Review Bioactive glass in tissue engineering Mohamed N. Rahaman a,���, Delbert E. Day a, B. Sonny Bal b, Qiang Fu c, Steven B. Jung a,d, Lynda F. Bonewald e, Antoni P. Tomsia c a Department of Materials Science and Engineering, and Center for Bone and Tissue Repair and Regeneration, Missouri University of Science and Technology, Rolla, MO 65409, USA b Department of Orthopaedic Surgery, School of Medicine, University of Missouri, Columbia, MO 65211, USA c Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA d Mo-Sci Corporation, Rolla, MO 65409, USA e Department of Oral Biology, School of Dentistry, University of Missouri���Kansas City, Kansas City, MO 64108, USA a r t i c l e i n f o Article history: Received 20 January 2011 Received in revised form 10 March 2011 Accepted 16 March 2011 Available online 21 March 2011 Keywords: Bioactive glass Tissue engineering Bone repair Angiogenesis Soft tissue repair a b s t r a c t This review focuses on recent advances in the development and use of bioactive glass for tissue engineer- ing applications. Despite its inherent brittleness, bioactive glass has several appealing characteristics as a scaffold material for bone tissue engineering. New bioactive glasses based on borate and borosilicate compositions have shown the ability to enhance new bone formation when compared to silicate bioac- tive glass. Borate-based bioactive glasses also have controllable degradation rates, so the degradation of the bioactive glass implant can be more closely matched to the rate of new bone formation. Bioactive glasses can be doped with trace quantities of elements such as Cu, Zn and Sr, which are known to be ben- eficial for healthy bone growth. In addition to the new bioactive glasses, recent advances in biomaterials processing have resulted in the creation of scaffold architectures with a range of mechanical properties suitable for the substitution of loaded as well as non-loaded bone. While bioactive glass has been exten- sively investigated for bone repair, there has been relatively little research on the application of bioactive glass to the repair of soft tissues. However, recent work has shown the ability of bioactive glass to pro- mote angiogenesis, which is critical to numerous applications in tissue regeneration, such as neovascu- larization for bone regeneration and the healing of soft tissue wounds. Bioactive glass has also been shown to enhance neocartilage formation during in vitro culture of chondrocyte-seeded hydrogels, and to serve as a subchondral substrate for tissue-engineered osteochondral constructs. Methods used to manipulate the structure and performance of bioactive glass in these tissue engineering applications are analyzed. �� 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Tissue engineering has emerged as a promising approach for the repair and regeneration of tissues and organs lost or damaged as a result of trauma, injury, disease or aging [1,2]. It has the potential to overcome the problem of a shortage of living tissues and organs available for transplantation. In the most common approach, a bio- material scaffold with a well-defined architecture serves as a tem- porary structure for cells and guide their proliferation and differentiation into the desired tissue or organ. Growth factors and other biomolecules can be incorporated into the scaffold, along with the cells, to guide the regulation of cellular functions during tissue or organ regeneration [3���6]. The overall purpose of this scaf- fold-based tissue engineering approach is to provide the temporary support structure for tissue forming cells to synthesize new tissue of the desired shape and dimensions. The last two decades have seen a dramatic growth in the field of tissue engineering. These efforts have resulted in cell-based regen- eration of individual tissues such as skin [7���10], bone [11���13] and cartilage [14,15]. Recent work in cell-based restoration of multiple tissue phenotypes by composite tissue grafts, such as osteochon- dral and fibro-osseous grafts, have shown promising results for the tissue-engineered regeneration of complex anatomical struc- tures such as the synovial joint condyle, bone���tendon complex, bone���ligament junction and the periodontium [16]. These advances would not have been possible without the inno- vative design and fabrication of biomaterials and scaffolds. Bioma- terials used for creating scaffolds are designed to meet a set of stringent requirements that are either essential or desirable for optimized tissue formation [17]. Scaffolds, as mentioned earlier, must provide a temporary structure for cells to synthesize new tissue but must undergo degradation upon neogenesis of that 1742-7061/$ - see front matter �� 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.03.016 ��� Corresponding author. Tel.: +1 573 341 4406. E-mail address: rahaman@mst.edu (M.N. Rahaman). Acta Biomaterialia 7 (2011) 2355���2373 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

tissue. The architecture of the scaffold is critical for providing cells with an optimized microenvironment to synthesize new tissue and to allow flow or diffusion of nutrients between the cells and the surrounding environment. Recent advances in innovative materials processing such as electrospinning, solid freeform fabrication (ra- pid prototyping) and unidirectional freezing of suspensions offer considerable promise for tissue regeneration using cell-based ther- apies [18���22]. The purpose of this article is to evaluate the role and impact of one particular subset of biomaterials in tissue engineering applica- tions, namely: bioactive glass for hard and soft tissue regeneration. The focus is on recent developments of new bioactive glasses and their formation into scaffolds with the requisite anatomical shape and architecture. Methods that can be used to manipulate the materials structure and the variables that affect the materials per- formance in these tissue engineering applications are analyzed. 2. Scaffolds for tissue engineering The ideal scaffold should (i) be biocompatible (not toxic) and should promote cell adhesion and proliferation (ii) exhibit, after in vitro tissue culture, mechanical properties that are comparable to those of the tissue to be replaced (iii) have a porous three- dimensional (3-D) architecture to allow cell proliferation, vascular- ization and diffusion of nutrients between the cells seeded within the matrix and the surroundings (iv) degrade at a rate that matches the production of new tissue, into nontoxic products that can be easily resorbed or excreted by the body and (v) be capable of being processed economically into anatomically relevant shapes and dimensions, and be sterilized for clinical use. Scaffolds for tissue engineering are commonly constructed from biodegradable polymeric materials, synthetic or natural [23���28]. However, for the regeneration of load-bearing bones, the use of biodegradable polymer scaffolds is challenging because of their low mechanical strength. Attempts have been made to reinforce the biodegradable polymers with a biocompatible inorganic phase, commonly hydroxyapatite (HA) [29���32]. Although brittle, scaffolds fabricated from inorganic materials such as calcium phosphate- based bioceramics and bioactive glass can provide higher mechan- ical strength than polymeric scaffolds. Biodegradable metals are currently under investigation [33], but their corrosion behavior in vivo remains a key concern. Calcium phosphate-based bioceramics, such as HA, Ca10 (PO4)6(OH)2, b-tricalcium phosphate (b-TCP), Ca3(PO4)2, and bipha- sic calcium phosphate (BCP), a mixture of HA and b-TCP, composed of the same ions as bone, are the inorganic materials which have received most attention for bone repair applications [34���37]. When compared to b-TCP, HA resorbs slowly and undergoes little conversion to a bone-like material after implantation [38,39]. However, for the same porosity, b-TCP scaffolds often have lower strength than HA scaffolds, so their use in the repair of load- bearing bones may be challenging. The use of BCP with different HA to b-TCP ratios allows manipulation of the degradation rate [40], as well as other properties [41]. Bioactive glass and glass���ceramics are also used in bone repair applications and are being developed for tissue engineering appli- cations [42���45]. Bioactive glass has an amorphous structure, whereas glass���ceramics are crystallized glasses, consisting of a composite of a crystalline phase and a residual glassy phase. There has been heightened interest in the science and biomedical appli- cation of bioactive glass over the last two decades, as evidenced by the growing number of publications in the field (Fig. 1). 3. Bioactive glass In a general sense, a bioactive material has been defined as a material that has been designed to induce specific biological activ- ity [46]. In a more narrow sense, a bioactive material has been de- fined as a material that undergoes specific surface reactions, when implanted into the body, leading to the formation of an HA-like layer that is responsible for the formation of a firm bond with hard and soft tissues [47]. The ability of a material to form an HA-like surface layer when immersed in a simulated body fluid (SBF) in vitro is often taken as an indication of its bioactivity [48]. Fur- thermore, it has been suggested that this in vitro bioactivity is an indication of the bioactive potential of a material in vivo [49]. However, this narrow definition of bioactivity has been called into question recently [50]. Dicalcium phosphate dihydrate, for exam- ple, shows the formation of an HA-like surface layer when im- mersed in an SBF in vitro, but no direct bone bonding in vivo [51���53]. Furthermore, b-TCP does not always lead to the formation of an HA-like material in an SBF despite its extensive bonding to bone [54]. 3.1. Silicate bioactive glass Since the report of its bone-bonding properties nearly 40 years ago [55], the bioactive glass designated 45S5, sometimes referred to by its commercial name Bioglass��, has been the most widely re- searched glass for biomedical applications [44]. This glass is a sili- cate glass based on the 3-D glass-forming SiO2 network in which Si is fourfold coordinated to O. The key compositional features that are responsible for the bioactivity of 45S5 glass are its low SiO2 content (when compared to more chemically durable silicate glasses), high Na2O and CaO (glass network modifiers) content, and high CaO/P2O5 ratio (Table 1). The mechanisms of bioactivity and bone bonding of 45S5 glass have been widely studied, and described in detail elsewhere [44,56,57]. Based on those studies, the bonding of 45S5 glass to bone has been attributed to the formation of a carbonate- substituted hydroxyapatite-like (HCA) layer on the glass surface in contact with the body fluid. Because this HCA layer is similar to the mineral constituent of bone, it bonds firmly with living bone and tissue. While some details of the chemical and structural changes are not clear, the HCA layer is generally believed to form as a result of a sequence of reactions on the surface of the bioactive glass implant, as described by Hench [44]: Fig. 1. Number of papers published per year in the field of ������bioactive glass������ (compiled from a literature search in Web of Science carried out in December 2010). 2356 M.N. Rahaman et al. / Acta Biomaterialia 7 (2011) 2355���2373

Stage 1: Rapid ion exchange reactions between the glass net- work modifiers (Na+ and Ca2+) with H+ (or H3O+) ions from the solution, leads to hydrolysis of the silica groups and the creation of silanol (Si���OH) groups on the glass surface: e.g. SiAOANa�� �� H�� ! SiAOH�� �� Na����aq�� ��1�� The pH of the solution increases due to the consumption of H+ ions. Stage 2: The increase in pH (or OH concentration) leads to at- tack of the SiO2 glass network, and the dissolution of silica, in the form of silicic acid, Si(OH)4, into the solution, and the contin- ued formation of Si���OH groups on the glass surface: SiAOASi �� H2O ! SiAOH �� OHASi ��2�� While the solubility of silica is low, the products of 45S5 glass and glass���ceramic dissolution in aqueous solutions have shown an increase in Si concentration [58], indicating that dissolution of silica is an important mechanism. However, other mechanisms could also contribute to the increase in Si concentration. Stage 3: Condensation and polymerization of an amorphous SiO2-rich layer (typically 1���2 lm thick) on the surface of the glass depleted in Na+ and Ca2+. Stage 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. Stage 5: The glass continues to dissolve, as the ACP layer incor- porates (OH) and (CO3)2 from the solution and crystallizes as an HCA layer. With the initial formation of an HCA layer, the biological mech- anisms of bonding to bone are believed to involve adsorption of growth factors, followed by attachment, proliferation and differen- tiation of osteoprogenitor cells [59]. Osteoblasts (bone-forming cells) create extracellular matrix (collagen), which mineralizes to form a nanocrystalline mineral and collagen on the surface of the glass implant while the degradation and conversion of the glass continues over time [60]. The biocompatibility of 45S5 glass has long been established [61]. As described above, upon implantation, 45S5 bioactive glass undergoes chemical degradation, releasing ions such as Na+ and Ca2+, and conversion to an HCA material. Silicon, presumably in the form of silicic acid, Si(OH)4, is also released during the degrada- tion by dissolution or by other mechanisms, such as small pieces of silica-rich material eaten by phagocytes and excreted out. The re- lease of Si from 45S5 granules implanted in the muscle and tibiae of rabbits has been studied to determine the pathway of silicon re- leased during the degradation of the glass in vivo [62]. By measur- ing the silicon released in urine and blood samples for up to 7 months post-implantation, and using chemical and histopatholo- gical analyses of bone tissue and several organs, it was found the silicon resulting from the 45S5 degradation was harmlessly ex- creted in soluble form through the urine. 45S5 glass remains the gold standard for bioactive glass but, as a scaffold material, it has several limitations. One limitation is the difficulty of processing 45S5 glass into porous 3-D scaffolds. Porous bioactive glass scaffolds are commonly prepared by heat- ing (sintering) glass particles, already formed into the desired 3-D geometry, to bond the particles into a strong glass phase containing an interpenetrating network of pores. Because of the limited ability of 45S5 glass to sinter by viscous flow above its glass transition temperature (Tg), and the narrow window between Tg and the onset of crystallization, severe difficulties are encountered in sintering the particles into a dense network. Consequently the scaffold often has low strength [63]. Commonly, the glass devitrifies during sintering to form a predominantly combeite crystalline phase (Na2O���2CaO���3SiO2). While devitrification does not inhibit the ability of 45S5 glass to form an HA-like surface layer, it has the effect of reducing the rate of conversion to HA [64]. Another limitation of 45S5 glass is its slow degradation rate and conversion to an HA-like material [56,57], which makes it difficult to match the degrada- tion rate of the scaffold with the rate of new tissue formation. The conversion of the scaffold to an HA-like material is often incomplete, so therefore a portion of unconverted glass contain- ing SiO2 could remain in the scaffold, raising uncertainty about the long-term effects of SiO2 in vivo. A complication with the use of 45S5 glass and other bioactive glasses and other biodegradable materials is that the local biolog- ical microenvironment can be influenced significantly by their deg- radation. Increases in the concentration of ions, such as Na+ and Ca2+, and changes in the pH occur as a result of the degradation, particularly in the early stages when the degradation rate is fast [56,57,65,66]. The biological effects of these changes are difficult to predict from in vitro experiments. Furthermore, the biological roles of these soluble species, their toxicity and their removal are often not clearly understood. A silicate bioactive glass designated 13-93 [67,68] is based on the 45S5 composition, but it has a comparatively higher SiO2 con- tent and additional network modifiers, such as K2O and MgO, when compared to 45S5 (Table 1). Products of 13-93 glass have been ap- proved for in vivo use in Europe. Because 13-93 has better process- ing characteristics by viscous flow sintering (larger window between Tg and the onset of crystallization), the glass phase in por- ous 3-D scaffolds can be sintered to high density without crystalli- zation. In vitro cell culture showed no marked difference in the proliferation and differentiated function of osteoblastic MC3T3- E1 or MLO-A5 cells between dense disks of 45S5 and 13-93 glass [69]. However, 13-93 glass degrades (and converts to an HA-like material) more slowly than 45S5 glass. 3.2. Borate bioactive glass More recent work has shown that certain compositions in other glass-forming systems, such as borate glass [70���74], are also bioactive (Table 1). Because of their lower chemical durabil- ity, some borate bioactive glasses degrade faster and convert more completely to an HA-like material, when compared to sil- icate 45S5 or 13-93 glass [56,57,65,66]. The conversion of borate Table 1 Compositions of various bioactive glasses. Composition (wt.%) 45S5 13-93 6P53B 58S 70S30C 13-93B1 13-93B3 P50C35N15 Na2O 24.5 6.0 10.3 0 0 5.8 5.5 9.3 K2O 0 12.0 2.8 0 0 11.7 11.1 0 MgO 0 5.0 10.2 0 0 4.9 4.6 0 CaO 24.5 20.0 18.0 32.6 28.6 19.5 18.5 19.7 SiO2 45.0 53 52.7 58.2 71.4 34.4 0 0 P2O5 6.0 4.0 6.0 9.2 0 3.8 3.7 71.0 B2O3 0 0 0 0 0 19.9 56.6 0 M.N. Rahaman et al./ Acta Biomaterialia 7 (2011) 2355���2373 2357