Bioactive Glass-Biopolymer Composites … · Bioactive Glass-Biopolymer Composites Yaping Dinga, Marina T. Souzab, Wei Lic, Dirk W. Schuberta, Aldo R. Boccaccinic and Judith A. Roethera*

Bioactive Glass-Biopolymer Composites

Yaping Dinga, Marina T. Souzab, Wei Lic, Dirk W. Schuberta, Aldo R. Boccaccinic and Judith A. Roethera*aInstitute of Polymer Materials, University of Erlangen-Nuremberg, Erlangen, GermanybDepartment of Materials Engineering, Federal University of São Carlos, São Carlos, SP, BrazilcInstitute of Biomaterials, University of Erlangen-Nuremberg, Erlangen, Germany

Abstract

Tissue engineering (TE) is a biomedical field in continuous expansion. However, there are still manychallenges, and the further development of TE approaches requires interdisciplinary interaction andcollaboration among various research areas with a notable contribution expected from biomaterialsscience. In the last couple of decades, significant advances in the development of biomaterial-basedscaffolds for hard and soft tissue regeneration have been accomplished, including the manufacture ofbiocomposites that combine natural or synthetic polymers with bioactive glasses or glass-ceramics. Thesenovel biomaterials present the possibility of tailoring a variety of parameters and properties such asdegradation kinetics, mechanical properties, and chemical composition according to the aimed applica-tion. This chapter presents a concise update of the field of biopolymer–bioactive glass composite scaffolddevelopment for TE covering several popular processing techniques for biocomposite fabrication,namely, microsphere processing, solvent casting-particulate leaching method, electrospinning, freeze-drying, and rapid prototyping techniques, which lead to scaffolds exhibiting a variety of 3Dmorphologiesand different pore structures.

Keywords

Composite materials; Bioactive glass; Biodegradable polymers; Scaffolds; Bone tissue engineering; Softtissue engineering; Porosity; Fabrication techniques; Degradation; Cell biocompatibility

Introduction

In the last decades enormous advances in the biomaterials field have been made especially considering thedevelopment of bioactive materials for tissue engineering (TE) applications. These materials should beable to restore the function of a body part that has been compromised in structure and function, as a resultof disease or trauma, but also they must be capable of actively inducing the regeneration of new tissues.Such bioactive materials in the form of “scaffolds” are essential for the advancement of the TE andregenerative medicine fields being central actors of approaches being put forward for the regeneration ofpractically every part of the human anatomy ranging from hard tissues such as the bone to soft tissues suchas the skin, heart, and blood vessels [1–8]. In this interdisciplinary approach, cells and three-dimensionalbioactive and bioresorbable structures, the “scaffolds,” are combined aiming to mimic the natural bodystructures and to accelerate tissue regeneration.

A large number of different materials are being investigated in this context. For many tissues, inparticular for the bone, the combination of a bioresorbable polymer and a bioactive glass or glass-ceramic

*Email: [email protected].

Handbook of Bioceramics and BiocompositesDOI 10.1007/978-3-319-09230-0_17-1# Springer International Publishing Switzerland 2015

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in a composite material is a promising approach as the unique properties of each material phase can becombined in one structure, while at the same time, the disadvantageous properties of the individual phasescan be minimized [1].

Specific properties required for a successful scaffold material have been reported previously in theliterature [9, 10]. These include properties such as biocompatibility, adequate pore sizes, a high degree ofinterconnected porosity of at least 60 %, mechanical properties closely matched to those of the tissue thatneeds to be regenerated, and adequately tailored biodegradability [11, 12]. Another relevant property forthe regeneration of hard and soft tissues is bioactivity, that is, the biomaterials’ ability to interact with orbind to living tissues [13].

The combination of biopolymers and inorganic fillers to develop tissue engineering scaffolds has beeninvestigated during the last 10 years with major efforts devoted to increase the bioactivity of otherwisebioinert polymers, including synthetic and natural polymers [1, 9, 10, 14]. A frequently consideredinorganic filler or additive in biopolymer composites is bioactive glass in its many chemical compositionsand morphologies,such as particles, granules or fibers [1]. The field of bioactive glasses started in the1970s with the development of Na2O–CaO–SiO2–P2O5 glasses [13]. In this system, one of the mostbioactive glasses, currently known as 45S5 Bioglass®, was developed with the specific composition of46.1 SiO2, 24.4 Na2O, 26.9 CaO, and 2.6 P2O5 (mol.%) [13, 15]. 45S5 Bioglass® can induce a beneficialbiological response to human host tissue, which is revealed by the formation of a surface hydroxyapatite(HA)-like layer when implanted, similar in composition to the mineral phase of the human bone. ThisHA-like layer is able to bond firmly to hard and soft tissues [9, 16]. Bioactive glasses have been applied inclinical treatments for many years in different product forms, such as granules and particulates, dense orporous implants. For example, bioactive glass granules or particulates have been widely used to fill spaceswhere bone grafting is needed and to enhance the natural repair process. These applications includegeneral orthopedic, craniofacial, and maxillofacial prosthesis, chronic osteomyelitis treatment, soft tissueregeneration, and wound healing [8, 9, 13, 16].

In addition to the above mentioned oxides, varying amounts of other oxides can also be incorporatedinto glass compositions to impart specific properties; for example, SrO could improve bone healing [17]and Ag2O leads to antibacterial features [10]. Furthermore, some trace elements when incorporated intobioactive glass compositions are shown to provide additional properties that can be beneficial for tissueregeneration. For instance, zinc is responsible for vitamin A and E metabolism; magnesium can activatethe phosphate-transferring enzyme; copper can help the formation of myelin sheaths for neurologicalsystem, and B can enhance angiogenesis [18]. Therefore, numerous ion-doped bioactive glasses are beingdeveloped, which exhibit high biological activity and tissue regeneration capability [10, 33]. Table 1summarizes relevant previous studies on metallic ion-doped bioactive glasses showing the different cellbiology effects they provide. It is important to observe the fact that the excessive release of these elementsin body fluids could harm healthy tissues; thus, the release of ions from bioactive glasses should be keptbelow the toxic level which may vary for different applications and conditions.

The traditional and first generation of bioactive glasses was mainly manufactured by melt-quenchingmethod at temperatures above 1300 �C, from which the silica content was limited within 60 % foreffective bonding with the host tissue [37]. Melt-quenched glasses usually stay in dense state and a stableoxide network occurs; hence, the bioactivity is strongly dependent on the silica content. The secondgeneration of bioactive glasses was invented in the early 1990s through the sol–gel route, in which a gelstructure is formed and self-assembled through hydrolysis and polycondensation of compositional pre-cursors at room temperature followed by a drying or low-temperature sintering process [38]. The reactedsilica network is abundant in Si–OH groups, therefore enabling much higher amount of silica (>90 %) foreffective HCA formation [37]. In addition, incorporation of sodium is not necessary in sol–gel approachessince it basically reduces the melting point and enhances processability in melt-quenched glasses. Many


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ternary or binary sol–gel-derived bioactive glasses without sodium have been reported for excellentHCA-forming ability when immersed in simulated body fluid (SBF), such as 58S, 77S, or 70S30C[9]. Mesoporous bioactive glasses can be regarded the third generation of bioactive glasses. They aredeveloped based on the sol–gel method but with involvement of a supermolecular surfactant as templateof ordered mesoporosity, typically in the 2–50 nm size range. Typical surfactants are triblock copolymers,such as P123 and F127 [38]. The mesopore size can be adjusted by altering the surfactant type andconcentration, solvents, pH, and temperature. In comparison to dense bioactive glasses and sol–gel-derived glasses, highly ordered pore channels in mesoporous glasses enormously increase their surfacevolume ratio and porosity, which makes mesoporous bioactive glasses ideal carriers in sustained drugdelivery combined with bioactivity [36]. Nevertheless, the low processing temperatures, wet-chemicalreactions, and high porosity of sol–gel-induced bioactive glasses lead to a significant reduction of theirmechanical properties. Thus, only small dimensional products can be produced, for example,nanoparticles, microspheres, powders, thin films and coatings, or monoliths of size below 1 cm [37]. Inaddition, although bioactive glasses show impressive bioactivity for bone repair and tissue regeneration,their brittleness limits their clinical applications, especially when there is a significant stress and/orcycling load-bearing demand, in cases of some bone and joint grafts, or when flexible structures forsoft tissue regeneration are required. Thus, the development of composites, with the incorporation ofbioactive glass in biopolymer matrices, represents an interesting approach, not only to develop bioactivescaffolds with suitable mechanical properties but also to create a better environment for cell attachmentand growth for TE applications.

Early reports on the combination of bioactive glasses and biodegradable polymers to produce tissueengineering scaffolds were published at the end of the twentieth century, as reviewed elsewhere [1], and

Table 1 Summary of some of the most investigated ion-doped bioactive glasses

Elements Expected effect Ref.

Zinc Antibacterial effectAids in protein and DNA synthesis and cell proliferation

[19–21]

Magnesium Directly stimulates osteoblast proliferation [19, 20,22]

Silver Antimicrobial effect [23, 24]

Iron Drug delivery and hyperthermia treatment of cancer [25, 26]

Fluoride Positively influences bone densityForms fluorapatite (FAp), which is the main component of enamel and dentine and has higheracid resistance than HCA

[27, 28]

Copper Enhances vascularization, osteostimulation, and bone-related gene expressionInduces differentiation of stem cells and osteoblastic cellsAntibacterial property

[29, 30]

Strontium Accelerates bone healing processIncreases bone mass and mechanical strengthEnhances bone formation and decrease osteoporosis

[19, 31]

Cerium,gallium

Induce positive response to osteoblastsIncrease bone calcium contentInhibit osteoclast activity

[32]

Cobalt Mimics hypoxia and induces angiogenesis [33, 34]

Boron Helps new bone formation and maintenanceStimulates RNA synthesis in fibroblast cells

[10, 18,33, 35]

Lithium Enhances remyelination of peripheral nerves [36]


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tremendous research efforts have been devoted to these composite systems in the last 15 years. Indeednumerous different compositions of bioactive glasses are now available and suitable for combination withbiopolymers for the design of composite scaffolds for tissue engineering.

In this chapter a concise update of the field of biopolymer–bioactive glass composite scaffolds for tissueengineering is presented, focusing on several established processing techniques, namely, microsphereprocessing, solvent casting-particulate leaching method, electrospinning, freeze-drying, and rapidprototyping methods. These processing techniques were chosen for the present discussion as they enablethe fabrication of bioactive composites of different morphologies, including microspheres, fibers, foams,and architectured scaffolds, illustrating the variety of systems currently available for TE.

Composites as Useful Materials for Tissue Engineering

When a polymer and an inorganic phase are combined, it is possible to tailor the properties of the resultingcomposite for the intended applications. For a successful composite with desired mechanical properties,for example, it is necessary to control the interface between the polymer and the inorganic phaseadequately, being thus possible to fabricate composites which combine the favorable properties of eachphase while at the same time minimizing the negative aspects of each phase. In addition, using thecomposite approach, it is possible to mimic the natural structure of the bone and other complex organs inthe human body. In this framework, it is possible to balance the physical, mechanical, and biologicalproperties of each of the individual phases and also to manufacture multilayer, gradient, and hierarchicalstructures. The addition of inorganic nanoparticles can also induce a nanoscale roughness on the surfaceof the scaffold that has been shown to stimulate the adhesion, proliferation, and maturation of differentcell types [39–42]. Finally, the combination of polymeric and inorganic phases can also be convenientlyused to control the overall degradation rate of the composite, which is of paramount importance to designscaffolds with tuned degradation kinetics.

Development of Bioactive Glass–Biopolymer Composites

Dense or porous polymer–inorganic phase composite materials can be fabricated using different produc-tion methods such as solvent casting, thermally induced phase separation processes, freeze casting,electrospinning, freeze-drying, and rapid prototyping techniques, to mention a few. Several review papersand book chapters are available describing traditional methods employed to fabricate bioactiveglass–biopolymer composites [1, 7, 8, 11, 14, 39, 42–46]. In some cases, both phases interact on amolecular level; thus strong bonds exist between the phases forming organic–inorganic hybrids[37]. Bilayer or multilayer structures are also being developed, which result from the combination ofdifferent composite parts on the macro-scale. These types of scaffolds can be used to regenerate defectswhere one or more tissue layers with different mechanical properties and biochemical composition needto be regenerated, such as osteochondral defects [47]. In this section several common processing methodswith great potential to develop bioactive glass–biopolymer composite scaffolds are described, as theydeliver different scaffold architectures useful in tissue engineering, namely, microspheres, (nano)fibers,and 3D porous structures.

Biocomposite MicrospheresPolymer microspheres have been widely used as a delivery vehicle of drugs, proteins, and cells for therepair and regeneration of damaged tissue [48, 49]. These microspheres can encapsulate bioactive


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molecules and release them at controlled rates for relatively long periods of time, which makes thismethod preferable over traditional drug administration. Their spherical form enables them to easily filldefects of irregular and complex shapes and sizes; thus, they are promising as injectable and/or defectivesite-filling materials [49, 50].

Awide range of natural or synthetic polymers can be used to manufacture these structures. However,microspheres made from polymers alone are not the optimal material of choice for bone tissue engineeringapplications because of the absence of bioactivity. Therefore, bioactive glass particles can be dispersed orencapsulated in polymer microspheres, mainly to impart a bioactivity property, i.e., the microspheres willhave the ability to develop a CaP layer on their surface enabling better integration with the bonetissue [50].

A variety of techniques are currently available for preparing microspheres. The most widely known isthe emulsification–solvent evaporation process. Briefly, in this technique, a polymer solution is made witha volatile organic solvent, and during this process, it is possible to add drugs or other bioactive moleculessuch as proteins or growth factors. Then, the organic phase is emulsified to obtain an oil/water emulsionthat can be stabilized as droplets. After that, the microspheres can be collected by filtration or centrifu-gation. Other available techniques for microsphere manufacture are coacervation (polymer phase sepa-ration), spray drying, milling, and supercritical fluid techniques [51]. The manufacturing process isselected depending on the nature of the polymer, the intended application, and the requireddegradation rate.

During these processes bioactive glass particles can be incorporated aiming a higher control of thepolymer microsphere degradation, the increase of the reactivity of the microsphere, and acceleration of thehealing process by enhancing the osteogenic and angiogenic potential.

In 2002, Qui et al. [52] incorporated colloidal hydroxyapatite (HA) or modified bioactive glass (mBG),of size under 20 mm, into degradable poly(lactic acid) (PLA) microspheres in different ratios (1:1, 1:3, and1:9). After SEM, EDX, FTIR, and ion quantification characterizations, the obtained results indicated asignificantly enhanced surface reactivity of the microspheres containing bioactive glass. Also, themBG-containing microspheres showed greater bioactivity compared to microspheres containing HA asthe filler material.

Keshaw et al. [53] prepared PLGA microporous spheres containing 10 % w/w 45S5 bioactive glass(BG) using a thermally induced phase separation (TIPS) method, and the typical microstructure of themicrospheres is shown in Fig. 1. The produced composite spheres were 1.82 mm in diameter and had aporosity of 82.6%. Both the neat PLGA and PLGA/45S5 BGmicroporous spheres integrated rapidly withthe host tissue in a subcutaneous wound model. Moreover, 45S5 BG-containing composite spheresshowed a significant increase in vascular endothelial growth factor secretion from myofibroblasts.

In addition to the melt-derived bioactive glass, sol–gel-derived bioactive glasses including mesoporousglasses have also been incorporated into polymer microspheres [54, 55]. For example, Wu et al. [54]developed mesoporous glass/alginate composite microspheres, shown in Fig. 2, with controllable dexa-methasone delivery ability. The obtained composite microspheres were shown to be bioactive, and theincorporation of mesoporous glass in the alginate microspheres enhanced the drug-loading capacity andaltered the drug release profile. In order to treat osteoporosis-like bone defects, Mondal et al. [55] preparedpoly(lactide-co-caprolactone) microspheres loaded with alendronate sodium and bioactive glass-ceramicusing an oil-in-water emulsion solvent evaporation method, which are shown in Fig. 3. The compositemicrospheres were found to be bioactive, non-cytotoxic, and able to promote cell adhesion. The localdelivery of alendronate sodium by microspheres represents a superior strategy to the oral administration,which is associated with oral discomfort and low bioavailability.


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Fig. 2 SEM images of alginate microspheres (a), alginate/mesoporous BG microspheres (b), and alginate/non-mesoporousBG microspheres (c) developed by Wu et al. [54] (Reproduced from Ref. [54] with permission from Wiley Periodicals)

Fig. 1 SEM images of highly porous PLGA/45S5 BG microspheres obtained by TIPS according to Kershaw et al. [53]: (a)macroview of microsphere, (b) higher magnification image, (c) higher resolution image showing the internal tubular structurewith bioactive glass particles being distributed in the interconnected tubular structure (Reproduced from Ref. [53] withpermission from Mary Ann Liebert, Inc. Publishers)


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Composites from Solvent Casting-Particulate Leaching ProcessAs a simple, straightforward and inexpensive method, solvent casting-particulate leaching (SCPL)process involves water-soluble porogens (sugar, salt, etc.) mixing into a polymer/monomer solutionfollowed by mold casting into desired shape. Then, the solvent could be removed by evaporation orlyophilization and polymerization in the case of monomer mixtures. Afterwards, the porogens are leachedaway by water immersion and pores or pore channels are formed [56, 57]. Figure 4 schematicallyillustrates the process [58]. Porosity and pore sizes can be precisely adjusted by altering porogen amountand sizes. In this manner, scaffolds with porosity of up to 93 % and pore sizes of up to 500 mm can beprepared. The simple operation and the adequate control of porosity confer the method’s wide applica-tions and suitability for use with various polymers. However, pore shapes depend on the porogen shape,which are usually cubic-like, equiaxed, or spherical. Besides full interconnectivity is difficult to achieve inthese scaffolds, which limits the actual application fields.

As a conventional process for porous structure fabrication, diverse single polymers, blend polymers,and polymer/inorganic filler systems have been explored for porous scaffold fabrication in tissueengineering [59]. Here, only specific examples relevant to bioactive glass containing composites willbe introduced in the following paragraphs.

Li et al. [60] have incorporated both 40 wt% of normal sol–gel-derived bioactive glass (BG) andmesoporous bioactive glass (MBG) powders with a composition ratio of 80S15C into PCLmatrix throughSCPL method. Both PCL/BG and PCL/MBG scaffolds with porosity of around 90 % were successfullyprepared. Wettability studies on the two scaffolds types implied that MBG not only greatly improved thehydrophilicity of the PCL matrix but also imparted better wettability than normal BG. The investigationinvolving ion release and SBF incubation for 3 weeks indicated that MBG released Si ions faster than BG,therefore leading to higher HCA formation ability. SEM images show that a dense and continuous apatitelayer was formed on MBG-containing samples, but only scattered and discrete apatite appeared on

Fig. 3 SEM images of P(LLA-co-CL) microspheres, scale of 200 mm (a); P(LLA-co-CL) microspheres loaded with 50 wt%bioactive glass, scale of 200 mm (b); and P(LLA-co-CL) microspheres loaded with 50 wt% bioactive glass and alendronatesodium, scale of 100 mm (c); developed by Mondal et al. [55] (Reproduced from Ref. [55] with permission from Elsevier)


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BG-containing samples. The highly enhanced bioactivity of MBG-containing samples was attributed tothe ordered channels and high surface to volume ratio of MBG particles, which accelerated the ionexchange during SBF incubation.

The ability to bond to bone tissue and promote bone regeneration is already well known for bioactiveglasses, as discussed above. However, a recent study of PHBV/Bioglass scaffolds by SCPL methoddemonstrated promising application in cartilage tissue engineering as well [61]. Wu et al. incorporated20 wt% 45S5 Bioglass® powders into PHBV matrix for PHBV/BG porous scaffolds by theabovementioned SCPL method and conducted in vitro and in vivo tests by culturing chondrocytes andimplanting into nude mice. Results showed that not only the hydrophilicity and compressive strengthwere highly enhanced, but also the chondrocyte’s penetration length, thickness of cartilage-like tissue ofin vivo constructs, and the mechanical strength of the formed cartilage tissue were all promoted.

In a very recent study reported by Niu et al. [62], 30 wt% mesoporous bioactive glass particles wereincorporated into PLLA scaffolds by SCPL method using NaCl as pore-forming agent. Figure 5 displays thetypical porous microstructure of the obtained scaffolds with different MBG ratios: PLLA, PLLAwith 15 wt%of MBG (15BPC), and PLLAwith 30 wt% of MBG (30BPC). A detailed investigation on wettability, waterabsorption, degradability, in vitro culture of MC3T3-E1 cells, and in vivo implantation in rabbits wasdisclosed. Results demonstrated that hydrophilicity, water absorption, degradation rate, HCA formationability, cell proliferation, and ALP activity were all significantly increased by MBG incorporation, whichwere bothMBG content dependent and time dependent. In addition, bone growth in a femur defect in a rabbitmodel was fully developed and recovered when 30BPCwas used within 12 weeks. Especially the histologicalanalysis of in vivo implantation demonstrated that the PLLA scaffolds with 30 wt% MBG incorporationproduced by SCPL have promising potential in clinical trials and further in bone tissue engineering.

Fig. 4 Illustration of the fabrication steps of macroporous scaffolds by solvent casting-particle leaching method (Reproducedfrom Ref. [58], Open Access article distributed under the terms of the Creative Commons Attribution Non-CommercialLicense (http://creativecommons.org/licenses/by-nc/3.0/))


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http://creativecommons.org/licenses/by-nc/3.0/

Electrospun Composite FibersElectrospinning has experienced a rapid and continuous development as a convenient process for micro-or nanofiber production for numerous applications including tissue engineering scaffolds [63, 64]. Themethod is based on the principle that under an electrostatic field, a continuous fiber jet is ejected from thetip of a charged solution when the electrostatic force exceeds the surface tension, and after solidification,the fibers will pile up on a grounded collector [63]. The diameter and morphology of the fibers aredetermined by the balance between electrostatic forces and surface tension. This technique makespossible the production of continuous fibers with diameters in the submicrometric scale by adjustingparameters regarding the solution, operation, and ambient conditions. Some other intrinsic parametersthat can also affect the fiber manufacture are the polymer’s molar mass, concentration, and solventcomposition and concentration in the polymer solution. On the other hand, the applied voltage, collectingdistance, and the feed rate are extrinsic operational variables also affecting the process outcomes. Inaddition, humidity and temperature can also alter the morphology of the product [63, 65]. Figure 6 showsa schematic representation of the electrospinning setup.

The simplicity of the electrospinning technique enables a broad applicability on various kinds ofpolymers. As the demand of biomaterial scaffolds for tissue engineering has dramatically increased inrecent years, intense research on electrospinning of different biopolymers is being conducted, not only

Fig. 5 SEM images of the surface morphology of PLLA (a, b), 15BPC (c, d), and 30BPC (e, f) scaffolds at differentmagnifications [62] (Reproduced with permission from Royal Society of Chemistry (RSC) publishing)


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concerning natural polymers such as collagen, chitosan, gelatin, etc., but also synthetic biopolymers suchas PLA, PLGA, PCL, PHB, PHBV [67]. To overcome the limitations of a single polymer or polymerblend, organic–inorganic composite and hybrid fibers are being also developed to combine and balancetheir physiochemical and biological properties [63, 68].

Many efforts have been devoted to the development and improvement of the electrospinning process ofbiopolymer–bioactive glass suspensions aiming a better integration of the brittle and bioactive inorganicphase with the elastic and bioinert organic phase for hard and soft tissue regeneration. Basically, there aretwo strategies for bioactive glass particle incorporation; the first one is to associate melt or sol–gel-derivedglass particles or fillers with the polymer solution with and without surfactants in an ultrasonic bath. Nohet al. [69], for example, fabricated bioactive glass nanofiller-reinforced PLA electrospun scaffolds, inwhich the nanofillers were chopped from fiber mats of electrospun sol–gel-derived bioactive glass.Results indicated a significant enhancement of the in vitro apatite formation on the composite nanofibersurface in SBF solution, and it was reported that osteoblastic cells could adhere and proliferate well on thecomposite nanofibers.

Kouhi et al. [70] and Lin et al. [71] both fabricated sol–gel-derived bioactive glass/PCL fibrousscaffolds through electrospinning from a solution of PCL and glass nanoparticles. The study of Kouhiet al. showed that bioactivity and cell differentiation activity were significantly improved due to thenanosized bioactive glass addition. Lin et al. fabricated a multifunctional bioactive scaffold (shown inFig. 7) with controlled simvastatin-releasing property. Due to the suitable physical interactions between

Fig. 6 Schematic representation of the electrospinning setup [66] (Reused with the permission of Elsevier)

Fig. 7 (a) SEM of electrospun PCL fiber mats with 10 wt% mesoporous BG powder fabricated by the direct mixing method,scale of 30 mm, and (b) TEM image of a single fiber which shows that the mesoporous glass nanoparticles were attached to thefiber surfaces [71] (Reproduced with permission from Springer)


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the polymer and inorganic particles, the BG loading up to 15 wt% led to the improvement of the tensilestrength however simultaneously to a decrease in strain [71].

Ren et al. [72] obtained biocomposite scaffolds incorporating strontium-substituted bioactive glass(SrBG) particles in PCL (10 wt%) forming highly porous fibrous sheets. The scaffolds demonstrated to benon-cytotoxic in vitro, and, when compared to PCL-only scaffolds, they could enhance alkaline phos-phatase activity and increased osteoblast differentiation through upregulation of gene expression (ALPand OCN) inMC3T3-E1 cells. The fibrous sheets also facilitated cellular attachment and proliferation andimproved collagen deposition.

Another approach for inorganic phase incorporation in electrospun fibers is to combine the bioactiveglass precursor with the polymer solution to prepare a hybrid solution for electrospinning. The obtainedhybrid fibers have a uniform structure, where the organic and inorganic phases interacted on a molecularlevel. Allo et al. [73] reported a single-phase electrospun 70SiO2–26CaO–4P2O5 bioactive glass/PCLscaffold using electrospinning as well as the sol–gel method without using any coupling agent. Thechemical characterization of the scaffolds revealed that natural polymers which possess multiple func-tional groups can easily be covalently bonded to sol–gel-derived bioactive glass; however, syntheticpolyesters could only link to sol–gel-derived bioactive glass through hydrogen bonding, which is nothelpful for the enhancement of all properties simultaneously.

Han et al. [74] developed a one-step solution, in which a mesoporous bioactive glass precursor(80SiO2–15CaO–5P2O5) was dissolved in PLA followed by the electrospinning process. The obtainedscaffolds presented a hierarchical porous structure with macro- and mesoporous structure (10 mm and5 nm, respectively). The biocomposites demonstrated high bioactivity, wettability, and rapid hydroxyap-atite (HAP)-inducing mineralization in SBF solution as well as good cell attachment (for HeLa cellsin vitro tests) and controllable drug release (in this study ibuprofen was used).

In another relevant study, Gao et al. [75] successfully prepared 70SiO2–25CaO–5P2O5 bioactive glass/gelatin hybrid fibrous scaffolds using the coupling agent 3-glycidoxypropyltrimethoxysilane (GPTMS)through a combination of the sol–gel method and electrospinning. GPTMS provided linkage for gelatinand hydrolyzed silanol groups and led to covalent bond formation, which enabled the manufacture of anorganic–inorganic hybrid scaffold with dramatic enhancement of tensile strength and elongation at break(Fig. 8).

A different reported approach involving electrospinning is to reinforce electrospun-bioactive glass fibermats with a polymer solution or the polymer melt. Kim et al. [76] prepared bioactive glass nanofiber-filledPLA composites, which proved to be highly bioactive and possessed a favorable osteoblastic response.Unlike the aforementioned strategies, in their study, the 58SiO2–38CaO–4P2O5 bioactive glass nanosizedfiber mats were first fabricated through the sol–gel electrospinning method followed by sintering at700 �C. Subsequently, different fractions of fiber mats were fully soaked and infiltrated with PLA/THFsolutions and then dried. These procedures were followed by a thermal-compression process at 130 �C toobtain a homogeneous dense plate material. A schematic drawing of this process is shown in Fig. 9. Theobtained nanocomposites had a higher in vitro bioactivity, and osteoblast cells attached and grew well ontheir surface as well as secreted collagen protein and improved the expression of ALP when compared tothe pure PLA nanocomposites.

In another study using the same BG composition, Kim et al. [77] prepared a biocomposite for boneregeneration with this bioactive glass nanofiber (BGNF) and collagen. The BGNF and self-assembledcollagen sol were combined in aqueous solution and cross-linked to produce a BGNF–collagennanocomposite thin membrane or a macroporous scaffold. These biocomposites showed rapid formationof bone-like apatite minerals on their surfaces in SBF solution and exhibited good bioactivity in vitro. Cellviability test indicated that osteoblasts presented favorable growth, and the alkaline phosphatase activitywas significantly higher for the cells exposed to the nanocomposite.


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In a related study, Lee et al. [78] obtained membranes of nanofibrous bioactive glass and PCL. Sol–gel-derived bioactive glass (70SiO2–25CaO–5P2O5) nanofibers were added to a PCL solution at 20 wt%. Themanufactured nanocomposite membranes induced rapid formation of apatite-like layer on the biomate-rials’ surface in SBF solution, and in vitro tests showed that murine-derived osteoblasts (MC3T3-E1)grew actively over the nanocomposite indicating a significant improvement on cell viability whencompared to pure PCL membrane. Furthermore, the osteoblastic activity, as assessed by the expressionof alkaline phosphatase, was significantly higher on the nanocomposite membrane than on the pure PCLmembrane.

Fig. 9 Process chart for the fabrication of PLA-reinforced bioactive glass fiber mats via electrospinning (Adapted from Ref.[76], reproduced with permission from Wiley Periodicals)

Fig. 8 (a) SEM and (b) TEM of electrospun sol–gel-derived BG/gelatin hybrid fiber mats as prepared by Gao et al. [75] byassociating the sol precursor and gelatin solution with the addition of a cross-linking agent; (c) the sol–gel fiber mats showedenhanced mechanical properties compared to gelatin alone (Reproduced from Ref. [75] with permission from WileyPeriodicals)


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Jo et al. [79] also fabricated PCL/sol–gel-derived BG nanofiber (BGNF, 20 wt%) biocomposites andinvestigated their biocompatibility and mechanical properties in comparison with composites containingbioactive glass in the microparticulated form. The obtained microstructure, showed in Fig. 10, resulted inan improvement of the biological and mechanical properties, presenting a superior bioactivity and ahigher elastic modulus. Moreover, in vivo animal experiments using Sprague–Dawley albino ratsrevealed that the PCL/BGNF biocomposites had a satisfactory biocompatibility and enhanced boneregeneration capability of calvarial bone defects.

In a recent study, Castaño et al. [80], using electrospinning as a suitable method to create fibers thatmimic the extracellular matrix (ECM), produced hybrid fibrous mats with three different contents of Si(40 %, 52 %, and 70 % – compositions defined by the authors as ormoglass), using anormoglass�polycaprolactone blend approach. These obtained mats showed a homogeneous structure,and the material and biological characterization suggested that the hybrid biomaterials with a Si contentequivalent to 40 % released a fair quantity of calcium ions and were satisfactorily osteogenic, enhancingMC3T3-E1 cell differentiation (Fig. 11). These biocomposites also showed a greater ability to form bloodvessels, since they could modulate the release of Ca2+ in a rate suitable to activate the VEGF productioncascade, thus demonstrating an angiogenic-promoting feature.

Fig. 10 Surface (a) and cross-sectional (b) images of PCL/BGNF biocomposites containing 20 wt% of BGNF (Adapted fromRef. [79], reproduced with permission from Wiley Periodicals)

Fig. 11 Cell adhesion to the fibrous scaffolds developed by Castaño et al. [80]. In this image cells and filopodia wereartificially colored in order to calculate the biocomposites’ surface occupation byMC3T3-E1 cells (Reproduced from Ref. [80]with permission from ACS Publications)


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Composite Scaffolds by Freeze-DryingFreeze-drying, which sometimes is also termed cryodesiccation or lyophilization, belongs to thethermally induced phase separation techniques and was described by Ezekwo et al. in 1980[81]. The technique has found increasing interest in the last years for fabrication of highly porousscaffolds with interconnected and tailored architecture [82–84]. This process has been investigated by anumber of research groups pursuing the fabrication of polymer matrix composites with the incorporationof inorganic phases such as HA [82, 83], demineralized bone [85], calcium phosphates [86, 87], andbioactive glass particles [88–91].

In this technique, a solution, dispersion, or emulsion consisting of a solvent, the polymer, and theinorganic nanoparticles (and in some cases a cross-linking agent such as glutaraldehyde, genipin, or other)is subjected to rapid gelation, which causes the solids or the solute to be displaced by an advancing icefront into the interstitial spaces between ice crystals [88–90]. Once fully frozen, the freeze-drying processitself takes place, which typically employs temperatures of �20 to �80 �C for different periods of time.The suspension is then sublimated in vacuum for varying lengths of time at a temperature suitable for thesublimation of the solvent from the mixture.

The overall porosity, pore structure, and mechanical properties of the scaffolds can be tailored byadjusting processing parameters such as solvent concentration, concentration of solid component in theslurry, and processing time, among other parameters; hence, different scaffold morphologies can beattained [92]. This process can be simplified into four steps, as shown in Fig. 12, and typical microstruc-ture of polymer/bioactive glass scaffolds made by freeze-drying are shown in Fig. 13 [93].

Therefore, just like in the case of electrospinning, freeze drying also allows the incorporation ofbioactive glass particles for the obtainment of reinforced scaffolds. For example, fibrillar collagen/bioactive glass porous composites have been developed using a multistep sol–gel procedure, whichwere subsequently adsorbed from an acidic suspension using freeze-drying technology at differentcollagen/bioactive glass ratios (wt%): 20:80, 50:50, and 80:20 [94]. Mesoporous glasses in combinationwith silk have also been used as tissue engineering scaffolds. These scaffolds showed higher mechanical

Fig. 12 Schematic diagram of the freeze-drying process for the fabrication of composite scaffolds

Fig. 13 SEM images showing PLGA and PLGA/Bioglass® scaffolds by freeze-drying method (Reproduced from Ref. [93],with permission from Elsevier)


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strength, in vitro apatite mineralization, silicon (Si) ion release, and higher pH stability compared toscaffolds fabricated by the same technique but using melt-derived bioactive glass. In vivo studies ofcalvaria defects in SCID mice also showed that the new bone apposition was slightly higher for thescaffolds containing mesoporous glass.

Hunger et al. [95] investigated the structure–property–processing correlation in composite scaffoldsfabricated by freeze-drying using a blend of chitosan and gelatin with 75 v.% of alumina particlereinforcement as a model system. They found that the particle size of the inorganic reinforcing phaseplayed a deciding role in determining the elastic modulus, compressive yield strength, and the failuremode of the composite scaffolds. Whereas small particles and bimodal particle size distribution led tothicker polymer walls and brittle failure, the incorporation of larger alumina particle sizes led to thinnerpore walls with alignment of the particles in the polymer phase forming a structure type of “beads on astring.” The difference in failure modes was explained by the difference in the structure of the lamellarwalls, which were more porous for smaller and bimodal particle incorporation. The study also found thatwith increase of the rate of freezing the mechanical strength of the composites increased, while thelamellar spacing and the pore aspect ratio were reduced. Both the rate of freezing and the effect of particlesize have also been investigated, and it was confirmed that these two factors must be considered to designscaffolds with compatible mechanical and morphological properties for a given application [88, 92,95]. Compressive strength values as high as 11.5 MPa and elastic moduli as high as 215 MPa have beenreported for freeze-dried BG-filled scaffolds [88].

Another important advantage of the freeze-drying technology is that bioactive molecules such asgrowth factors [96] or drugs [97] can be easily incorporated into the scaffolds since no high-temperatureprocessing is required. Additionally, it is possible to make use of the ions leached from bioactive glasses toprovide a favorable environment for the cells.

For novel materials and scaffold development, different single techniques can be combined leading tomodifications of the microstructure and porosity of the final composite material. Qu et al. [98], forexample, combined sol–gel method, freeze-drying, and particulate leaching process to fabricate nano-structured gelatin/silicate bioactive glass (SBG) hybrid scaffolds. By utilizing sol–gel technique, amor-phous SBG was uniformly distributed into the gelatin matrix acting as a reinforcing phase. In comparisonto directional pore arrangement by conventional freeze-drying process, the pores were round with size inthe range 250–420 mm, which was determined by the leached particulates. Hierarchical pore structureswith macropores of around several hundred micrometers and nanopores of around several hundrednanometers were generated from the particulate leaching and freeze-drying processes, respectively. Themorphologies of gelatin/SBG with different SBG content are shown in Fig. 14 [98]. Dental pulp stem cell(DPSCs) cultures evidenced that the gelatin/SBG hybrid scaffolds were more favorable for cell adhesion,proliferation, and osteogenic differentiation than the gelatin-only scaffolds. Moreover, gene expressionwas highly promoted by SBG addition. The hybrid scaffolds showed a high potential for regeneration ofdecayed dentin/pulp structure.

Rapid PrototypingThe advances of rapid prototyping (RP) (or solid freeform fabrication (SFF), additive manufacturing)techniques are bringing a wide range of possibilities to the biomedical field. These techniques permit themanufacture of customized components conferring precise control of their architecture [12, 99]. Thesemethods are based on computer-aided design to manufacture custom-made devices (scaffolds) directlyfrom digital data. The most widely used RP technologies for manufacturing scaffolds for tissue


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Fig. 14 SEM micrographs of gelatin/SBG hybrid scaffolds with SBG ratios of 0 % (a, d, g), 5 % (b, e, h), and 10 % (c, f, i);ALP activity and gene expression of gelatin scaffolds and gelatin/SBG (5 %) scaffolds (Reproduced from Ref. [98] withpermission from Royal Society of Chemistry (RSC) publishing)


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engineering are stereolithography (SL or SLA), selective laser sintering (SLS), fused deposition modeling(FDM), 3D printing (3DP), multi-jet modeling (MJM), and laminated object manufacturing (LOM)[12]. Figure 15 shows representative schemes of different SFFmethods, or reported in literature [99, 100].

SFF techniques’ main advantages include reproducibility and precise control over the architecture of3D scaffolds, mainly concerning the scaffold’s internal structure, geometry, pore sizes and distribution,Scaffolds exhibiting high, controlled shape porosity and full interconnectivity can be obtained. SFF alsoallows the fabrication of scaffolds with high complexity, which can be based on patient-specific data andwith anisotropic or graded microstructures. Generally, SFF techniques are versatile requiring a relativelylow processing time, in comparison to conventional methods, generating scaffolds with enhancedmechanical and biological capabilities [12]. RP technologies are demonstrating a great potential inhealthcare applications, and studies worldwide start to emerge which show the advantages of combiningbiopolymers and bioactive glasses through these modern techniques.

Stereolithography (SLA)SLA is a traditional RP technique based on the deposition of a photosensitive liquid resin in a movableplatform, which is irradiated with an ultraviolet laser to cure and solidify the polymer. In this method,scaffolds are made through a layer-by-layer process, normally reaching a spatial resolution of approxi-mately 50 mm [12].

Fig. 15 (continued)


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SLA can be used with a range of biopolymers and also to build bioceramic devices with a photosen-sitive polymer as a binder. The fabrication of polymer/ceramic composite scaffolds is more complicateddue to the high viscosity of the final suspension and other limitations intrinsic to the technique. Somestudies have shown the fabrication of Bioglass® scaffolds using a combination of SLA and castingtechniques [101, 102]. In this indirect process, a polymer mold is obtained by SLA, and after casting thebioceramic upon it, a thermal treatment is placed resulting in a ceramic scaffold. Also, Gmeineret al. [103] successfully fabricated 3D dense bioactive glass-ceramic structures with increased mechanicalproperties and scaffolds with an accuracy of up to 25 � 25 � 25 mm3 using ceramic and glass-ceramicslurries and the SLA technique. Tesavibul et al. [104] obtained a highly bioactive glass scaffold usinglithography-based additive manufacturing. The technique allowed the tailoring of the scaffold’s macro-scopic and microstructural features, developing scaffolds with a pore size of 500 mm.

Most recently, Elomaa et al. [105] developed a 3D scaffold with highly interconnected pores, combin-ing photocrosslinkable PCL resin and S53P4 bioactive glass. The addition of the inorganic phaseimproved the compression modulus of the scaffolds and also their bioactivity, enhancing the attachmentand the proliferation of human fibroblasts on the 3D structures.

Selective Laser Sintering (SLS)SLS uses a laser beam to selectively sinter or fuse some regions of a powder surface. This technique isused when high-complexity porous materials are desired. Due to limitations intrinsic to this techniquesuch as optimal powder composition, particle size range, speed, and laser intensity, biopolymer–bioactive

Fig. 15 Schematic diagram of SFF technologies: (a) stereolithography, (b) fused deposition modeling, (c) mini-extruder inprecision extrusion deposition, (d) selective laser sintering, (e) 3D printing (Reproduced from [99] and [100] with permissionsfrom Royal Society of Chemistry (RSC) publishing and IOP publishing, respectively)


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glass composites are still to be realized with this method [12]. However, some studies successfullyobtained PLLA/HA [106] biocomposite scaffolds using this technique, thus opening the possibility for thefabrication of biopolymer–bioactive glass composites.

3D Printing (3DP)3D printing has been widely used for the fabrication of scaffolds using different biopolymers andceramics. This technique incorporates ink-jet technology to precisely place a liquid binder on a powerbed, adhering the powder together and forming a cross-sectional layer. After this step, another powderlayer is spread over the surface, and this process occurs until the predesigned device is obtained.Following this procedure, the unfused excess powder is removed by compressed air or manually brushed.

Bergmann et al. [107] employed the 3D printing process to fabricate a composite of b-TCP and abioactive glass (similar to 45S5 Bioglass®), using orthophosphoric acid (H3PO4) and pyrophosphoricacid (H7P2O7) as binders. Results showed that the glass phase had no effect on the cement reaction and themaximum resolution obtained (a layer thickness) was approximately 50 mm. The achieved bendingstrength was approximately 15 MPa and XRD analysis revealed the presence of two phases, CaNaPO4

and CaSiO3, both biocompatible and feasible of biodegradation.Zhao et al. [108] developed scaffolds with high osteogenic capability combining mesoporous bioactive

glass (MBG) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) via 3D printing. Thebiocomposite scaffolds had uniform interconnected macropores and high porosity, and their compressivestrength was approximately 200 times higher than that of polyurethane foam templated MBG scaffolds.These scaffolds also presented high bioactivity tested by the proliferation and differentiation of humanbone marrow-derived mesenchymal stem cell (hBMSC). In vivo tests revealed that MBG/PHBHHxbiocomposite scaffolds had good osteogenic capability and stimulated bone regeneration in critical-sizerat calvarial defects within 8 weeks. The morphologies of the 3D plotted scaffolds with varying weightratio are shown in Fig. 16 [108].

Serra et al. [109] presented studies incorporating calcium phosphate glasses to PLA. In the first study, in2013, they fabricated biodegradable 3D-printed PLA/CaP glass scaffolds via nozzle-based rapidprototyping. As reported, the addition of 5 % PEG to the PLA matrix allowed the fabrication of high-resolution scaffolds without affecting the polymer blending properties. The chosen technique alsopermitted the manufacture of highly porous scaffolds with satisfactory mechanical properties. Theaddition of the glass (and PEG) to the PLA matrix induced a more positive biological response, allowingcell (mesenchymal stem cells) adhesion and spreading on the biomaterial’s surface [109]. The secondstudy was aimed at identifying the optimal conditions for manufacturing PEG/PLA/CaP glass scaffoldsvia 3D printing [110]. Results showed that the incorporation of PEG in the PLAmatrix could improve thematrix’s wettability and elastic modulus. In vitro tests indicated that the inclusion of PEG significantlyaccelerated the biomaterial’s degradation rate and improved the processing features [110]. Almeidaet al. [111] studied PLA/CaP glass and chitosan scaffolds. While all scaffolds supported monocyte/macrophage adhesion and stimulated cytokine production, PLA-based scaffolds induced higher produc-tion of interleukin (IL)-6, IL-12/23, and IL-10. This study also draws attention to the scaffold’s geometryimpact on macrophages morphology and cytokine secretion profile. Recently, Zhang et al. [112] fabri-cated strontium-containing mesoporous bioactive glass and PVA scaffolds by 3D printing. Resultsindicated that the scaffolds had uniform interconnected macropores and high porosity. Also, theircompressive strength was approximately 170 times that of polyurethane foam templated MBG scaffolds.The manufactured scaffolds possessed good apatite-forming ability and stimulated osteoblast cellsproliferation and differentiation. In this study, dexamethasone was also used as a model drug. TheSr-MBG/dexamethasone scaffolds exhibited sustained drug delivery capability for use in local drugdelivery therapy.


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Fused Deposition Modeling (FDM)This technique employs the concept of extruding a melt through a movable nozzle with a small orificeonto a substrate platform, depositing the material in parallel form, creating the layer. Normally, thisprocess uses thermoplastic polymers, and the combination with bioceramics can produce highly porous3D scaffolds. Korpela et al. [113] have reported the manufacture of highly porous biocomposite scaffoldsbased on bioactive glass S53P4/poly(e-caprolactone) via FDM process. SEM images showed that BGparticles were present at the scaffold’s surface. Also, the study indicated that this technique was suitablefor developing mechanically durable and biocompatible biocomposites.

The studies discussed in this section can confirm that SFF offers a series of advantages when comparedto scaffold conventional fabrication routes, and there are a range of processing techniques being exploredin order to create polymer, ceramic, or composite highly complex scaffolds with precise control of poresize and spatial distribution, high reproducibility, and flexibility in shape and size. Currently, the SLA,SLS, 3DP, and FDM techniques are used for the fabrication of polymer/bioactive glass biocompositescaffolds. The results obtained so far indicate that the newly developed biomaterials have achievedsatisfactory mechanical properties and enhanced biological performance, making possible the manufac-ture of scaffolds with excellent accuracy and good surface quality. These relatively novel techniques bringthus new perspectives and opportunities for tissue engineering when combined with biopolymer–bioac-tive glass systems, they are expected to lead to improved scaffolds with tuned properties and porestructure for different applications.

Fig. 16 SEM images of 3D-printed MBG-based composite scaffolds: a1 (MBG: PVA = 7:1(w/w)); b1, c1, and d1 representthe scaffolds with MBG: PHBHHx = 7:1, 5:1, and 3:1 (weight ratio) (Reproduced from Ref. [108] with permission fromRoyal Society of Chemistry (RSC) publishing)


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Summary

In the last 15 years, great advances in the development of tissue engineering scaffolds have beenaccomplished. A variety of combinations of natural or synthetic polymers with bioactive glasses orglass-ceramics have been explored. These composite structures have many advantages, including thepossibility of tailoring the degradation kinetics, mechanical properties, and chemical compositionaccording to the characteristics of the surrounding tissue where the scaffold will be applied.

The addition of biologically active ions to bioactive glass, such as strontium, boron, copper, etc. is aninteresting approach being increasingly considered to provide enhanced functionalities to compositescaffolds. The fact that some bioactive glasses are able to bond to soft tissues offers great new pathwaysfor the use of these types of composites in soft tissue engineering, especially for the regeneration of tissueswith higher complexity, as recently reviewed [16].

However, some major challenges remain to achieve clinical applications of the different scaffoldsdeveloped over the years in particular considering the required balance between (time-dependent)physical, mechanical, and biological properties. The optimal scaffold needs to mimic the ECM as closelyas possible. One of the most difficult challenges is to analyze the interaction between cells and scaffoldsin vitro and in vivo (animal models) and to translate these findings successfully to the clinic, consideringthe time-dependent change of the scaffolds’ structure and properties. In a composite biomaterial, this isparticularly complex as the multiple phases are varying with time, in mechanical properties and compo-sition, at different degradation rates.

As discussed in this chapter, biopolymer–BG composites, includingmesoporous BG and nanoscale BGparticles, represent promising materials for superior scaffolds for TE. With the expansion and improve-ment of processing techniques for such composites (some of which were discussed in this chapter indetail), new possibilities for the incorporation of the BG phase in different concentrations and in differentgeometrical arrangements become possible which should lead to tailored mechanical, biological anddegradation behavior of the scaffolds. It is anticipated that further research in the field, likely involvingcombination of polymers, BGs (in different morphologies), and the smart addition of bioactive molecules[97], will lead to superior biomimetic composites for TE scaffolds with opportunities for clinical use.

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