotoxic effects on scaffolds. However, at higher concentrations it showed cytotoxity.

Cobalt



Incorporating low concentrations of cobalt (0.5 wt%) showed no cytotoxic effects on scaffolds.


25, 83, 144

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6.2. Silanization In the recent years several of studies have suggested using silanization approach to enhance the physicochemical properties of biomaterials which contain a rich source of hydroxyl groups in their surface

145.

Owing to the presence of a rich source of OH at

bioactive glass surface using this approach could be useful in modifying its physicochemical surface properties and subsequently its biocompatibility

145-146.

This

approach helps in increasing the interactions between the biomaterials surface and biomolecules to improve the dispersion stability of biomaterials in biological fluids and also the drugs’ immobilization 147. It has been shown that the silanol groups of hydrolyzed silicon alkoxides which possess functional groups such as amino, chloro, carboxyl could potentially interact with the OH groups of the material’s surface

148.

As previously

mentioned the adsorption of proteins on the biomaterial’s surface plays a key role in determining following cell responses to the surface. Some studies have revealed that using this method could have some positive effects on protein adsorption to the surface of biomaterials. It has been reported that 3-aminopropyltriethoxysilane (APTES) as a wellknown silane could potentially improve the surface properties of bioactive glasses without 47 ACS Paragon Plus Environment


decreasing its bioactivity 149. Ling et al. 150 have recently treated the surface of bioactive glasses by using APTES as a silane agent. Their results indicated that the stretching vibration related to saturated C-H seemed in Fourier-transform infrared spectroscopy (FTIR) spectrum of treated sample, and also significant mass loss achieved from combustion of organic group was detected in the differential thermogravimetry (DTG) curve, which proved the interactions between the APTES and bioactive glass surface. In addition, the MC-3T3 cell growth studies revealed that the cell responses to saline-treated sample was significantly higher than untreated ones. Additionally, some researchers by the implantation of APTES in a short-time chemical reaction have recently designed a novel amino-functionalized mesoporous bioactive glass, which based on their results had a great drug loading capacity alongside with extended drug release time. They also showed that the surface area of treated samples with amino groups meaningfully increased in comparison with untreated samples. In addition, the in vitro bioactivity studies revealed that the modified samples were suitable for biological applications 151.

6.3. Biological Surface Functionalization of Bioactive Glasses 48 ACS Paragon Plus Environment

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As previously mentioned the adsorption of proteins on the biomaterials’ surface plays a key role in determining following cell responses to the surface. It has been widely shown that the chemical composition of substrate could highly have effects on the absorption of proteins. Physical and chemical immobilization of biological molecules and cells on the bioactive glass surface is another approach which has recently attracted a great attention among scientists for improving the protein adsorption on the surface of glass. Salinization is an example of chemical immobilization which in the previous section was explained. In addition, the bone integration of bioactive glasses could be enhanced by functionalization of their surface through proteins. In fact, the adsorbed proteins on the surface of glass could have prohibiting or enhancing influence on the HCA-like layer formation on the surface. For instance, the Fg and FN potentially stimulate HCA-like layer formation on bioactive glass surface

26, 152-153.

Some researchers have reported that the

expansion of cells on bioactive glass was only five percent in the absence of FN, however, its expansion increased to 100 percent in the existence of this protein 154. However, some serum proteins negatively retard the formation of HA-like layer on the surface of bioactive glass

155.

In addition, as cells through integrins interact with the adsorbed proteins on 49 ACS Paragon Plus Environment


biomaterial surface, using these receptors on the surface of bioactive glass could also enhance its biocompatibility

26.

Moreover, forming a calcium-phosphate on the surface

could significantly enhance the protein adsorption on it. It has been demonstrated that the concentration of adsorbed proteins on the surface of modified surfaces was meaningfully greater than untreated ones

154.

Additionally, another study reported that the porous

bioactive glasses which were treated with calcium-phosphate selectively adsorbed greater concentrations of FN from serum than the HA or untreated glasses 156.

7. Bioactive Glasses in the Market Over the past three decades, Hench’s original 45S5 Bioglass® has been clinically used in at least 1.5 million patients for bone tissue engineering applications 12, 157-158. The first 45S5 Bioglass® implant was clinically used for substituting the small bones of the middle ear in the USA 159. FDA later approved using this implant, and it was commercially used under the name of “Bioglass® Ossicular Reconstruction Prosthesis” or “Middle Ear Prosthesis” MEP®. However, short- and mid-term outcomes proved the better performance of MEP® in comparison with nearly-inert scaffolds 160. The long-term clinical 50 ACS Paragon Plus Environment

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investigations demonstrated that this class of materials could show advanced dissolution and fragmentation in the body

161.

Hence, these implants were collected from the U.S.

market few years later. However, its reformed version known as Douek-MEDTM is still commercially existing in some countries. Another glass-based scaffold materials, known as Ceravital® glass-ceramics, was also available for the middle ear small bones applications for a long time; however, more recently its production has been stopped owing to its dissolution in the biological conditions over time

162.

Bioglass®-EPI

(extracochlear percutaneous implant) is also another bioglass-based scaffold that commercialized for anchoring cochlear implants to the temporal bone of severely deaf patients 163. After some years, the fabrication of this product has been also stopped owing to the dissolution risks

164.

Subsequently, Endosseous Ridge Maintenance Implant

(ERMI®) has been introduced to the market for using in dental regeneration applications. A five-year follow-up investigation of this implant proved its high stability and safety

165.

However, because of their rigidity, the widespread clinical applications of these products are still limited. Some long-term follow-up studies have shown that custom-made monolithic bioactive glasses were more appropriate than the above mentioned scaffolds 51 ACS Paragon Plus Environment


for orbital bone fractures therapy

166.

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In addition, bioactive glass particles such as

PerioGlas® (NovaBone Products LLC, Alachua, FL, USA) that could be shaped into different bone defects (especially tooth and jaw ones) have been introduced to the market 167.

Moreover, Biogran® (Biomet 3i, Palm Beach Gardens, FL, USA), has been also

commercially employed for jaw bone regeneration. This scaffold showed a fine particle size distribution compare to that of PerioGlas®

168.

In addition to these 45S5 Bioglass®

implants, some other commercially available bioactive glass are accessible in the market. Most commercially accessible bioactive glasses have demonstrated a SiO2-based composition, comprising some extra modifiers in particular quantities

169.

3–93 (53SiO2–

6Na2O–12K2O–5MgO–20CaO–4P2O5 wt %) and S53P4 are some of the most studied bioactive glasses which their potential in bone regeneration have been wellacknowledged

170.

However, a quicker bone regeneration has been detected in

BoneAlive® in comparison with 13–93, which is owing to the existence of magnesium oxide. TheraGlass®, MedCell, Burgess Hill, UK, as bioactive glass powders fabricated by the sol-gel process demonstrated quicker bone regeneration rate compared to that of melt-derived samples, which is because of its inherent nanoporosity. Additionally, 45S552 ACS Paragon Plus Environment

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based implants for oral regeneration have been commercially presented. Some years ago, a 45S5 Bioglass® particulate known as NovaMin® (NovaMin Technology, FL, USA; now owned by GlaxoSmithKline, Brentford, UK) was added to a toothpaste to treat dental hypersensitivity

171-172.

Furthermore, there are some bioactive glass products that owing

to their angiogenesis abilities, can be used in both soft and hard tissues. For example, bioactive glass nanofibers (basic 13-93B3 composition: 53B2O3–6Na2O–12K2O–5MgO– 20CaO–4P2O5 wt %), commercially known as DermaFuse™/Mirragen™, could potentially improve the regeneration of long-term venous stasis ulcers in diabetic patients

173-174.

“RediHeal” (Avalon Medical, Stillwater, MN, USA) is a commercially available product that owing to doping copper in its structure has demonstrated potential angiogenesis effects 175-176.

As mentioned, many studies can be found in the literature regarding doping

different ions within the microstructure of bioactive glasses. The research on this topic is still active and there is hope to see more products in the market for a wide range of tissue engineering and regenerative medicine applications.

8. Discussion 53 ACS Paragon Plus Environment


Over the past few decades, bioactive glasses have gained an increasing attention among biomedical scientists and surgeons as promising candidates for tissue engineering applications. However, despite their current wide applications, some challenges particularly with respect to their biomechanical and also biocompatibility properties still remain unclear. In addition, however, silicate-based glasses have been broadly studied, borate and phosphate-based structures have recently suggested new chances for tissue engineering applications. It has been more recently proposed that the bioactive glass materials could also have proangiogenic properties, which make them suitable for soft tissue regeneration. However, more in vitro and in vivo studies should be done on studying the suitability of them for soft tissue regeneration. In addition, although, in the recent years many surface modification strategies have been rapidly proposed for improving antibacterial, bioactivity, and subsequently biocompatibility of bioactive glasses, more deeply in vitro and in vivo examinations should be done on investigating their efficacy in biological conditions. As several of studies have demonstrated, a great caution should be done during doping different metallic ions into the bioactive glasses owing to their dose-dependent cytotoxicity. Additionally, more in vitro and in vivo 54 ACS Paragon Plus Environment

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examinations on the toxicity of each type of modifiers should be done before suggesting them for clinical usage. Regarding ion-doped bioactive glass systems introducing some novel controlled release systems or even modifying the present ones should be considered. Additionally, as bioactive glasses are innately brittle, modifying the current processing methods or even suggesting some novel ones for enhancing their mechanical properties should be taken into account in the future research. Moreover, in the current years some studies have suggested designing composite materials for improving the biocompatibility and biomechanical properties of bioactive glasses, which also need more investigation. Furthermore, some other modifiers such as zirconia and alumina have been currently suggested for improving the physicochemical properties of bioactive glasses, which should be taken into consideration by more investigating their effects on the system 177-178.

Moreover, additional investigation is essential to create a sure place for bioactive

glass materials in long-term clinical use. In other hands, additional investigations should be done to examine all parameters which could have impacts on the biological response. There is still much to learn about the responses of key proteins to bioactive glass surfaces and further specific studies regarding protein adsorption on this class of biomaterials 55 ACS Paragon Plus Environment


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should be conducted. The literature still also suffers from not having a valid and strong database regarding precise mechanisms involved in the FBRs which should be also taken into considerations by biologists, and immunologists. If we had the ability to completely comprehend the principles of cell response to specific materials, and ions, then we could suggest more promising biomaterials for the future. Additionally, recently Shah et al.

179

have reported that the medium conditions such as its pH, composition, and the presence of proteins could highly have influences on the biological properties of bioactive glasses. Hence, the different medium characteristics and their effects on these biomaterials should also more deeply be examined. Many groups are working on this subject from different countries around the world. A Scopus search on "bioactive glass" (in TITLE-ABS-KEY), shows that the most active researchers are from China, United States, United Kingdom, Germany, Italy, Finland, Brazil, Spain, India, Iran, Japan, France, Portugal, South Korea and others (sorted by the number of publications). This is suggesting that Iran is the only country in the Middle East that extensively works on different aspects of bioactive glasses. Another search focusing on Iran shows that our research team is the leading group in the country on this subject. 56 ACS Paragon Plus Environment

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We are now expanding the research on bioactive glasses with a special focus on innovative applications of this class of materials through better understanding the key molecular and cellular pathways. It is expected that bioactive glass materials in different form can not only play a key role in the future of bone tissue engineering but also play a critical role in soft tissue engineering.

9. Conclusions Over the past few decades, substantial attentions have been paid to bioactive glasses as promising biomaterials that can chemically make connections with the neighboring tissues. Many studies have examined the biological responses to bioactive glasses as appropriate biomaterials for tissue regeneration. In this regard, different ions and additives have been employed to modify the molecular and cellular pathways. However, in some cases there is a lack of sufficient information regarding the exact cellular and molecular behavior of this class of biomaterials. There is still much to learn about the biological responses at the surface of bioactive glasses and more specific 57 ACS Paragon Plus Environment


studies should be conducted in this regard. The literature still suffers from not having a valid and strong database concerning the mechanisms involved in the FBRs which should be also taken into considerations.


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Abbreviations: Dendritic Cells (DCs); Foreign Body Responses (FBRs); Hydroxycarbonate Apatite (HCA); Fibronectin (FN); Fibrinogen (Fg); Albumin (ALB); Extracellular Matrix (ECM); Simulated Body Fluid (SBF); Glass Transition Temperature (Tg); Bioactive Glass Microspheres (BGMs); Irregular Bioactive Glass (IBG); Human Mesenchymal Stem Cells (hMSCs); Methicillin-Resistant Staphylococcus Aureus (MRSA); Alkaline Phosphatase (ALP); Fluorapatite (FA); Magnesium (Mg); Strontium (Sr); Silver (Ag); Copper (Cu); Human Umbilical Vein Endothelial Cells (HUVECs); Zinc (Zn); Cobalt (Co); HypoxiaInducible Factor-1 (HIF-1); 3-aminopropyltriethoxysilane (APTES); Calcium Sulfate (CS); Osteoid Tissues (OT); X-ray emission/Rutherford Backscattering Spectrometry (PIXERBS); Scanning electron microscope/Energy dispersive X-Ray (SEM/EDX); Colorimetric Cell Viability Kit I (WST-8).



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For Table of Contents Use Only:



Figure. 1. A) Surface morphology of irregular bioactive glass (IBG) after immersion in simulated body fluid (SBF) for a) 1 day, b) 7 days. B) Morphology of bioactive glass microspheres (BGMs) after immersion in SBF after c, d) 1 day, e, f) 7 days. C) Cells morphology after seeding on IBG for g) 4 hours, h) 7 days and BGM for i) 4 hours, and j) 7 days. Reproduced with the permission from 41. Copyright 2018 Elsevier.


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Figure. 2. A) Radiographic images of rats before and after implantation. Illustrative preoperative radiographs presenting tibial osteomyelitis in the 4 various groups (1a, 2a, 3a, 4a), categorized by bone damage (arrows), periosteal new bone development (arrowhead) and sequestral bone formation (asterisk). (1b) The images of Group 1 after surgery indicated worsening of osteomyelitis combined with periosteal new bone formation (arrowhead), damage of bone (arrows) and sequestral bone formation (asterisk). (2b) The images of Group 2 after surgery indicated that the osteomyelitis was partially well-ordered. (3b, 4b) The images of Group 3 and 4 after surgery indicated that the osteomyelitis was treated. B) Illustrative images of H&E stained segments of rabbit tibiae at two months after implantation. (a) Group 1 (without scaffold): Characteristic symbols of osteomyelitis, comprising damage of bone (DB), intramedullary abscess (IA), with practically no new bone development; (b) Group 2 (intravenous injection of drug): no distinctive marks of osteomyelitis and tiny new bone development; (c) Group 3 (drug-encapsulated CS cement): the CS cement was commonly degraded with inadequate new bone development (white arrow) neighboring it, and some macrophages near the degraded material (CS); (d) Group 4 (vancomycin-loaded borate BG cement): a substantial quantity of new bone (white arrow) was shaped which was in direct interaction with the degraded implant (BG). C) SEM images of the cement after surgery for eight weeks in rabbit tibiae infected with MRSA-induced osteomyelitis. The BG implant became porous (d), was attached to osteoid tissues (OT) at the interface (arrow) (e), and some cells (f) and fibrous tissue (F) connected to the substrate (c). Reproduced with the permission from 46. Copyright 2018 Elsevier.



Figure. 3. Confocal laser scanning microscopy remark, for silicate glass scaffolds encapsulated with human bone marrow stem cells (hBMSCs) and cultured for a) one hour, b, c) 24 hours, d) 7 days, e) 14 days and f) 21 days. For borate glass composite g) one hour, h, i) 24 hours, j) 7 days, k) 14 days and l) 21 days. Reproduced with the permission from 49 . Copyright 2018 Elsevier.


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Figure. 4. A) Microscopic images of Eosin-stained protoscoleces after exposing to FBG nano-powders in different times. B) Disk diffusion. Different samples of FBG were placed on Mueller–Hinton agar plate cultured with E. coli. C) Counting inflammatory cells seven days after implantation. (D, E) The H&E stained samples that received subcutaneous injection of normal saline (as negative control) and 20%-FBG seven days after implantation. (F) Counting inflammatory cells 28 days after implantation. (G, H) The H&E stained samples that received subcutaneous injection of normal saline (as negative control) and 20%-FBG 28 days after implantation Reproduced with permission from 71. Copyright 2018 Elsevier.



Figure. 5. A) Three dimensional recreated images (column a, b and c) and sagittal images (column d) by micro-CT imaging of the region neighboring the scaffold, presentation freshly shaped bone at dissimilar spaces from the edge of the scaffold after surgery for four weeks and eight weeks in critical-sized rabbit femoral condyle defects. B) Histological test of freshly shaped bone and bone−implant contact (BIC) index for BBG and Sr-BBG scaffolds fixed for four weeks and eight weeks in critical-sized rabbit femoral condyle defects. e) Un-decalcified sections stained with Van Gieson’s picrofuchsin. The red color shows the new bone, and the cement seems black. B: bone; yellow arrow: BIC part; (f) BIC index for the BBG and Sr-BBG scaffolds defined by the histomorphometric measurements. Reproduced with the permission from 90. Copyright 2018 Elsevier.


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Figure. 6. A) Field-emission scanning electron microscope images of hBMSC cells cultured on the 4 substrates (BG, BG–0.5%Cu, BG–1%Cu and BG–3%Cu) for 48 h: a) BG; b) BG–0.5%Cu; c) BG–1%Cu; (d) BG–3%Cu. B) Micro-CT assessment of bone repair in the rat calvarial defects treated with the BG–3Cu ceramics and the BG ceramics and in the unoccupied defect at eight weeks after surgery. e) Top, bottom and cross-sectional views of rebuilt images; f) and g) bone mineral density (BMD) and bone volume/total volume (BV/TV) in the defects treated with the ceramics and in the empty defects. C) h) Transmitted light images of van Gieson picrofuchsin stained segments of rat calvarial defects treated with BG–3Cu and BG scaffolds and the unoccupied defects at eight weeks after surgery. Red color is the new bone while the scaffold seems black. i) Percentage of new bone part in the defects treated with the scaffolds and in the empty defects. Reproduced with the permission from 103 . Copyright 2018 Elsevier..



Figure. 7. A) Essential scattering in the cross section of the scaffolds after responses to SBF for seven days. A) 1393 glass scaffold (53 wt % SiO2, 6 wt % Na2O, 12 wt % K2O, 5 wt % MgO, 20 wt % CaO, and 4 wt % P2O5), B) 1393-1Co scaffold, C) 1393-5Co scaffold. Reproduced with the permission from 116. Copyright 2018 Elsevier.. 145x136mm (72 x 72 DPI)


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