Novel Techniques of Scaffold Fabrication for Bioactive Glasses

Chapter 18

Novel Techniques of Scaffold Fabrication for Bioactive Glasses Zohaib Khurshid*, Shehriar Husain†, Hessah Alotaibi‡, Rabiya Rehman‡, Muhammad S. Zafar§, Imran Farooq¶, Abdul S. Khan‖ ⁎

Department of Prosthodontics and Implantology, College of Dentistry, King Faisal University, Al-Ahsa, Saudi Arabia, †Department of Dental Materials Science, Sindh Institute of Oral Health Sciences, Jinnah Sindh Medical University, Karachi, Pakistan, ‡Department of Biomedical Engineering, School of Engineering, King Faisal University, Al-Ahsa, Saudi Arabia, §Department of Restorative Dentistry, College of Dentistry, Taibah University, Almadinah Almunawwarah, Saudia Arabia, ¶Department of Biomedical Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ‖Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia

18.1 INTRODUCTION From a materialist’s perspective, the human body comprises a plethora of remarkable structures that invoke both awe and wonder in the casual and trained observer alike. The dearth of textures, components, and properties on display are best understood as a continuum of toughness, stiffness, flexibility, strength, and softness of biological components that may be lightweight or boast a dense convoluted hierarchy while highlighting isomerism and/or metamerism. The process and the subsequent regulation of biomineralization is a key stakeholder in the ongoing success of hard/mineralized tissues. The ability of this remarkable process to imbue connective tissues with an array of mechanical properties is exemplified by its critical function and abundance in organic life forms. Bone defects in general and critical/large bone defects in particular as a result of traumatic insult, surgery, pathological process, and genetic mutation induced deformities, test the management skills of even the most seasoned clinicians to date. The gold standard for producing templates conducive to bone regeneration encompasses complex spatial three-dimensional structures that aside from having an interconnected, reproducible, and quantifiable pore morphology should be significantly bioactive and biodegradable coupled with hard tissue isomechanical loading traits for bone tissue engineering (Sheikh et al., 2015; Zafar et al., 2015). A range of techniques are currently in use for synthesizing scaffolds such as particulate leaching, porogen templating, gel-cast foaming, Biomedical, Therapeutic and Clinical Applications of Bioactive Glasses https://doi.org/10.1016/B978-0-08-102196-5.00018-5 © 2019 Elsevier Ltd. All rights reserved.

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and freeze gelation. Although techniques such as porogen templating and gelcast foaming may yield porous templates exhibiting high values of mechanical strength, their utilization does not ensure effective tailoring of precise pore characteristics. Since the advent of 3D printing as a formidable and effective tool for fabricating biomimetic templates, a precise control over the parameters that govern pore architecture, which includes their size, interconnectivity, morphology, and surface area, is achievable (Samuels and Flowers, 2015; Ventola, 2014; Zafar et al., 2016). The discovery and subsequent introduction of bioactive glasses in the realm of biomaterials by Hench in the 1970s revolutionized our perception of the bone repair sequalae. The inherent ability of Bioglass and other clinically used bioactive ceramics such as sintered hydroxyapatite (HA), sintered β-tricalcium phosphate (TCP), HA/TCP biphase ceramic and glass ceramic A-W to initiate and maintain a chemical union with host mineralized tissue while predictably forming a carbonated HA layer on the surface post implantation has been exploited in numerous clinical scenarios that include intervertebral disc and periodontal bone repair along with use as bone fillers, spacers, and for endodontic treatment (Danial Khalid et al., 2017). In this backdrop, the quest for a silicon containing bioceramic and/or a silicate cement material that would submit itself to 3D printing at room temperature and emerge as a stable scaffold form with dictated pore architecture seems a justified and economically feasible one. This chapter/book aims to embolden the reader by equipping them with an up-to-date knowledge of some of the scaffold synthesis techniques commonly employed in the laboratory for devising a viable tissue-emulating scaffold system primed for delivery to the clinic. The desired outcome in this instance would be to establish a grounding concept of the evolution and modification of scaffold design over the years. Topics considered as front-runners in bioactive glass scaffold synthesis techniques—sol-gel synthesis, mesoporous scaffold design, and variants of 3D printing have been given special focus as an acknowledgement of their pivotal role in designing modern bioactive nanostructured scaffolds.

18.2  BIOACTIVE GLASS Bioactive glass is an inorganic, synthetic, and biocompatible material. Its bioactivity comes from its ability to bond to the bone by the formation of carbonated HA when it reacts with physiological fluid (Chen et al., 2008). Hench developed bioactive glass in 1969, which has many clinical applications such as being used as a scaffold in tissue engineering, as bone grafts, and as a coating material for dental implants (Farooq et  al., 2012). Bioactive glass are of different types, and each type has a unique composition. The rate of bone bonding varies based on compositions of bioactive glass (Hench, 2013). The most investigated type of bioactive glass is the bioactive silicate, which is made of 46.1 mol% SiO2, 24.4 mol% Na2O, 26.9 mol% CaO, and 2.6 mol% P2O5. A bioactive glass

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dissolves gradually when it is implemented. Because of the dissolution, ions are released and prompt the formation of a carbonated HA layer at the surface of the bioactive glass. It was indicated that high amounts of Na2O, CaO, and Cao/P2O5, which are being called as the modifiers, could motivate the glass reactivity in the physiological environment. One of the characteristics of bioactive glass is its kinetics of surface modification as a function of time when it is implanted in the body. The network activity of bioactive glass, which can be utilized to assess the bioactivity, surface reactivity, and solubility, is based on the number of bridging oxygen atoms. In vitro, the ability of bioactive glass to form the carbonated HA is studied by using simulated body fluid (SBF), which can be prepared using ionic compositions that are similar to the blood plasma (Farooq et al., 2012; Zeimaran et al., 2015). Transition temperature (Tg) is a critical parameter of bioactive glass. It is indicated when the amorphous solid is changed into a supercool liquid by heating. Tg is used to characterize a bioactive glass because it is an indirect mean for measuring the glass network, which is related to several important properties of glass such as solubility, degradation mechanical properties, and crystallization. Tg has a linear relationship with hardness; a low value of Tg shows a low level of bioactive glass hardness and vice versa (Stanić, 2017). Peak crystallization temperature is another important parameter of bioactive glass. A proper coordination between Tg and Peak crystallization temperature ensures that the glass sinters without any formation of crystals. Crystallization of bioactive glass reduces its bioactivity as the process of ion exchange between the bioactive glass and the formed crystals will restrict the biological solution.

18.3  TYPES OF BIOACTIVE GLASS 18.3.1  Silicate Bioactive Glass The first bioactive glass was designed by Hench is 45S5, which has the commercial name Bioglass. It is the most investigated type of bioactive glass by researchers. It is a silicate glass based on the 3D glass-forming SiO2 network in which Si fourfold coordinated to O. The bioactivity of silicate bioactive glass comes from the low content of SiO2, high amounts of modifiers (Na2O and CaO) and Cao/P2O5 ratio (Humagain et  al., 2013). The formation of a carbonated HA layer is a result of several reactions occurring on the surface of the silicate bioactive glass when it is implanted in the body as described by Hench: (1) The ions in the glass modifiers (Na+ and Ca+) will be exchanged rapidly with H+ or (H3O+) in the physiological solution. At the surface of the glass, the silica group will be hydrolyzed, and Si–OH group will be formed because of the rapid ion exchange. Si - O - Na + + H + ® Si - OH + Na + ( aq )

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(2) As the consumption of H+ ions increase, the PH of the solution will increase and will lead to the attack of SiO2 glass network and the dissolution of silica in the form of Si(OH)4, which will result in the continuous formation of SiOH groups on the glass surface: Si - O - Si + H 2 O ® Si - OH + OH - Si (3) There will be condensation and polymerization of an amorphous SiO2-rich layer (typically 1–2 μm thick) on the surface of the glass depleted in Na+ and Ca2+. (4) Further dissolution of the glass, coupled with migration of Ca2+ and (PO4)3− ions from the glass through the SiO2-rich layer and from the solution, leading to the formation of an amorphous calcium phosphate (ACP) layer on the surface of the SiO2-rich layer. (5) The glass continues to dissolve, as the ACP layer incorporates (OH)− and (CO3)2− from the solution and crystallizes as an HCA layer. 45S5 bioactive glass has several limitations when it is used as a scaffold. For example, it is very difficult to process 45S5 bioactive glass into 3D porous scaffold due to the limited ability of 45S5 bioactive glass to sinter by viscous flow above its glass transition temperature, which is the conventional method of processing 3D porous scaffold. This limitation results in low strength scaffold. 45S5 bioactive glass also has slow degradation rate and conversion to HA-like material, which makes the match between the rate of scaffold degradation and rate of new tissue generation difficult (Humagain et al., 2013). The slow and incomplete conversion to the HA-like material is due to noncongruent dissolution (Ojha, 2016).

18.3.2  Borate Bioactive Glass Using borate bioactive glasses in tissue engineering has several advantages. In the past, borate and borosilicate bioactive glass was prepared by replacing SiO2 with P2O3 partially or completely. Replacing SiO2 with P2O3 completely in 45S5 for the first time was studied by Richard. At a temperature of 37°C, the borate-based 45S5 glass was added into a solution of K2PHO4 and as a result of this, a HA, Ca10 (PO4)6 (OH)2 layer was formed (Margha and Abdelghany, 2012). Borate bioactive glasses have promising applications in bone regeneration and angiogenesis. Due to the low chemical durability, borate bioactive glass degrades faster than silicate-based bioactive glass and converts completely into a HA layer. The conversion process to the HA-like material is similar to the process in 45S5 but without the formation of the SiO2 layer. Borate bioactive glass enhances the proliferation and differentiation processes that occurs in vitro and tissue infiltration in vivo. They can be made into glass particulate at relatively low temperature without crystallization. The degradation rate can be controlled by replacing SiO2 with P2O3 in the silicate bioactive glass (Ojha, 2016).

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One limitation can be observed when borate bioactive glass is used, which is the release of toxic borate ions (Humagain et al., 2013). However, studies have shown that toxicity with an acceptable rate was detected when borate bioactive glass was used in a rat (Ojha, 2016).

18.3.3  Phosphate Bioactive Glass Phosphate bioactive glass has been developed to be used in different biomedical applications due to its chemical resistance. It is a potential substitute for silicate bioactive glass and a promising material for bone reconstruction and repair treatments as it has a high level of bioactivity (Mishra et al., 2016). It also has unique dissolution properties in the aqueous-based fluid. Its glass compositions can be changed to control the rate of degradation, which can be varied from several hours to weeks. Phosphate-based glasses are in the Ca-Na-P2O5 system, and its elements naturally exist in the body. Therefore, the normal physiological process is enough to excrete them. Silica-based glass can be used in much specialized applications due to its high melting temperature, which is considered in the manufacturing process while phosphate-based glasses can be prepared at relatively low temperature. Therefore, it is very difficult to make fibers from ­silica-based glasses, whereas phosphate-based glasses can be quickly drawn into fibers. To lower the melting temperature in silica-based glasses, metal oxides like CaO and Na2O can be added. However, the bioactivity of silica glass can be affected adversely as the crystallization increases. Phosphate bioactive glasses have a low chemical durability. Therefore, their use in the technological applications is limited. On the other hand, they are preferred to be employed in the applications like the release of oligo-elements in soil (Neel et al., 2009). The main difference between these glasses (silicate, borate, and phosphate) is in the way they dissolve and react in  vitro and in  vivo. While silicate-based glasses dissolve in a noncongruent manner, borate and phosphate-based glasses dissolve in a congruent manner (Ojha, 2016).

18.4 SCAFFOLDS/TEMPLATES 18.4.1  What is a Scaffold/Template? Tissue engineering is considered a rapidly emerging field that draws on novel concepts entrenched in traditional engineering and medical science together in order to overcome biological constraints—the penultimate aim being restoration, maintenance, or improvement of tissue function (Langer and Vacanti, 1993). At its core, the concept of tissue engineering is focused on the regeneration of biological structures that entails neofunctional tissue and/or entire organs. New tissue synthesis usually kicks off with the stimulation of cells—the underlying process often combining many molecular and mechanical signals.

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Biomaterials can be broadly conceptualized as the medium through which these signals are delivered. They can be essentially designed as 3D constructs boasting variable concepts and designs tailored-to-deliver therapeutic elements necessary for regulating functions of living tissues or to regenerate damaged tissue. One of the ways in which this may come to pass is if the biomaterial is released in the guise of a bioartificial scaffold (Langer and Vacanti, 1993). A scaffold is a natural/synthetic support used for designing a biological substitute that aims to deliver an elevated level of satisfactory performance than the damaged tissue owing to superior mechanical and structural properties (Zohora et al., 2014). Scaffolds can be potentially tailored to imitate the same ECM environment that is necessary to cell’s adhesion and movement in order to support the growth of new tissue of a particular phenotype (Zippel et al., 2010). During the synthesis of extracellular matrix (ECM), the cells are key building blocks of new tissue material. In the process of reproduction, the scaffold is populated by cells in due course, eventually leading to the inception of a three-dimensional tissue construct. As new tissue formation proceeds, the scaffold degrades itself as blood vessels start growing inside the new tissue (Zippel et al., 2010). Important key factors concerning scaffolds for use in tissue engineering are as follows: (i) Biocompatibility: An ideal scaffold must have an excellent biocompatibility to ensure cell survival and minimal immune response after implantation (Bitar and Zakhem, 2014). (ii) Biodegradability: The scaffold material must possess controlled biodegradability in order to degrade on time to ensure proper remodeling of the tissue (Bitar and Zakhem, 2014; Sachlos and Czernuszka, 2003). (iii) Mechanical Properties: The scaffold material must have sufficient mechanical strength, stiffness, and elasticity in order to provide support for the target cells. (Chen et al., 2008; Sachlos and Czernuszka, 2003).

18.4.2  Materials of Scaffolds Many types of porous scaffolds including polymeric, ceramic, and composite materials are widely used in tissue engineering process (Zohora et al., 2014). There are many synthetic and natural inorganic ceramic materials available for making scaffolds. HA and TCP are considered as best scaffold materials because of their superb osteoconductivity. However, they are considered as brittle and have poor mechanical properties. Also they are not appropriate to work with soft tissues (Sachlos and Czernuszka, 2003). Bioactive glasses are favorable biomaterials for tissue engineering due to their ability to support bone cell growth, bonding to both hard and soft tissues, capability to repair defect sites and controllable degradation rate in vivo (Zohora et  al., 2014). Millions of composites have already been discovered: synthetic polymers with natural polymers, synthetic polymers with bioceramics, polymers with metals, metals with ceramics, etc. Composites are necessary

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to obtain optimal biological, structural, mechanical, and chemical properties of scaffolds. For example, in bone tissue engineering, bioceramics/polymers are commonly used composites (Zohora et al., 2014).

18.4.3  2D and 3D Scaffolds Methods 18.4.3.1  Salt Leaching This is achieved by adding porogen or salt crystals and polymer in a mold. A hardened polymer with pores will be formed once all the salt leaches out by the dissolution of the polymer in an inorganic solvent. The main advantage of this technique is that it does not require any specialized equipment. However, the interpore openings and pore shape of scaffolds produced by this method is not controllable (Loh and Choong, 2013). 18.4.3.2 Freeze-Drying This happens when synthetic polymers are dissolved in glacial acetic acid or benzene. The resultant solution is then frozen and freeze-dried to yield porous scaffolds. The porosity and pore sizes of the scaffolds fabricated are largely dependent on the parameters such as ratio of water to polymer solution and viscosity of the emulsion. The pore structure of the scaffolds can be controlled by varying the freezing temperature. The advantages of this process are the elimination of several rinsing steps since dispersed water and polymer solvents can be removed directly. Moreover, polymer solutions can be used directly, instead of the need to cross-link any monomers. However, the freeze-drying process should be controlled to reduce heterogeneous freezing to increase scaffold homogeneity (Loh and Choong, 2013).

18.5  NOVEL TECHNIQUES FOR THE SYNTHESIS OF SCAFFOLDS FOR BIOACTIVE GLASS Bioactive glass has become a very attractive prospect to explore in the realm of tissue regeneration. Its inherent ability to achieve a chemical union with the host tissue is a sought-after trait in this still very infantile branch of modern medicine. Upon implantation, there is a gradual dissolution of ions, which subsequently contribute to the growth of a surface hydroxylcarbonate apatite (HCA) layer. The presence of sodium and calcium within the structure of bioglass, appropriately referred to as network modifiers, contributes to an overall low connectivity value of SiO2 network due to formation of silicon-oxygen bonds of the nonbridging variety. This leads to an enhanced dissolution index, which translates to increased probabilities of ionic exchange events at the implant-tissue interface. As Na and Ca cations and/or phosphate ions are initially exchanged with H+ ions in the biologic fluid. A concurrent rise in pH contributes to hydrolysis of SiOSi bonds, which in turn brings about an increased frequency of SiOH bonds. This leads to a deposit of NaCa exhausted surface glass.

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Surface migration of Ca2+ and PO 4 3- induces formation of an ACP layer. The eventual crystallization of this layer into biomimetic HA follows post addition of hydroxide and carbonate ions from native biological fluid (Hench, 1991; Vichery and Nedelec, 2016; Gunawidjaja et al., 2010). Following is an overview of some of the popular and emerging techniques regarding how bioglass scaffolds may be fabricated with defined parameters for designing bespoke tissue emulating 3D constructs.

18.5.1  Melt Quenched Method The melt quenching technique is the most common technique for the production of bioactive glass (Kaur et al., 2014). The procedure includes melting oxides of silica, calcium, phosphate, and sodium (45S5, 46.1% SiO2, 24.4% Na2O, 26.9% CaO, and 2.6% P2O5, in mol%) at >1300 °C in a platinum crucible and quenched in a graphite mold (for rods or monoliths) or in water (frit) (Ignatius et al., 2001). First, the materials are grinded into powdered form by a ball mill. The ground mixture is then melted in a furnace and the melted material is poured into molds to produce rods/cylinders or any other desired shape. Copper plates can also be used to get the frits in order to obtain the quenched of melt in air. To remove the internal stresses from the glasses, it is required to anneal the quenched glass at 500°C (Kaur et al., 2014). The batch of glass is preheated to evaporate the water of hydration or hydroxyl groups. In melt quench method, the silica content must be <60 mol% to obtain a bond with bone (Kaur et al., 2014). The scaffolds fabricated by melt quench technique are dense (Fig. 18.1), representing the schematic steps for the development of bioactive glass particles (Jones, 2015).

18.5.2  Sol-Gel Method The sol-gel method can produce different kinds of useful morphologies with the formation of solid materials through gelation of solutions (Owens et al., 2016a). This soft chemistry strategy provides a very versatile method that can be merged with many other synthesis techniques for fabricating bioactive glass nanoparticles (Vichery and Nedelec, 2016). A very attractive feature of this method is that bioactive glasses of compositions like the original version proposed by Hench can be released on the nanoscale at significantly lower temperatures. The formative processes and the coming together of precursors occurs in many steps such as hydrolysis, condensation, polymerization, gelation, drying, and a dehydration process (Kaur et al., 2014). Glass derived via the sol-gel method boasts nanopores with a large surface area. As a result, degradation and a faster conversion of these glasses into HA is obtained as compared to the scaffold prepared by melt quenched method with the same composition (Kaur et al., 2014). Under an acidic condition, the compositional precursors are first hydrolyzed to a solution. Using an alkaline solution, the substance is then condensed and

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Melt quenched method

Oxides / chemical compounds

Melting

Casting in mold

Fritting into water

Annealing

Rinsing and drying

Monolithic glass

Ground powder

Heat treatment Glass

Densification and crystallization

FIG. 18.1  Schematic representation of melt quench method for the development of glass particles.

participated to gel particles and a colloidal sol is produced (Boccaccini et al., 2016). The nanoparticles assembled together to form a silica-based gel network. After the addition of prehydrolyzed precursor to colloidal sol, the process of gelation continues for few more hours. The excess water is then removed by a drying process. To remove the residues of precursor the gel is heated above 680°C, which decomposes the gel network (Martin et al., 2012). Tetraethyl orthosilicate (TEOS) can be used as silicate-based precursor. Whereas calcium nitrate tetrahydrate and triethylphosphate, are also commonly used precursors for calcium and phosphate respectively (Boccaccini et al., 2016). The sol-gel method can be controlled by altering the initial precursors, time allowed for gelation, catalysts, degree of solvation, gelation conditions, or physical processing of the gel itself (Owens et al., 2016a). The sol-gel technique yields particles that have a massive one up on the conventional melt derived technique in terms of a higher pore volume and specific surface area (Sepulveda et al., 2001). This is directly proportional to the level of bioactivity observed, which in turn will almost certainly impact glass dissolution and apatite formation rates. Some bioactive glasses have been prepared as porous scaffolds by the solgel process such as 58S with the composition (mol%): 60% SiO2, 36% CaO, 4% P2O5. Moreover, there is a high incidence of SiOH bonds in the glass matrix after formation. The reason in large part is the low elaboration temperatures, which hinders oxolation reactions from proceeding. Hence, the resulting

506  PART | III  Bioactive Glasses for Tissue Engineering and Regenerative Medicine Mixing

Molecular precursors Hydrolysis and condensation

Solution (nano-particles in solution)

Gelation

Gel particles colloidal solution is produced Strengthening and shrinking

Ageing

Removal of by-products

Sol-gel glass

Aged gel

Dried gel Stablization

Drying

(Sol-gel method) FIG. 18.2  Schematic representation of the sol-gel method for the development of glass.

reduced connectivity of sol-gel glasses in contrast to glasses prepared via the melt quenched method contribute to higher dissolution rates and concurrently bioactivity. Therefore, this method is credited with providing high purity glasses with more homogeneity but processing should be done at lower temperatures. Although scaffolds derived from sol-gel method may exhibit low strength values, the process is found to be highly versatile in that particle size and morphology can be significantly altered by fine tuning of parameters such as the water/ alcohol ratio, type of alcohol and precursor, and/or the concentration and type of catalyst. Thus it can be stated that the bioactivity of glasses can be controlled by both composition and the process when harnessing sol-gel chemistry (Schubert, 2015). Sol-gel method derived bioactive glass are considered near ideal materials for substituting defects in low-load bearing sites and have enhanced bioactivity, compared to melt derived glasses because of the highly porous nature of this material (Kaur et al., 2014; Ignatius et al., 2001) (Fig. 18.2).

18.5.3  Mesoporous Bioactive Glass Scaffolds The design of mesoporous structures for fabricating bioactive glass scaffolds is an attractive prospect given the innumerable advantages associated with it. These include an ordered mesoporous structure, the ability to customize pore size, pore volume, and surface properties (Zhu et al., 2008). For many tissue engineering applications, scaffolds possessing a macroporous structure with an average pore size of 100 μm or larger are deemed adequate for facilitating nutrient delivery and concomitantly tissue in growth toward the center of the regenerated tissue (Okii et al., 2001). However, this is not to imply that scaffolds with an average pore size at the nanoscale level have had a falling out of late. On the contrary, mesoporous nanoscale scaffold materials having pore size ranging between 2 and 50 nm are center stage when it comes to addressing pitfalls associated with drug delivery and bone regeneration. The logic in the latter case being clear documented advantages in terms of enhanced levels of cell adhesion

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and metabolite adsorption (Owens et al., 2016b). The following are some of the important and popular techniques by which 3D mesoporous glass scaffolds are fabricated.

18.5.3.1  Polyurethane Sponge Template (PUST) Method The development of a 3D mesoporous bioactive glass scaffold (MBAGS) using a polyurethane (PU) sponge has been reported (Zhu et al., 2008). Combined with a P-123 surfactant (the structure direction determinant), PU sponges are used as co-templates prior to an evaporation induced self-assembly (EISA) process. The average pore diameter of this macroporous structure spans across 200–400 μm, with a mesopore wall size of around 5 nm. In vitro analysis of these MBAGS involved assessments with varying chemical compositions in SBF. Cell culture studies using human bone cells revealed better cell attachment levels that were observed with the 80S15C along with formation of a thick layer of HCA post immersion in SBF. PUST method can be very effective for fabricating hierarchical macro and mesoporous bioactive glass scaffolds (MMBAGSs) of the CaOSiO2-P2O5 and CaO-MO-SiO2-P2O5 systems. A modification of this approach infuses the scaffolds with essential trace elements like Mg, Zn, or Sr. After which they are fabricated using combinations of block copolymer and PU sponges as cotemplates. The technique entails immersing the PU sponges in the CaO-MO-SiO2-P2O5 sol. Following uniform coating of the sponges with the sol, drying at room temperature and squeezing out the excess sol, the PU sponge support is decomposed by raising the temperature to around 700°C at a rate of 1°C min−1 leaving behind an intact mesoporous MBAG template. Another variant of this technique involves the use of silk as an additive to the mesoporous bioglass scaffolds (MBGs) by the preparation and addition of silk protein-based solutions (using water as the solvent) of the order 2.5 and 5.0% (w/v) and subsequent immersion of the previously calcined MBGs (Wu et al., 2010; Jiang et al., 2009; Zhao et al., 2009). Silk had a significantly positive effect in terms of defining the pore morphology and imparting greater mechanical strength to the scaffolds. The treatment of MBG scaffolds with silk solution is a logical approach as it offers favorable biocompatibility, a slow rate of degradation, a uniform pore network (without affecting porosity) and imparts mechanical properties conducive for bone tissue engineering. Enhanced cellular activity of the seeded cells was observed on PUST-derived scaffolds in terms of attachment, proliferation, and differentiation for all MBAG templates synthesized using the PUST method. This was expressed in terms of elevated alkaline phosphatase (ALP) levels. In the case of trace element infused scaffolds, increased cellular proliferative activity was also attributed in part to the release of Ca, P, Si, Mg, Zn, and Sr into the cell culture medium (Wang et al., 2011). Moreover, no aberrations were detected with respect to changes in phase composition from the MMBAGS without the trace elements (Wang et al., 2011). Hence, this could prove to be a reliable technique by which nontoxic MMBAGS can be predictably synthesized, provided their trace element

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leaching properties can be quantified and subsequently realized in terms of a clinically viable release profile.

18.5.3.2  One-Pot Synthesis Method for Synthesis of Magnetic and MMBGCs Synthesis of magnetic mesoporous materials is another technique gaining significant traction in terms of multiple biomedical applications. The system basically comprises magnetic particles suspended in a matrix of materials such as polymer (Zhang et al., 2007), lipid (Cinteza et al., 2006), and silica (Kim et al., 2006) for tissue engineering purposes in general and for fabricating bioactive glass composite scaffolds in particular. MMBGCs have been drawing center stage attention of late owing to substantial head ways in the realms of drug delivery (Ruiz-Hernandez et al., 2007), tumor therapy (Veverka et al., 2007), and imaging techniques (Bull et al., 2005). These applications are predicated on the aggregation and subsequent rapid biodegradation of magnetic nanoparticles at the target site. Based on these principles, magnetic and MMBGCs have been successfully synthesized by Li et al. (2008). They made use of a novel one-pot synthesis technique to inculcate all precursors into the composite via simultaneous EISA process. The basic technique for synthesizing MMBGCs using the one-pot synthesis route involves making use of a block copolymer template (EO20PO70EO20) (P123) and the precursors P123, tetra ethyl orthosilicate (TEOS), Ca(NO3)2·4H2O, Fe(NO3)3·9H2O, and tetraethyl phosphate (TEP). A solution of these additives is made with 1.0 g of 0.5 M HCl after dissolution in 60 g of ethanol. The solution is then stirred at room temperature for at least 24 h. The resultant sol is subjected to the EISA process. The dried gel is calcined at 700°C for 3 h in air followed by further calcination at 380°C for 3 h in a hydrogen/argon atmosphere in order to yield the final products. These scaffolds were further assessed for potential applications in drug delivery. For drug loading, ibuprofen solutions were prepared in determined concentrations derived from ibuprofen hexane solutions with 1-g MMBGCs with a concentration of 40 mg mL−1 at room temperature. Separation of the ibuprofen samples from solution by centrifugation preceded vacuum drying at 60°C. The synthesized MMBGC-based drug storage materials were then compacted into disks for a simultaneous assessment of bioactivity (in terms of HCA-forming ability)—visible as phosphate, carbonate, and hydroxyl absorption bands on FTIR spectra (Rehman and Bonfield, 1997), and drug release profile after performing SBF immersion studies. SBF solution preparation with tris-(hydroxymethyl)-aminomethane and HCl was performed. Based on deductions made from EDX, XRD, and TEM results, the synthesized MMBGCs boasted an ordered mesoporous structure with a homogenous distribution of Fe3O4 nanoparticles suspended in an amorphous silicate matrix comprising Ca and P. The primary elemental composition of the MMBGCs was Si, O, Ca, and Fe with the chemical composition of the final product corresponding with the values of the precursors.

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Therefore, this is a very attractive approach for designing MBAGSs boasting high specific surface area and pore volume with elevated levels of HA formation tendencies and simultaneous drug loading. The implications are substantial for developing novel bioactive materials as amenable drug delivery systems in the guise of a synergistic targeted tissue/organ therapy modality such as for cancer treatment of bone marrow cells (Knežević et al., 2013).

18.5.3.3  3D Printing and Synergy With the Spin Coating Technique 3D printing, also referred to as additive manufacturing, solid free—form fabrication and prototyping, is yet another useful and convenient approach for the manufacture of hierarchal scaffolds for the purpose of tissue engineering (An et  al., 2015). The parameters that define an ideal 3D scaffold include a greatly enhanced porosity index with intricate pore networks that play host to a variety of cellular functions and activities such as proliferation, migration, differentiation, and infiltration (Loh and Choong, 2013). Conventional manufacturing techniques such as solvent casting, particulate leaching, and foaming allow for restricted levels of tinkering and tweaking of pore size and network. One of the most sought after characteristics in scaffold design are consistency and ­reproducibility—elusive traits that prove to be a precarious undertaking if one resorts to conventional techniques alone. In order to surpass these glaring roadblocks in the face of inevitable progress in scaffold science, 3D printing has emerged in recent years as a viable and formidable tool for fabricating bespoke scaffolds with controlled pore morphology (Leong et al., 2003). Currently there are >30 different kinds of documented protocols addressing techniques for 3D printing. Out of all these stereolithography, selective laser sintering (SLS), color jet printing, and fused deposition modeling (FDM) techniques are the most frequently utilized, and therefore the principal fabrication method in many scaffold design investigations. This is also in large part due to the ability of these techniques to successfully utilize plastic material. Design of scaffold architecture has a strong bearing on both the final mechanical property and cell behavior near the construct. The addition of a nano-structured mesoporous bioactive glass (MBG) coating on the surface of β-TCP scaffolds for improving upon the surface chemistry and topography of the templates have been investigated (Zhang et al., 2015). A synergistic effect in terms of enhanced mechanical and physicochemical properties comparable to the top range of cancellous bone, having a compressive strength index of up to 12 MPa, was observed. This was coupled with promising levels of angiogenesis, osteogenesis, and protein expression, desirous for upregulating bone formation levels significantly. The 3D printing and spin coating technique synergy system makes use of the 3D plotting principle using β-TCP pastes for designing nanolayer functionalized scaffolds for bone regeneration (Zhang et al., 2015). Pastes are initially prepared by mixing β-TCP powders with 6 wt.% polyvinyl alcohol (PVA) solution in fixed mass ratios. Following loading of the homogenous pastes in the

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printing tubes—β-TCP scaffolds are printed. In this manner, pure β-TCP scaffolds are obtained after thorough drying and subsequent sintering at 1100°C for 3 h. The synthesized β-TCP scaffolds are then coated uniformly with MBG utilizing a spin coating technique. The MBG precursor solution was prepared using a similar protocol as in a study based on copper containing variants (Wu et  al., 2013) and as described above. The resulting MBG precursor solutions are coated onto the struts of the 3D printed β-TCP scaffolds using spin-coating at 500 rpm for initial 10 s followed by 2000 rpm for 20 s. After the elapse of an overnight evaporation period, the MBG dry layers underwent consolidation via the EISA process. The MBG coating was performed a further 9 times. The samples were annealed at 650°C for 5 h after heating at the rate of 1°C min−1. This yielded MBG-coated β-TCP scaffolds. By virtue of combining 3D printed β-TCP templates with nano-mesoporous bioactive glass modification, the resulting hybrid MBG-β-TCP scaffolds were found to accommodate enhanced levels of osteogenic- and angiogenic-related gene expression. Moreover, evident key markers for osteoblastic differentiation and initial mineralization of ECM of rabbit bone marrow stromal cells (rBMSCs) such as elevated levels of ALP activity and angiogenic differentiation in the guise of increased levels of vascular endothelial growth factor (VEGF) are also upregulated. SEM images and EDS analysis post SBF immersion studies showed full surface coverage of MBG-β-TCP scaffolds with apatite crystals in contrast to pure β-TCP scaffolds. These results have proved to be elusive when 3D printing alone is applied as the technique for generating scaffolds due to limitations encountered when preparing functional nanostructures (Samuels and Flowers, 2015; An et al., 2015; Zhang et al., 2015; Lemu, 2012).

18.5.3.4  Cetyltrimethylammonium Bromide (CTAB) Template Method In order to induce bespoke shaping of particles while simultaneously preventing their agglomeration during synthesis of MBG, the use of additives and surfactants has been advocated by some groups. The shaping of particles using the surfactant CTAB as a templating agent has been investigated of late. Fine tuning the CTAB concentrations in solution from 1 to 5 mM for example, can induce a change in particle shape in solution from a spherical to rod-like morphology (Li et al., 2015a). Another study, more importantly, reported a change in the internal particle structure by altering CTAB concentrations. Case in point being a CTAB concentration hovering around 3.5 to 6.0 mM reduced particle size from 294 to 187 nm, and a concentration of 3.5 and 4.5 mM produced hollow particles while dense particles were observed with CTAB concentrations of around 6.0 ± 0.1 mM (Hu et  al., 2014). The use of CTAB along with other additives such as poly(styrene-b-acrylic acid) (Li et al., 2015b) and ethyl acetate (Liang et al., 2015) have yielded average particle sizes in the range of 250 and 180 nm, respectively. Surfactants like CTAB or P123 self-organize into micelles after they have been mixed with the sol in appropriate concentrations.

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However, it is worth noting here that despite similar preparation protocols, concentrations of the precursors, nature and concentrations of the solvents used were variable. CTAB concentration therefore, is not the only parameter influencing particle morphology and structure. Thus, considering this, further systematic enquiries are warranted if one is to fully realize the role and subsequent influence of altering individual parameters on particle morphology. It may be stated here that although all bioactive glasses prepared via the sol-gel route acquire an inherent mesoporosity, the combination of this process with surfactant supramolecular chemistry allows for the realization of an ordered mesoporous structure that will ultimately translate to more predictable and tailored reproductions of bioactive glass templates for bespoke hard tissue defect resolution in vivo according to its site, size, and shape.

18.6  APPLICATION OF BIOACTIVE GLASS IN BIOMEDICAL SCIENCES 18.6.1 Dentistry In the biomaterial science, oral cavity is extremely complex and a challenging environment since it includes both soft and hard tissues in an environment where microorganisms are present. Several studies show that a proper material that can be used in bone replacement applications should have the following properties: ● ● ● ● ●

Easy for handling. It is not a toxic or does not lead to a foreign body reaction. It should avoid chances for transmission of infectious diseases. There should be no donor site morbidity. The material should be economical with no additional cost or prolong the operation time.

Due to the ability of bioactive glass to support bonding and adherence to the biological tissues, simulate the growth of tissues, inhabit the growth of microorganisms, and have the appropriate biomechanical properties such as strength, stuffiness, and hardness, it is considered as a promising material for dental applications (see Fig. 18.3) (Profeta and Prucher, 2015).

18.6.1.1  Treatment of Dental Hypersensitivity The sharp pain of dental hypersensitivity is due to the flow of fluid through the opened dentinal tubules into the pulp of the tooth. Aqueous solutions or pastes that contain fine grained particles of S53P4 bioactive glass was used to treat the dental hypersensitivity. Other studies also examined the effect of 45S5 bioactive glass on the hypersensitivity. It was found that the opened tubules were sealed with an HAP participate. Therefore, the level of hypersensitivity decreased. In addition, the bioactive glass in the form of powder can be used to strengthen the

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Bone regeneration Toothpastes for inhibiting caries and tooth remineralization

Bone grafts

Antibacterial and disinfectant during root canal treatment

Reduces dentine hypersentivity

Implant coating to enhances osseointegration

Bioactive glass in dentistry

Use as periodontal diseases treatment materials

Abrasive materials in dental air abrasion machine

FIG. 18.3  Representation of different uses of bioactive glass in dentistry.

tubules during the treatment of enamel and root carries (Van Meerbeek et al., 2014). The bioactive glass in the toothpastes can be used for other purposes. For instance, the bioactive glass in the toothpastes can be formulated to release therapeutically active ions such as zinc fluoride and potassium. It is also used as an aid in remineralization of apatite in tooth enamel after attack of acidic aqueous solution or bleaching treatment (Van Meerbeek et al., 2014).

18.6.2  Bone Regeneration Bioactive glass has different bone regeneration applications that were studied in vivo: ● ●

●

Graft material to prevent the loss of bone after removal of tooth. Graft material with capability to regenerate tissues in periodontal disease induces defects around roots of healthy teeth and metal implants. Graft material guiding and inducing bone regeneration before denture replacement.

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● ●

Filling materials for voids in implant placement. Components in glass ionomer cement to remineralize damaged and decayed dentine (Van Meerbeek et al., 2014).

The current commercial bioactive glass that is ready to be used in dental applications is based on 45S5 bioglass formulation. Generally, the results of the conducted studies support the use of bioactive glass in bone grafting. Using a bioactive glass as a grafting material succeeded as it guides and enhances the bone growth that is required for stable anchorage of implants in the jaw. The first commercial application of bioactive glass in dentistry was cones of 45S5 bioglass, which was used to prevent resorption of alveolar. Usually, when the tooth is removed, the bone around the cavity starts to resorb because it is not mechanically stressed. 45S5 particles, commercially known as PerioGass is used for bone grafting in anchorage of denture. It is also found that these particles are suitable to be used for healing the bone in periodontitis (Van Meerbeek et al., 2014).

18.6.3  Coating Enhancing Osseointegration One of the first applications of bioactive glass was coating on metals prostheses. It was studied to examine the potential of enhancing the attachment of metals and polymers prostheses to the bone. Several in  vivo studies have shown that bioactive glass 1–98 coating on titanium implants improved the initial attachment of tissues, supported the growth of bones, and enhanced the osseointegration process. In addition, the results of coating bioactive glass on fiber reinforced polymer composites, which was used for dental prosthetic devises, showed that the osseointegration of the fiber reinforced polymer composites implants improved (Van Meerbeek et al., 2014). Coating bioactive glass on metals has some limitations. The bioactive glass may separate from the metal surface as the brittleness of bioactive glass cannot resist the tension or the bending of the metal. Also, the bioactive glass cannot provide a permanent fixation as it dissolves gradually (Van Meerbeek et al., 2014).

18.6.4  Wound Healing and Skin Repair Bioactive glass can accelerate the early stages of wound closure and therefore reduces the potential of having infections. Bioactive glasses have potential uses in accelerating the healing of wounds that are compromised by special conditions such as diabetes, cancers, and others which make the healing process very slow compared to the normal conditions. Micron-sized fibers, which were produced from 45S5 and borate bioactive glass, were studied by Zhao et  al. (2015) to examine the potential of using bioactive glass as gauzes for treating full thickness wound in a rat model. The results showed that borate bioactive glass resulted in faster reduction of the wound size as more blood vessels formed compared to that of 45S5 bioactive glass (Boccaccini et al., 2016).

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The potential of bioactive glass to support hemostasis process and avoid infections is also being examined. A composition of bioactive glass based on 45S5 is used in China as a product to prevent ulceration of the skin and accelerate the tissue repair (Boccaccini et al., 2016).

18.6.5  Nerve Tissue Regeneration Several studies have shown the potential of bioactive glass to treat the damaged nerves due to diseases or trauma. Koudehi et al. (2014) investigated the ability of nanosized bioactive glass and gelatin that were fabricated as composite conduits to guide the growth of neurons in male Wistar rats. The studies were conducted in vitro and in vivo and both results showed that the developed structure was biocompatible and helped in the growth of myelinated axon fibers. The results of in vivo study that was conducted on carbon nanotubes interfaced phosphate glass fibers scaffold showed that the developed structure helped in the regeneration of transected sciatic nerve at the interface between nerve conduit and peripheral neural tissues (Boccaccini et al., 2016). Bioactive glasses have other biomedical applications that were not mentioned in the previous sections including the treatment of frontal sinusitis, repair nasal septal perforations, and reconstruction of facial bone defects (Boccaccini et  al., 2016). The bioactive glass S53P4 in head and neck surgery has shown successful rate in reconstruction of craniofacial defect (Van Meerbeek et al., 2014).

18.7  FUTURE TREND OF BIOACTIVE GLASS SCAFFOLD Bioactive glass enjoys several advantages over other scaffold materials such as enhanced angiogenesis (Kent Leach et al., 2006), superior bone bonding properties, and effective release of ions that stimulate expression of osteogenic genes (Xynos et al., 2001). Although bioactive glasses are ideal material for the preparation of scaffolds, they have certain limitations as well such as their low toughness, particularly in load-bearing bone areas. To overcome this in the future, preparation of bioactive glass scaffolds with an outer coating of a biocompatible polymer should be encouraged, as it will improve its strength by contributing crack bridging system by polymer layer for energy distribution (Fu et al., 2011). One example of biocompatible polymer is gelatin, which has been reported to promote cell migration (Elzoghby, 2013). Synthesizing techniques like freeze casting and solid freeform fabrication (SFF) could also help in the development of future scaffolds with high porosity and increased strength (Liu et al., 2013). Preparation of bioactive glass scaffold usually involves steps like firing and sintering, which can lead to crystallization and low bioactivity (Fagerlund et al., 2012). With the recent introduction of new compositions like borate-based bioactive glass compositions, this problem is tackled as these borate-based glasses contain no or low content of silica, thus they are formed at a temperature which is below their crystallization domain and they also possess better degradation rates (Massera et al., 2015). The incorporation of ZnO in bioactive glass

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c­ omposition not only improves its strength (Shuai et al., 2015), but also encourages bone growth (Grandjeanlaquerriere et al., 2006). Therefore, future research on new bioactive glass compositions is needed as it can overcome problems like low strength, and low bioactivity encountered with the conventional bioactive glass compositions.

ACKNOWLEDGMENT We are thankful to Dr. Sana Zohaib and Dr. Shariq Najeeb for their support and guidance for the compilation of this chapter.

REFERENCES An, J., Teoh, J.E.M., Suntornnond, R., Chua, C.K., 2015. Design and 3D printing of scaffolds and tissues. Engineering 1, 261–268. https://doi.org/10.15302/J-ENG-2015061. Bitar, K.N., Zakhem, E., 2014. Design Strategies of Biodegradable Scaffolds for Tissue Regeneration. Biomed. Eng. Comput. Biol. 13–20. https://doi.org/10.4137/BECB.S10961.RECEIVED. Boccaccini, A.R., Brauer, D.S., Hupa, L. (Eds.), 2016. Bioactive Glasses: Fundamentals, Technology and Applications. Royal Society of Chemistry, Cambridge, UK. Bull, S.R., Guler, M.O., Bras, R.E., Venkatasubramanian, P.N., Stupp, S.I., Meade, T.J., 2005. Magnetic resonance imaging of self-assembled biomaterial scaffolds. Bioconjug. Chem. 16, 1343–1348. https://doi.org/10.1021/bc050153h. Chen, Q., Roether, J., Boccaccini, A., 2008. Tissue engineering scaffolds from bioactive glass and composite materials. In: Topics in Tissue Engineering. Vol. 4, pp. 1–27. https://doi. org/10.1586/17434440.2.3.303. Cinteza, L.O., Ohulchanskyy, T.Y., Sahoo, Y., Bergey, E.J., Pandey, R.K., Prasad, P.N., 2006. Diacyllipid micelle-based nanocarrier for magnetically guided delivery of drugs in photodynamic therapy. Mol. Pharm. 3, 415–423. https://doi.org/10.1021/mp060015p. Danial Khalid, M., Khurshid, Z., Muhammad Sohail Zafar, M., Farooq, I., Sannam Khan, R., Najmi, A., 2017. Bioactive glasses and their applications in dentistry. J. Pak. Dent. Assoc. 26, 32–38. Elzoghby, A.O., 2013. Gelatin-based nanoparticles as drug and gene delivery systems: ­reviewing three decades of research. J. Control. Release 172, 1075–1091. https://doi.org/10.1016/j.­ jconrel.2013.09.019. Fagerlund, S., Massera, J., Hupa, M., Hupa, L., 2012. T–T–T behaviour of bioactive glasses 1–98 and 13–93. J. Eur. Ceram. Soc. 32, 2731–2738. https://doi.org/10.1016/j.jeurceramsoc.2011.10.040. Farooq, I., Imran, Z., Farooq, U., Leghari, A., Ali, H., 2012. Bioactive glass: a material for the future. World J. Dent. 3, 199–201. https://doi.org/10.5005/jp-journals-10015-1156. Fu, Q., Saiz, E., Rahaman, M.N., Tomsia, A.P., 2011. Bioactive glass scaffolds for bone tissue engineering: state of the art and future perspectives. Mater. Sci. Eng. C 31, 1245–1256. https://doi. org/10.1016/j.msec.2011.04.022. Grandjeanlaquerriere, A., Laquerriere, P., Jallot, E., Nedelec, J., Guenounou, M., Laurentmaquin, D., et al., 2006. Influence of the zinc concentration of sol–gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes in  vitro. Biomaterials 27, 3195–3200. https://doi.org/10.1016/j.biomaterials.2006.01.024. Gunawidjaja, P.N., Lo, A.Y.H., Izquierdo-Barba, I., García, A., Arcos, D., Stevensson, B., et  al., 2010. Biomimetic apatite mineralization mechanisms of mesoporous bioactive glasses as

516  PART | III  Bioactive Glasses for Tissue Engineering and Regenerative Medicine probed by multinuclear31P,29Si,23Na and13C solid-state NMR. J. Phys. Chem. C 114, 19345– 19356. https://doi.org/10.1021/jp105408c. Hench, L.L., 1991. Bioceramics: from concept to clinic. J. Am. Ceram. Soc. 74, 1487–1510. https:// doi.org/10.1111/j.1151-2916.1991.tb07132.x. Hench, L.L., 2013. Chronology of bioactive glass development and clinical applications. New J. Glass Ceram. 3, 67–73. https://doi.org/10.4236/njgc.2013.32011. Hu, Q., Li, Y., Zhao, N., Ning, C., Chen, X., 2014. Facile synthesis of hollow mesoporous bioactive glass sub-micron spheres with a tunable cavity size. Mater. Lett. 134, 130–133. https://doi. org/10.1016/j.matlet.2014.07.041. Humagain, M., Nayak, D.G., Uppoor, A.S., Hum, J., Boccaccini, A.R., Huhtinen, R., et al., 2013. Sol-gel based fabrication and characterization of new bioactive glass-ceramic composites for dental applications. J. Mater. Sci. Mater. Med. 24, 669–676. https://doi.org/10.1016/­j.­ actbio.2011.03.016.Bioactive. Ignatius, A.A., Ohnmacht, M., Claes, L.E., Kreidler, J., Palm, F., 2001. A composite polymer/tricalcium phosphate membrane for guided bone regeneration in maxillofacial surgery. J. Biomed. Mater. Res. 564–569. https://doi.org/10.1002/jbm.0000. Jiang, X., Zhao, J., Wang, S., Sun, X., Zhang, X., Chen, J., et al., 2009. Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs. Biomaterials 30, 4522–4532. https://doi.org/10.1016/j.biomaterials.2009.05.021. Jones, J.R., 2015. Reprint of: Review of bioactive glass: from Hench to hybrids. Acta Biomater. 23, S53–82. https://doi.org/10.1016/j.actbio.2015.07.019. Kaur, G., Pandey, O.P., Singh, K., Homa, D., Scott, B., Pickrell, G., 2014. A review of bioactive glasses: their structure, properties, fabrication and apatite formation. J. Biomed. Mater. Res. Part A 102, 254–274. https://doi.org/10.1002/jbm.a.34690. Kent Leach, J., Kaigler, D., Wang, Z., Krebsbach, P.H., Mooney, D.J., 2006. Coating of VEGFreleasing scaffolds with bioactive glass for angiogenesis and bone regeneration. Biomaterials 27, 3249–3255. https://doi.org/10.1016/j.biomaterials.2006.01.033. Kim, J., Lee, J.E., Lee, J., Yu, J.H., Kim, B.C., An, K., et al., 2006. Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals. J. Am. Chem. Soc. 128, 688–689. https://doi.org/10.1021/ ja0565875. Knežević, N.Ž., Ruiz-Hernández, E., Hennink, W.E., Vallet-Regí, M., 2013. Magnetic mesoporous silica-based core/shell nanoparticles for biomedical applications. RSC Adv. 3, 9584. https://doi. org/10.1039/c3ra23127e. Koudehi, M.F., Fooladi, A.A.I., Mansoori, K., Jamalpoor, Z., Amiri, A., Nourani, M.R., 2014. Preparation and evaluation of novel nano-bioglass/gelatin conduit for peripheral nerve regeneration. J. Mater. Sci. Mater. Med. 25, 363–373. Langer, R., Vacanti, J.P., 1993. Tissue engineering. Science 260, 920–926. https://doi.org/­ 10.1007/978-3-642-02824-3. Lemu, H.G., 2012. Study of capabilities and limitations of 3D printing technology. AIP Conf. Proc. 1431, 857–865. https://doi.org/10.1063/1.4707644. Leong, K.F., Cheah, C.M., Chua, C.K., 2003. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials 24, 2363–2378. https://doi. org/10.1016/S0142-9612(03)00030-9. Li, X., Wang, X., Hua, Z., Shi, J., 2008. One-pot synthesis of magnetic and mesoporous bioactive glass composites and their sustained drug release property. Acta Mater. 56, 3260–3265. https:// doi.org/10.1016/j.actamat.2008.03.013.

Novel Techniques of Scaffold Fabrication for Bioactive Glasses  Chapter | 18  517 Li, Y., Chen, X., Ning, C., Yuan, B., Hu, Q., 2015a. Facile synthesis of mesoporous bioactive glasses with controlled shapes. Mater. Lett. 161, 605–608. https://doi.org/10.1016/j.­ matlet.2015.09.057. Li, Y., Bastakoti, B.P., Yamauchi, Y., 2015b. Smart soft-templating synthesis of hollow mesoporous bioactive glass spheres. Chem. Eur. J. 21, 8038–8042. https://doi.org/10.1002/ chem.201406570. Liang, Q., Hu, Q., Miao, G., Yuan, B., Chen, X., 2015. A facile synthesis of novel mesoporous bioactive glass nanoparticles with various morphologies and tunable mesostructure by sacrificial liquid template method. Mater. Lett. 148, 45–49. https://doi.org/10.1016/j.matlet.2015.01.122. Liu, X., Rahaman, M.N., Hilmas, G.E., Bal, B.S., 2013. Mechanical properties of bioactive glass (13-93) scaffolds fabricated by robotic deposition for structural bone repair. Acta Biomater. 9, 7025–7034. https://doi.org/10.1016/j.actbio.2013.02.026. Loh, Q.L., Choong, C., 2013. Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng. Part B Rev. 19, 485–502. https://doi.org/10.1089/ten. TEB.2012.0437. Margha, F.H., Abdelghany, A.M., 2012. Bone bonding ability of some borate bio-glasses and their corresponding glass-ceramic derivatives. Process. Appl. Ceram. 6, 183–192. Martin, R.A., Yue, S., Hanna, J.V., Lee, P.D., Newport, R.J., Smith, M.E., et  al., 2012. Characterizing the hierarchical structures of bioactive sol-gel silicate glass and hybrid scaffolds for bone regeneration. Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 370, 1422–1443. https://doi. org/10.1098/rsta.2011.0308. Massera, J., Shpotyuk, Y., Sabatier, F., Jouan, T., Boussard-Plédel, C., Roiland, C., et  al., 2015. Processing and characterization of novel borophosphate glasses and fibers for medical applications. J. Non-Cryst. Solids 425, 52–60. https://doi.org/10.1016/j.jnoncrysol.2015.05.028. Mishra, A., Rocherullé, J., Massera, J., 2016. Ag-doped phosphate bioactive glasses: Thermal, structural and in-vitro dissolution properties. Biomed. Glass 2, 38–48. https://doi.org/10.1515/ bglass-2016-0005. Neel, E.A.A., Pickup, D.M., Valappil, S.P., Newport, R.J., Knowles, J.C., 2009. Bioactive functional materials: a perspective on phosphate-based glasses. J. Mater. Chem. 19 (6), 690–701. Ojha, N., 2016. Borosilicate Glass With Enhanced Hot Forming. Okii, N., Nishimura, S., Kurisu, K., Takeshima, Y., Uozumi, T., 2001. In Vivo Histological Changes Occurring in Hydroxyapatite Cranial Reconstruction. Case Report. vol. 41. Owens, G.J., Singh, R.K., Foroutan, F., Alqaysi, M., Han, C., Mahapatra, C., et al., 2016a. Sol-gel based materials for biomedical applications. Prog. Mater. Sci. 77, 1–79. Owens, G.J., Singh, R.K., Foroutan, F., Alqaysi, M., Han, C.M., Mahapatra, C., et  al., 2016b. Sol-gel based materials for biomedical applications. Prog. Mater. Sci. 77, 1–79. https://doi. org/10.1016/j.pmatsci.2015.12.001. Profeta, A.C., Prucher, G.M., 2015. Bioactive-glass in periodontal surgery and implant dentistry. Dent. Mater. J. 34, 559–571. https://doi.org/10.4012/dmj.2014-233. Rehman, I., Bonfield, W., 1997. Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy. J. Mater. Sci. Mater. Med. 8, 1–4. https://doi.org/10.1023/A: 1018570213546. Ruiz-Hernandez, E., Lopez-Noriega, A., Arcos, D., Izquierdo-Barba, I., Terasaki, O., Vallet-Regi, M., 2007. Aerosol-assisted synthesis of magnetic mesoporous silica spheres for drug targeting. Chem. Mater. 19, 3455–3463. https://doi.org/10.1021/cm0705789. Sachlos, E., Czernuszka, J.T., 2003. Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 5, 29–39. 39–40.

518  PART | III  Bioactive Glasses for Tissue Engineering and Regenerative Medicine Samuels, K., Flowers, J., 2015. 3D printing: exploring capabilities. Technol. Eng. Teach. 74, 17–21. Schubert, U., 2015. Chemistry and fundamentals of the sol-gel process. In: Sol-Gel Handbook. vol. 1–3, pp. 1–28. https://doi.org/10.1002/9783527670819.ch01. Sepulveda, P., Jones, J.R., Hench, L.L., 2001. Characterization of melt-derived 45S5 and sol-gelderived 58S bioactive glasses. J. Biomed. Mater. Res. 58, 734–740. https://doi.org/10.1002/ jbm.10026. Sheikh, Z., Najeeb, S., Khurshid, Z., Verma, V., Rashid, H., Glogauer, M., 2015. Biodegradable materials for bone repair and tissue engineering applications. Materials (Basel) 8, 5744–5794. https://doi.org/10.3390/ma8095273. Shuai, C., Deng, J., Gao, C., Feng, P., Peng, S., Xiao, T., et al., 2015. Mechanisms of tetraneedlelike ZnO whiskers reinforced forsterite/bioglass scaffolds. J. Alloys Compd. 636, 341–347. https:// doi.org/10.1016/j.jallcom.2015.02.077. Stanić, V., 2017. Variation in properties of bioactive glasses after surface modification. In: Clinical Applications of Biomaterials. Springer International Publishing, Cham, pp. 35–63. https://doi. org/10.1007/978-3-319-56059-5_2. Van Meerbeek, B., Van Landuyt, K., Mine, A., Yoshihara, K., Poitevin, A., De Munck, J., Peumans, M., 2014. Bonding in dentistry. In: Handbook of Oral Biomaterials. first ed. Pan Stanford ­Publishing, Singapore, pp. 1–56. Ventola, C.L., 2014. Medical applications for 3D printing: current and projected uses. P T 39, 704–711. Veverka, M., Veverka, P., Kaman, O., Lančok, A., Závěta, K., Pollert, E., et al., 2007. Magnetic heating by cobalt ferrite nanoparticles. Nanotechnology 18, 345704. https://doi.org/10.1088/09574484/18/34/345704. Vichery, C., Nedelec, J.-M., 2016. Bioactive glass nanoparticles: From synthesis to materials design for biomedical applications. Materials (Basel) 9, 288. https://doi.org/10.3390/ma9040288. Wang, X., Li, X., Ito, A., Sogo, Y., 2011. Synthesis and characterization of hierarchically macroporous and mesoporous CaO-MO-SiO2-P2O5 (M = mg, Zn, Sr) bioactive glass scaffolds. Acta Biomater. 7, 3638–3644. https://doi.org/10.1016/j.actbio.2011.06.029. Wu, C., Zhang, Y., Zhu, Y., Friis, T., Xiao, Y., 2010. Structure-property relationships of silk-­modified mesoporous bioglass scaffolds. Biomaterials 31, 3429–3438. https://doi.org/10.1016/j.biomaterials.2010.01.061. Wu, C., Zhou, Y., Xu, M., Han, P., Chen, L., Chang, J., et al., 2013. Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity. Biomaterials 34, 422–433. https://doi.org/10.1016/j.biomaterials.2012.09.066. Xynos, I.D., Edgar, A.J., Buttery, L.D.K., Hench, L.L., Polak, J.M., 2001. Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass® 45S5 dissolution. J. Biomed. Mater. Res. 55, 151–157. https://doi.org/10.1002/10974636(200105)55:2<151::AID-JBM1001>3.0.CO;2-D. Zafar, M.S., Khurshid, Z., Almas, K., 2015. Oral tissue engineering progress and challenges. J. Tissue Eng. Regen Med. 12, 387–397. https://doi.org/10.1007/s13770-015-0030-6. Zafar, M., Najeeb, S., Khurshid, Z., Vazirzadeh, M., Zohaib, S., Najeeb, B., et al., 2016. Potential of electrospun nanofibers for biomedical and dental applications. Materials (Basel) 9, 73. https:// doi.org/10.3390/ma9020073. Zeimaran, E., Pourshahrestani, S., Djordjevic, I., Pingguan-Murphy, B., Kadri, N.A., Towler, M.R., 2015. Bioactive glass reinforced elastomer composites for skeletal regeneration: A review. Mater. Sci. Eng. C 53, 175–188. https://doi.org/10.1016/j.msec.2015.04.035.

Novel Techniques of Scaffold Fabrication for Bioactive Glasses  Chapter | 18  519 Zhang, J.L., Srivastava, R.S., Misra, R.D.K., 2007. Core-shell magnetite nanoparticles surface encapsulated with smart stimuli-responsive polymer: Synthesis, characterization, and LCST of viable drug-targeting delivery system. Langmuir 23, 6342–6351. https://doi.org/10.1021/ la0636199. Zhang, Y., Xia, L., Zhai, D., Shi, M., Luo, Y., Feng, C., et al., 2015. Mesoporous bioactive glass nanolayer-functionalized 3D-printed scaffolds for accelerating osteogenesis and angiogenesis. Nanoscale 7, 19207–19221. https://doi.org/10.1039/C5NR05421D. Zhao, J., Zhang, Z., Wang, S., Sun, X., Zhang, X., Chen, J., et  al., 2009. Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines. Bone 45, 517–527. https://doi. org/10.1016/j.bone.2009.05.026. Zhao, S., Li, L., Wang, H., Zhang, Y., Cheng, X., Zhou, N., et al., 2015. Wound dressings composed of copper-doped borate bioactive glass microfibers stimulate angiogenesis and heal full-­ thickness skin defects in a rodent model. Biomaterials 53, 379–391. Zhu, Y., Wu, C., Ramaswamy, Y., Kockrick, E., Simon, P., Kaskel, S., et al., 2008. Preparation, characterization and in vitro bioactivity of mesoporous bioactive glasses (MBGs) scaffolds for bone tissue engineering. Microporous Mesoporous Mater. 112, 494–503. https://doi.org/10.1016/j. micromeso.2007.10.029. Zippel, N., Schulze, M., Tobiasch, E., 2010. Biomaterials and Mesenchymal Stem Cells for Regenerative Medicine. Recent Pat. Biotechnol. 1–22. Zohora, F.T., Yousuf, A., Azim, M.A., 2014. Biomaterials as porous scaffolds for tissue engineering applications: a review. Eur. Sci. J. 10, 1857–7881. https://doi.org/10.1089/ten.1995.1.41.