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Biomaterials 27 (2006) 34133431 www.elsevier.com/locate/biomaterials

Review

Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering
K. Rezwana, Q.Z. Chena, J.J. Blakera, Aldo Roberto Boccaccinia,b,
b

Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK Centre for Tissue Engineering and Regenerative Medicine, Imperial College London, London SW7 2AZ, UK Received 6 December 2005; accepted 31 January 2006 Available online 28 February 2006

Abstract Biodegradable polymers and bioactive ceramics are being combined in a variety of composite materials for tissue engineering scaffolds. Materials and fabrication routes for three-dimensional (3D) scaffolds with interconnected high porosities suitable for bone tissue engineering are reviewed. Different polymer and ceramic compositions applied and their impact on biodegradability and bioactivity of the scaffolds are discussed, including in vitro and in vivo assessments. The mechanical properties of todays available porous scaffolds are analyzed in detail, revealing insufcient elastic stiffness and compressive strength compared to human bone. Further challenges in scaffold fabrication for tissue engineering such as biomolecules incorporation, surface functionalization and 3D scaffold characterization are discussed, giving possible solution strategies. Stem cell incorporation into scaffolds as a future trend is addressed shortly, highlighting the immense potential for creating next-generation synthetic/living composite biomaterials that feature high adaptiveness to the biological environment. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Scaffolds; Bioactivity; Bone tissue engineering; Composites; Porosity; Biodegradability

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biodegradable polymer matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Saturated aliphatic polyesters (PLA, PGA and PCL). . . . . . . . 2.2. Polypropylene fumarate (PPF). . . . . . . . . . . . . . . . . . . . . . . . 2.3. Polyhydroxyalkanoates (PHB, PHBV, P4HB, PHBHHx, PHO) 2.4. Surface bioeroding polymers . . . . . . . . . . . . . . . . . . . . . . . . . Bioactive ceramic phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bioactive glasses and glass-ceramics . . . . . . . . . . . . . . . . . . . . 3.1.1. Composition and bioactivity . . . . . . . . . . . . . . . . . . . 3.1.2. Mechanical properties. . . . . . . . . . . . . . . . . . . . . . . . 3.2. Calcium phosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Composition and bioactivity . . . . . . . . . . . . . . . . . . . 3.2.2. Mechanical properties. . . . . . . . . . . . . . . . . . . . . . . . 3.3. Other bioactive ceramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3414 3414 3415 3416 3416 3417 3417 3418 3418 3419 3419 3419 3419 3419

3.

Corresponding author. Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK. Tel.: +44 207 594 6731; fax: +44 207 594 6757. E-mail address: a.boccaccini@imperial.ac.uk (A.R. Boccaccini).

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.01.039

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4.

Material processing strategies for composite scaffolds with interconnected pores . 4.1. Thermally induced phase separation (TIPS) . . . . . . . . . . . . . . . . . . . . . . 4.2. Solvent casting and particle leaching . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Solid freeform fabrication techniques (SFFT) . . . . . . . . . . . . . . . . . . . . . 4.4. Microsphere sintering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Coated scaffolds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Challenges and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Mechanical integrity of porous scaffolds. . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Incorporation of biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Long-term characterization of porous composite scaffolds . . . . . . . . . . . . 5.4. In vitro and in vivo characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Summary of current status and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Future trend: stem cells and ideal biomaterials . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Tissue engineering applies methods from materials engineering and life sciences to create articial constructs for regeneration of new tissue [1]. One common approach is to isolate specic cells through a small biopsy from a patient to grow them on a three-dimensional (3D) scaffold under controlled culture conditions. Subsequently, the construct is delivered to the desired site in the patients body with the aim to direct new tissue formation into the scaffold that can be degraded over time [13]. An alternative approach is to implant scaffolds for tissue ingrowth directly in vivo with the purpose to stimulate and to direct tissue formation in situ [2,4,5]. The advantage of this approach is the reduced number of operations needed, resulting in a shorter recovery time for the patient. Facing a complex biological and sensitive system as the human body, the requirements of scaffold materials for tissue engineering are manifold and extremely challenging. First, biocompatibility of the substrate materials is imperative; that is the material must not elicit an unresolved inammatory response nor demonstrate immunogenicity or cytotoxicity. In addition, the mechanical properties of the scaffold must be sufcient and not collapse during handling and during the patients normal activities. As with all materials in contact with the human body, tissue scaffolds must be easily sterilizable to prevent infection [6]. This applies notably for bulk degradable scaffolds, where both the surface and the bulk material must be sterile. A further requirement for a scaffold particularly in bone engineering is a controllable interconnected porosity to direct the cells to grow into the desired physical form and to support vascularization of the ingrown tissue. A typical porosity of 90% as well as a pore diameter of at least 100 mm is known to be compulsory for cell penetration and a proper vascularization of the ingrown tissue [710]. Other highly desirable features concerning the scaffold processing are near-net-shape fabrication and scalability for cost-effective industrial production.

Today, materials used for scaffolds are natural or synthetic polymers such as polysaccharides, poly(a-hydroxy ester), hydrogels or thermoplastic elastomers [2,4,11,12]. Other important categories of materials are bioactive ceramics such as calcium phosphates and bioactive glasses or glass-ceramics [8,13,14]. Currently, composites of polymers and ceramics are being developed with the aim to increase the mechanical scaffold stability and to improve tissue interaction [1419]. In addition, efforts have also been invested in developing scaffolds with a drug-delivery capacity. These scaffolds can locally release growth factors or antibiotics and enhance bone ingrowth to treat bone defects and even support wound healing [14,2023]. Aforementioned requirements for scaffold materials are numerous. To fulll as many requirements as possible, composite systems combining advantages of polymers and ceramics seem to be a promising choice, in particular for bone tissue engineering, as demonstrated by the increasing research efforts worldwide [2,1422,2429]. This paper reviews tissue engineering relevant biodegradable polymers and bioactive ceramics, including strategies for fabrication of composite scaffolds with interconnected pores. Microstructure and mechanical properties will be discussed and compared, evaluating open challenges in this eld of biomedical materials research. In vitro and in vivo characteristics of porous composite scaffolds, with focus on bone regeneration, will be discussed as well as summarizing the currently available literature and pointing to research and development needs.

2. Biodegradable polymer matrices There are two types of biodegradable polymers: The natural-based materials are one category, including polysaccharides (starch, alginate, chitin/chitosan, hyaluronic acid derivatives) or proteins (soy, collagen, brin gels, silk) and, as reinforcement, a variety of biobers such as lignocellulosic natural bers which are described in detailed studies and reviews elsewhere [3034].

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K. Rezwan et al. / Biomaterials 27 (2006) 34133431 Table 1 Physical properties of synthetic, biocompatible, and biodegradable polymers used as scaffold materials Polymer Melting point Tm (1C) Glass transition point Tg (1C) Biodegradation time (months) Compressive* or tensile strength (MPa) Pellet: 35150* Film or disk: 2935 Pellet: 40120* Film or disk: 2850 Fibre: 8702300 Fibre: 340920 41.455.2 230* 2043 2527 3040* 416* 0.141.4 2.54.4 Modulus (GPa) 3415

1. Bulk degradable polymers PDLLA [3638] Amorphous PLLA [36,38] 173178

5560 6065

1216 424

Film or disk: 1.92.4 Film or disk: 1.23.0 Fibre: 1016 Fibre: 714 1.42.8

PGA [3739] PLGA [31] PPF [31,40] PCL [41] PHA and blends [62]

225230 Amorphous 58 120177

3540 4555 72 2 to 4

612 Adjustable: 112 Bulk 424 Bulk Surface Surface

2. Surface erodative polymers Poly(anhydrides) 150200 [31,40,41] Poly(ortho-esters) 30100 [31,42] Polyphosphazene [43] 66 to 50

242

Surface

This review will focus on the second category, synthetic biodegradable polymers. Synthetic polymers can be produced under controlled conditions and therefore exhibit in general predictable and reproducible mechanical and physical properties such as tensile strength, elastic modulus and degradation rate. A further advantage is the control of material impurities. Possible risks such as toxicity, immunogenicity and favoring of infections are lower for pure synthetic polymers with constituent monomeric units having a well-known and simple structure. Table 1 gives an overview of the discussed polymers and their physical properties [31,3544].

tration [2]. PLA and PGA can be processed easily and their degradation rates, physical and mechanical properties are adjustable over a wide range by using various molecular weights and copolymers. However, these polymers undergo a bulk erosion process such that they can cause scaffolds to fail prematurely. In addition, abrupt release of these acidic degradation products can cause a strong inammatory response [47,48]. In general, PGA degrades faster than PLA, as found in Table 1. Their degradation rates decrease in the following order: PGA4PDLLA4PLLA4PCL: Biodegradable polyester degradation occurs by uptake of water followed by the hydrolysis of ester bonds. Different factors affect the degradation kinetics, such as: chemical composition and congurational structure, processing history, molar mass (Mw), polydispersity (Mw/ Mn), environmental conditions, stress and strain, crystallinity, device size, morphology (e.g. porosity) and chain orientation, distribution of chemically reactive compounds within the matrix, additives [49,50], presence of original monomers and overall hydrophilicity. PLGA, for instance, has a wide range of degradation rates, the degradation kinetics being governed by both hydrophobic/hydrophilic balance and crystallinity. Composition of chains (i.e. contents in L-LA and D-LA and/or GA units) determines the degradation rate of PLGA polymers. Blends containing the greatest amount of PGA have been shown to degrade faster [49]. Poly(e-caprolactone) (PCL) on the other hand, can take several years to degrade in vivo [38,51]. Thick samples of these polymers can lead to heterogeneous degradation, faster inside than at the exterior.

2.1. Saturated aliphatic polyesters (PLA, PGA and PCL) The most often utilized biodegradable synthetic polymers for 3D scaffolds in tissue engineering are saturated poly-a-hydroxy esters, including poly(lactic acid) (PLA) and poly(glycolic acid) (PGA), as well as poly(lactic-coglycolide) (PLGA) copolymers [2,31,45,46]. PLA exists in three forms: L-PLA (PLLA), D-PLA (PDLA), and racemic mixture of D,L-PLA (PDLLA). The chemical properties of these polymers allow hydrolytic degradation through de-esterication. Once degraded, the monomeric components of each polymer are removed by natural pathways. The body already contains highly regulated mechanisms for completely removing monomeric components of lactic and glycolic acids. PGA is converted to metabolites or eliminated by other mechanisms, and PLA can be cleared through the tricarboxylic acid cycle. Due to these properties PLA and PGA have been used in products and devices, such as degradable sutures which have been approved by the US Food and Drug Adminis-

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Heterogeneous degradation may be ascribed to two phenomena [46]: (i) easier diffusion of soluble oligomers from the surface into the external medium than from inside, and (ii) neutralization of carboxylic end groups located at the surface by the external buffer solution (in vitro or in vivo). These phenomena contribute to reduce the acidity at the surface whereas, in the bulk, degradation rate is enhanced by autocatalysis due to carboxylic end groups. Hydrolysis of amorphous polymers, e.g. PDLLA, is faster due to the lack of crystalline regions. In general, the amount of absorbed water depends on diffusion coefcients of chain fragments within the polymer matrix, temperature, buffering capacity, pH, ionic strength, additions in the matrix, in the medium and processing history. Different aliphatic polyesters can therefore exhibit quite distinct degradation kinetics in aqueous solutions. PGA, for example, is a stronger acid and is more hydrophilic than PLA, which is hydrophobic due to its methyl groups. The stereochemistry inuences the nal properties; better alignment of neighbors leads to higher crystallinity. In general, the initial degree of crystallinity of polyesters affects the rate of hydrolytic degradation, as the crystal segments are chemically more stable than amorphous segments and reduce water permeation into the matrix. Degradation takes longer with the stereoisomers of the polymer, e.g. PLA composed of L-lactic repeating units takes more than 5 years for total absorption, whereas only about 1 year is needed for amorphous PLA (or PDLLA) [51]. Of particular signicance for applications in tissue engineering are debris and crystalline by-products, as well as particularly acidic degradation products of PLA, PGA, PCL and their copolymers that have been implicated in adverse tissue reactions [18,38]. Several groups have incorporated basic compounds to stabilize the pH of the environment surrounding the polymer and to control its degradation. Bioactive glasses and calcium phosphates have been used [4951]. In fact, the possibility of counteracting the acidic degradation of biodegradable polymers is another reason given for the use of composites [52] as discussed further below. PDLLA has been extensively investigated as a biomedical coating orthopedic material because of its excellent features with respect to implant performance [31,53]. In addition to its high mechanical stability [54], PDLLA also shows excellent biocompatibility in vivo and a good osteoconductive potential [55]. PDLLA of low molecular weight can be combined with drugs like growth factors, antibiotics, or thrombin inhibitor [56] to establish a locally acting drug-delivery system. It is because of these desirable features that much more attention has recently been paid to

PDLLA for applying it as a scaffold material for tissue engineering. PCL is also an important member of the aliphatic polyester family. It has been used to effectively entrap antibiotic drugs and thus a construct made with PCL can be considered as a drug-delivery system, being used to enhance bone ingrowth and regeneration in the treatment of bone defects [57]. The degradation of PCL and its copolymers involves similar mechanisms to that of PLA, proceeding in two stages: random hydrolytic ester cleavage and weight loss through the diffusion of oligometric species from the bulk. It has been found that the degradation of PCL system with a high molecular weight (M n of 50,000) is remarkably slow, requiring 3 years for complete removal from the host body [58]. 2.2. Polypropylene fumarate (PPF) PPF is an unsaturated linear polyester. Like PLA and PGA, the degradation products of PPF (i.e. propylene glycol and fumaric acid) are biocompatible and readily removed from the body. The double bond along the backbone of the polymer permits cross-linking in situ, which causes a moldable composite to harden within 1015 min. Mechanical properties and degradation time of the composite may be controlled by varying the PPF molecular weight. Therefore, preservation of the double bonds and control of molecular weight during PPF synthesis are critical issues [59]. PPF has been suggested for use as a scaffold for guided tissue regeneration, often as part of an injectable bone replacement composite [60]. It also has been used as a substrate for osteoblast cultures [61]. The development of composite materials combining PPF and inorganic particles, e.g. hydroxyapatite (HA) or bioactive glasses, has not been investigated to a large extent, in comparison with the extensive research efforts dedicated to PLGA- and PLA-based composites. 2.3. Polyhydroxyalkanoates (PHB, PHBV, P4HB, PHBHHx, PHO) Polyhydroxyalkanoates (PHA) are aliphatic polyesters as well, but produced by microorganisms under unbalanced growth conditions [62,63]. They are generally biodegradable (via hydrolysis) and thermoprocessable, making them attractive as biomaterials for applications in medical devices and tissue engineering. Over the past years, PHA, particularly poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly3-hydroxyoctanoate (PHO) were demonstrated to be suitable for tissue engineering and are reviewed in detail in Ref. [35]. Dependent on the property requirement by different applications, PHA polymers can be either blended, surface modied or composed with other polymers; enzymes or

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inorganic materials to further adjust their mechanical properties or biocompatibility. The blending among the several PHA themselves can change dramatically the material properties and biocompatibility [35,62]. PHB is of particular interest for bone tissue application as it was demonstrated to produce a consistent favorable bone tissue adaptation response with no evidence of an undesirable chronic inammatory response after implantation periods of up to 12 months. Bone is formed close to the material and subsequently becomes highly organized, with up to 80% of the implant surface lying in direct apposition to new bone. The materials showed no evidence of extensive structural breakdown in vivo during the implantation period of the study [64]. However, a drawback of some PHA polymers is their limited availability and the time-consuming extraction procedure from bacterial cultures that is required for

obtaining sufcient processing amounts as described in the literature [35,65]. Therefore, the extraction process might be a challenge to a cost-effective industrial upscale production for large amounts of some PHA polymers. 2.4. Surface bioeroding polymers There is a family of polymers that undergoes a heterogeneous hydrolysis process which is predominantly conned to the polymerwater interface. This property is referred to as surface eroding as opposed to bulk degrading behavior. Polymers known to show this property are poly(anhydrides), poly(ortho-esters) and polyphosphazene. These surface bioeroding polymers have been intensively investigated as drug-delivery vehicles. The surface-eroding characteristics offer three key advantages over bulk degradation when used as scaffold materials: (1) retention of mechanical integrity over the degradative lifetime of the device, owing to the maintenance of mass to volume ratio, (2) minimal toxic effects (i.e. local acidity), owing to lower solubility and concentration of degradation products, and (3) signicantly enhanced bone ingrowth into the porous scaffolds, owing to the increment in pore size as the erosion proceeds [66]. This group of polymers can also be designed to be bulk degradable by introducing a high surface to bulk ratio to the scaffold. Their properties are summarized in Table 1. 3. Bioactive ceramic phases A common characteristic of bioactive glasses and ceramics is a time-dependent kinetic modication of the surface that occurs upon implantation. The surface forms a biologically active hydroxy carbonate apatite (HCA) layer which provides the bonding interface with tissues. The HCA phase that forms on bioactive implants is chemically and structurally equivalent to the mineral phase in bone, providing interfacial bonding [13,67]. The in vivo formation of an apatite layer on the surface of a bioactive ceramic can be reproduced in a protein-free and acellular simulated body uid (SBF), which is prepared to have an

Fig. 1. SEM micrographs illustrating the typical cauliower morphology of hydroxyapatite formed on the surface of a 45S5 Bioglasss-based foam after immersion in simulated body uid (SBF) for 28 days. The foam was sintered at 1000 1C for 1 h. The magnication of the framed area shown in the inlet picture reveals rod-shaped crystals of hydroxyapatite (adapted from Ref. [69]).

Table 2 Mechanical properties of dense and highly porous hydroxyapatite, 45S5 Bioglasss, A/W glass-ceramic, and human cortical bone Ceramics Compressive strength (MPa) 4400 $500 1080 2.25 0.2-0.4 0.210.41 130180 412 Tensile strength (MPa) $40 42 215 50151 Elastic modulus (GPa) Fracture toughness p (Mpa m) $1.0 0.51 2.0 68 References

Hydroxyapatite (HA) 45S5 Bioglasss Glass-ceramicA/W Porous bioactive glass70S30C (82%) Porous Bioglasss-derived glassceramic (490%) Porous HA (8286%) Cortical bone Cancellous bone

$100 35 118 0.831.6 103 1218 0.10.5

[70,71] [71,72,76] [73] [105] [69] [106] [31,74,75] [107,108]

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ion concentration nearly equal to that of human blood plasma. The composition of SBF can be found in Ref. [68]. As an example, the typical cauliower morphologies of HCA layers formed on a scaffold made of 45S5Bioglasss immersed in SBF are illustrated by SEM micrographs in Fig. 1 (adapted from Ref. [69]). Typical mechanical properties of the bioactive ceramic phases discussed in the following paragraphs can be found in Table 2, compiled from available literature data [31,7076]. 3.1. Bioactive glasses and glass-ceramics In 1969, Hench et al. discovered that certain glass compositions had excellent biocompatibility as well as the ability of bone bonding [77]. Through interfacial and cellmediated reactions, bioactive glass develops a calciumdecient, carbonated phosphate surface layer that allows it to chemically bond to host bone. This bone-bonding behavior is referred to as bioactivity and has been associated with the formation of a carbonated hydroxyapatite (HCA) layer on the glass surface when implanted or in contact with biological uids [13,71,7680] (see also Fig. 1). The stages that are involved in forming the bone bond of bioactive glasses and bioactive glass-ceramics were summarized by Hench [13,67]. Although some details remain unknown, it is clearly recognized that for a bond with bone tissue to occur, a layer of biologically active HCA must form. This conclusion is based on the nding that HCA is the only common characteristic of all the known bioactive implant materials [81]. Bioactivity, however, is not an exclusive property of bioactive glasses. HA and related calcium phosphates also show an excellent ability to bond to bone, as discussed further below. The capability of a material to form a biological interface with surrounding tissue is critical in elimination of scaffold loosening. Of great importance and impact for applications in tissue engineering is that bioactive glasses have also been found to support enzyme activity [8285]; vascularization [86,87]; foster osteoblast adhesion, growth, differentiation; and induce the differentiation of mesenchymal cells into osteoblasts [26,8890]. A signicant nding for the development of bone engineering is that the dissolution products from bioactive glasses, in particular the 45S5 Bioglasss composition, upregulate the gene expression that control osteogenesis and the production of growth factors [91]. Silicon has been found to play a key role in the bone mineralization and gene activation, which has led to an increased interest in the substitution of silicon for calcium into synthetic HA. Investigations in vivo have shown that bone ingrowth into silicon-substituted HA granules was remarkably greater than that into pure HA [92]. The above-mentioned advantages are the reasons why 45S5 Bioglasss is successfully used in clinical treatments of periodontal disease (PerioglasTM) and as a bone ller material (NovaboneTM) [13]. Bioglasss implants have also

been used to replace damaged middle ear bones, restoring hearing to patients [79]. Bioactive glasses have gained new attention recently as promising scaffold materials, either as ller or coatings of polymer structures, or as porous materials themselves, which involves melt-derived and solgel-derived glasses [52,69,9399]. 3.1.1. Composition and bioactivity The basic constituents of the most bioactive glasses are SiO2, Na2O, CaO, and P2O5. 45S5 Bioglasss contains 45% SiO2, 24.5% Na2O, 24.4% CaO and 6% P2O5, in weight percent. An overview of different bioactive glass compositions and their corresponding bioactivities are given in Ref. [67]. Hench and coworkers have systematically studied a series of glasses in the four-component systems with a constant 6 wt% P2O5 content, as summarized in Refs. [67,81] and they divided the compositions into three regions according to their bioactivity. Bioactive glasses (e.g. 45S5 Bioglasss) with compositions in the system SiO2Na2OCaOP2O5, having o55% SiO2, exhibit a high bioactivity index (in region A), and bond to both soft and hard connective tissues. The bioactive glasses (i.e. glasses in region A) are osteoproductive (bone grows on material surfaces due to enhanced osteoblast activity) and osteoconductive. Glasses of compositions in region B exhibit only osteoconductivity. Compositions in region C are resorbed within 1030 days in tissue [13]. It has been found that reactions on bioactive glass surfaces can release critical concentrations of soluble Si, Ca, P and Na ions, depending on the processing route and particle size. The released ions induce intracellular and extracellular responses [91,100]. For example, a synchronized sequence of genes is activated in osteoblasts that undergo cell division and synthesize an extracellular matrix, which mineralizes to become bone. In addition, bioactive glass compositions doped with AgO2 have been shown to elicit anti-bactericidal properties while maintaining their bioactive function [101]. In recent investigations, 45S5 Bioglasss has also been shown to increase secretion of vascular endothelial growth factor (VEGF) in vitro and to enhance vascularization in vivo, suggesting scaffolds containing controlled concentrations of Bioglasss might stimulate neo-vascularization which is benecial to large tissue engineered constructs [86]. One key reason that makes bioactive glasses a relevant scaffold material is the possibility of controlling a range of chemical properties and thus the rate of bioresorption. The structure and chemistry of glasses, in particular solgelderived glasses [78,79], can be tailored at a molecular level by varying either composition, or thermal or environmental processing history. It is possible to design glasses with degradation properties specic to a particular application of bone tissue engineering. However, it was reported that crystallization of bioactive glasses decreased the level of bioactivity [102] and even turned a bioactive glass into an inert material [103]. This is

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a disadvantage that limits the application of bioactive glasses as scaffold materials, as full crystallization happens prior to densication by viscous ow sintering [104]. Extensive sintering however is necessary to densify the struts of a scaffold to achieve the required mechanical stability. 3.1.2. Mechanical properties A drawback of bioactive glasses is their low fracture toughness and mechanical strength, especially in a porous form. Hence, bioactive glasses alone have limited application in load-bearing situations. Table 2 gives compressive strength, elastic modulus and some fracture toughness values for dense and porous bioactive ceramics, compiled from Refs. [31,6975,105108]. It can be seen that particularly porous scaffolds needed for tissue engineering exhibit very low mechanical properties compared to cortical and cancellous bone. Bioactive glass-ceramic materials can exhibit better mechanical properties than amorphous glass and calcium phosphate ceramics (Table 2). The high bending strength of apatite-wollastonite (A/W) glass-ceramic is due to the precipitation of the wollastonite as well as apatite phases and attributed to the high fracture toughness of the precipitation microstructure [109]. 3.2. Calcium phosphates 3.2.1. Composition and bioactivity Around 60 wt% of bone is made of HA Ca10 (PO4)6(OH)2 and therefore it is evident why HA and related calcium phosphates (e. g. a-TCP, b-TCP) have been intensively investigated as the major component of scaffold materials for bone tissue engineering [76,110112]. As expected, calcium phosphates have an excellent biocompatibility due to their close chemical and crystal resemblance to bone mineral [113]. Although they have not shown osteoinductive ability, calcium phosphates certainly possess osteoconductive properties and may bind directly to bone under certain conditions [114116]. Numerous in vivo and in vitro assessments have reported that calcium phosphates, no matter of which form (bulk, coating, powder, or porous) and of which phase (crystalline or amorphous), always support the attachment, differentiation, and proliferation of relevant cells (such as osteoblasts and mesenchymal cells), with HA being possibly the most efcient among them [117]. While the excellent biological performance of HA and related crystalline calcium phosphates has been well documented, their relatively slow biodegradation and in particular low mechanical strength limit their application in engineering of new bone tissue, especially at load-bearing sites. Crystalline calcium phosphates have long degradation times in vivo, typically in the order of months or even years. The dissolution rate of synthetic HA depends on the type and concentration of the buffered or unbuffered solutions, pH of the solution, degree of the saturation of

the solution, solid/solution ratio, and the composition and crystallinity of the HA phase. In the case of crystalline HA, the degree of micro- and macro-porosities, defect structure and amount and type of other phases present have also a signicant inuence [116]. Crystalline HA exhibits the slowest degradation rate, compared with other calcium phosphates. The dissolution rate decreases in the following order [118]: Amorphous HAba TCPbb TCPb crystalline HA: 3.2.2. Mechanical properties In the body, the mechanical properties of natural bone change with their biological location because the crystallinity, porosity, and composition of bone adjust to the biological and biomechanical environment. The properties of synthetic calcium phosphates vary signicantly with their crystallinity, grain size, porosity, and composition (e.g. calcium deciency) as well. In general, the mechanical properties of synthetic calcium phosphates decrease signicantly with increasing amorphous phase, microporosity and grain size. High crystallinity, low porosity and small grain size tend to give higher stiffness, compressive and tensile strengths, and greater fracture toughness. It has been reported that the exural strength and fracture toughness of dense HA are much lower in a dry condition than in a wet condition [119]. If we compare the properties of HA and related calcium phosphates with those of cortical bone (Table 2), we nd that bone has a reasonably good compressive strength though it is lower than that of HA. But bone has a signicantly higher fracture toughness than HA. The mechanical properties are even lower for porous HA structures. The high tensile strength and fracture toughness of bone are attributed to the tough and exible collagen bers reinforced by HA crystals. Hence, calcium phosphates alone cannot be used for load-bearing scaffolds despite their good biocompatibility and osteoconductivity. 3.3. Other bioactive ceramics Representative bioactive ceramics are, as mentioned, Bioglasss, HA and glass-ceramics containing HA or its components, such as CaO and P2O5. However, formation of HA does not seem to be limited to those ceramics only. As Kokubo et al. [68] reported, other materials can show degrees of bioactivity after a simple chemical heat treatment as well. Chemical treatment of metals and ceramics, e.g. by NaOH and heat treatments of titanium metal, titanium alloys, and tantalum metal, and by H3PO4 treatment of tetragonal zirconia, resulted in functional graded surfaces that induced HA formation on the surface in SBF studies. In vivo studies of NaOH and heat-treated titanium metals implanted in a rabbit femur showed detaching fracture loads of up to 4 times higher than the untreated implants [68].

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It has been suggested that the TiOH, ZrOH, NbOH, and TaOH surface groups in anatase or the tetragonal/ monoclinic structures may provide effective epitaxial nucleation sites for apatite crystals and that a negative surface charge increases Ca2+ adsorption from the solution. In addition, the chemical treatment induces the incorporation of ions (e.g. Na+) on the metal oxide surface and transforms the crystal phase to an amorphous phase resulting in an increased ion solubility [120124]. However, the suggested models have not been investigated in detail yet and more studies will be required to elucidate the mechanisms. 4. Material processing strategies for composite scaffolds with interconnected pores Development of composite scaffold materials is attractive as advantageous properties of two or more types of materials can be combined to suit better the mechanical and physiological demands of the host tissue. By taking advantage of the formability of polymers and including controlled-volume fractions of a bioactive ceramic phase, mechanical reinforcement of the fabricated scaffold can be achieved [52,125]. At the same time, the poor bioactivity of most polymers can be counteracted. Probably the most important driving force behind the development of polymer/bioactive glass composite scaffolds for bone tissue engineering is the need for conferring bioactive behavior to the polymer matrix, which is achieved by the bioactive inclusions or coatings. The degree of bioactivity is adjustable by the volume fraction, size, shape and arrangement of inclusions [24,52,126137]. It has been shown that increased volume fraction and higher surface area to volume ratio of inclusions favor

higher bioactivity, hence in some applications the incorporation of bers instead of particles is favored [27,138]. Addition of bioactive phases to bioresorbable polymers can also alter the polymer degradation behavior, by allowing rapid exchange of protons in water for alkali in the glass or ceramic. This mechanism is suggested to provide a pH buffering effect at the polymer surface, modifying the acidic polymer degradation [24,52,139]. Inclusion of bioactive glasses has been shown to modify surface and bulk properties of composite scaffolds by increasing the hydrophilicity and water absorption of the hydrophobic polymer matrix, thus altering the scaffold degradation kinetics. In particular, the inclusion of 45S5 Bioglasss particles was found to increase water absorption compared to pure polymer foams of PDLLA [133] and PLGA [24,52]. In related research, it has been reported that polymer composites lled with HA particles hydrolyzed homogeneously due to water penetrating the interfacial regions [140]. Ideally, the degradation and resorption kinetics of composite scaffolds are designed to allow cells to proliferate and secrete their own extracellular matrix while the scaffolds gradually vanish, leaving space for new cell and tissue growth. The physical support provided by the 3D scaffold should be maintained until the engineered tissue has sufcient mechanical integrity to support itself. There are numerous foaming techniques including solgel routes to obtain highly porous structures [141,142]. However, only relevant fabrication techniques leading to 3D composite scaffolds with highly interconnected pores are discussed in the following paragraphs and compared in Table 3, compiled with data available from the literature [133,135,143148]. A selection of dense and porous scaffold composites including their physical properties is given in

Table 3 Fabrication routes for 3D composite scaffolds with high pore interconnectivity and their advantages and disadvantages Fabrication route Thermally induced phase separation (TIPS) [133,143] Advantages High porosities ($95%) Highly interconnected pore structures Anisotropic and tubular pores possible Control of structure and pore size by varying preparation conditions Controlled porosity Controlled interconnectivity (if particles are sintered) Porous structure can be tailored to host tissue Protein and cell encapsulation possible Good interface with medical imaging Graded porosity structures possible Controlled porosity Can be fabricated into complex shapes Quick and easy Disadvantages Long time to sublime solvent (48 hours) Shrinkage issues Small scale production Use of organic solvents Structures generally isotropic Use of organic solvents Resolution needs to be improved to the micro-scale Some methods use organic solvents Interconnectivity is an issue Use of organic solvents Clogging of pores, sometimes organic solvents used, coating adhesion to substrate can be too weak

Solvent casting/particle leaching [144,145]

Solid free-form [146,147]

Microsphere sintering [148]

Scaffold coating [135]

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K. Rezwan et al. / Biomaterials 27 (2006) 34133431 Table 4 Selection of scaffold composites designed for bone tissue engineering and their properties Scaffold composite Percentage of ceramic (%) Polymer 1.752.47 103 6.412.8 103 1.1 103 5.18 103 191134 0.95.7 103 1218 103 Porosity (%) Pore size (mm) Compressive (C), tensile (T), exural (F) strength (MPa) Modulus (MPa) Reference 3421

Ceramic 1. Dense composites HA bre HA b-TCP A/W Cortical bone 2. Porous composites Amorphous CaP HA

PDLLA PLLA PLGA PLLA-co-PEH PPF PE

210.5 (vol.) 1070 (wt.) 4085 (vol.) 75 (wt.) 25 (wt.) 1050 (vol.)

45 (F) 5060 (F) 22 (F) 51 (F) 7.57.7 (C) 1828 (B) 50150(T) 130180 (C)

[149] [150] [29,151] [152,153] [154] [31,74,75]

Bioglasss

PLGA PLLA PLGA PLGA PLGA PLLA PLGA PDLLA

2875 (wt.) 50 (wt.) 6075 (wt.) 75 (wt.) 2050 (wt.) 0.11 (wt.) 529 (wt.) 40 (wt.) 2040 (wt.)

75 8596 8191 3040 43 7780

94 9397 85.595.2

4100 100 300 8001800 110150 89 $100 (macro) $10 (micro) 50300 $100 (macro) 1050 (micro) 98154

0.39 (C) 0.070.22 (C) 0.42 (C) 1.53.9 (T)

65 1014 27.5 3371459 51 137260

[28,155] [127] [156] [157] [93,148,158] [17] [15,148] [132,134,136]

0.070.08

0.651.2

Phosphate glass A/W Cancellous Bone

PLA-PDLLA PDLLA

0.0170.020 (C) 412 (C)

0.0750.12 100500

[159,25] [107,108]

Table 4, which represent typical systems reported in the literature [15,17,25,28,29,31,74,75,93,107,108,127,132, 134,136,148159]. For comparison, the mechanical properties of human cortical and cancellous bone are listed as well. Representative morphologies of the fabricated scaffolds are illustrated in Fig. 2, taken from Refs. [147,148,160162]. 4.1. Thermally induced phase separation (TIPS) 3D resorbable polymer scaffolds with very high porosities ($97%) can be produced using the TIPS technique to give controlled macro- and microstructures suitable as scaffolds for tissues such as nerve, muscle, tendon, ligament, intestine, bone, and teeth [52,143,163]. The obtained scaffolds are highly porous with anisotropic tubular morphology and extensive pore interconnectivity. Microporosity of TIPS produced foams, their pore morphology, mechanical properties, bioactivity and degradation rates can be controlled by varying the polymer concentration in solution, volume fraction of the secondary phase, quenching temperature and the polymer and solvent used as discussed in a previous review paper [52]. Briey, the polymer is dissolved in dimethylcarbonate and stirred overnight to obtain a homogeneous polymer solution. A given amount of glass or ceramic powder can be added into the polymer solution. The mixture is

transferred into a ask and sonicated. Hereafter, the ask is quenched in liquid nitrogen and maintained at 196 1C for 2 h. The frozen mixture is then transferred into a cooling bath at 10 1C and connected to a vacuum pump. The solvent is sublimated at 10 1C for 48 h and then at 0 1C for 48 h, followed by drying at room temperature in a vacuum oven until reaching a constant weight [24]. Maquet et al. [24,133] developed highly porous PDLLA/ Bioglasss composite scaffolds prepared by TIPS with bimodal and anisotropic pore structures composed of tubular macropores of $100 mm, interconnected with micropores of 1050 mm in diameter, as shown in Fig. 2a. The pore volume was shown to decrease from 9.5 to 5.7 cm3/g after including 40 wt% Bioglasss, with little change observed in the overall pore morphology [164]. In vitro studies in phosphate-buffered saline at 37 1C showed that addition of Bioglasss increased water absorption and weight loss in comparison to pure polymer foams [24,133]. The molecular weight was found to decrease less within the composite foams, possibly due to the dissolution of alkaline ions from the Bioglasss providing a pH buffering effect, as discussed above. Both the PDLLA/Bioglasss composites and the neat PDLLA foams retained their structural integrity until the end of the experiment (16 weeks), which means degradation was still in the early stages.

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Fig. 2. Typical morphologies of porous polymer foams produced by different techniques and structure of cancellous bone. (a) Thermal induced phase separation (TIPS, adapted from Ref. [160]), (b) solvent casting and particle leaching [161], (c) solid freeform fabrication technique [147], (d) microspheresintering [148], (e) cancellous bone [162]. (Micrographs (b), (c) and (e) reprinted with permission of Elsevier Ltd. Micrograph (d) reprinted with permission of John Wiley & Sons, Inc.)

It is well documented [52,163,165] that due to autocatalysis non-porous biodegradable polylactides undergo degradation more rapidly than the porous counterparts. The reason for this effect lies in the fact that porous materials are able to facilitate dissolving and spreading of degradation products throughout the aqueous medium, thereby preventing autocatalysis. PDLLA/Bioglasss composites exhibit high bioactivity, assessed by the formation of HA on the composite surfaces upon immersion in SBF [133,134]. It has also been shown that the foams support the migration, adhesion, spreading and viability of MG-63 cells (osteosarcoma cell line) [134]. The potential of these scaffolds in bone and soft-tissue engineering has been demonstrated in vitro with optimized concentrations of 45S5 Bioglasss added to PDLLA or

PLGA matrices [134,136]. Highly porous tubular scaffolds with oriented porosity have also been fabricated by exploiting the TIPS process [15,166]. These are candidate materials for soft-tissue engineering with potential application in the regeneration of tissues requiring tubular shapes such as the intestine, trachea and blood vessels. TIPS fabricated PDLLA foams with and without Bioglasss additions have been shown to exhibit mechanical anisotropy concomitant with the TIPS-induced pore architecture [132]. Polymer matrix composite lms containing nanosized titania and other inorganic particulate inclusions have demonstrated enhanced cell adhesion and a tendency to increased Ca-containing mineral deposition [167]. Recently, 3D PDLLA foams containing both TiO2

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nanoparticles and Bioglasss additions have been synthesized by TIPS. These foams demonstrate enhanced bioactivity and surface nanotopography [131]. 4.2. Solvent casting and particle leaching Solvent casting of biocomposite scaffolds involves the dissolution of the polymer in an organic solvent, mixing with ceramic granules, and casting the solution into a predened 3D mould. The solvent is subsequently allowed to evaporate. The main advantage of this processing technique is the ease of fabrication without the need of specialized equipment. The primary disadvantages of solvent casting are: (1) the limitation in the shapes (typically at sheets and tubes are the only shapes that can be formed); (2) the possible retention of toxic solvent within the polymer; and (3) the denaturation of the proteins and other molecules incorporated into the polymer by the use of solvents. The use of organic solvents to cast the polymer may decrease the activity of bioinductive molecules (e.g. protein). The detailed processing steps have been described in the literature [93]. Polymer-ceramic constructs can also be fabricated by the solvent aggregation method. The polymer microspheres are rst formed from traditional water oil/water emulsions. Solvent-aggregated polymer-ceramic scaffolds can then be constructed by mixing solvent, salt or sugar particles, ceramic granules, and pre-hardened microspheres [131]. A 3D structure of controlled porosity is formed based on this method combined with particle leaching and microsphere packing. Fig. 2b illustrates a typical pore morphology obtained by this technique. The method shares similar advantages and disadvantages with the solvent casting technique [93]. There has been little work done on producing bioactive polymer-ceramic scaffolds using particle leaching. Certainly, a drawback of this technique is achieving pore interconnectivity at low porogen (salt/sucrose) loadings, as many of the porogen particles may remain trapped. Nevertheless, composites based on calcium phosphate inclusions with variable and graded porosity have been produced using this route [168]. 4.3. Solid freeform fabrication techniques (SFFT) SFFT, such as fused deposition modeling, have been employed to fabricate highly reproducible scaffolds with fully interconnected porous networks [147,169] as shown in Fig. 2c. Using digital data produced by an imaging source such as computer tomography or magnetic resonance imaging enables accurate design of the scaffold structure [169]. Solid freeform (SFF) manufacturing coupled with conventional foam scaffold fabrication procedures (phase separation, emulsion-solvent diffusion or porogen leaching) may be used to develop scaffolds with controlled micro- and macroporous structures. Such biomimetic internal architectures may prove valuable for multi-tissue and structural tissue interface engineering.

To the authors knowledge, there is no literature available on degradable polymer/bioactive glass composites made by SFFT. This technique has been only applied for composites containing calcium phosphates as the bioactive phase [147,170]. For example, Xiong et al. [170] fabricated composites of PLLA/TCP with porosities of up to 90% and mechanical properties close to human cancellous bone by using low-temperature deposition based on a layer-by-layer manufacturing method of SFF fabrication (computer-driven by 3D digital models). PLLA was dissolved in dioxane and TCP powder mixed to prepare a slurry, which was formed into frozen scaffolds, and subsequently freeze-dried. Alternate parallel layers formed macropores (400 mm diameter) and sublimation of the solvent during freeze-drying formed micropores (5 mm diameter). Taboas et al. [147] produced PLA scaffolds with computationally designed pores (500800 mm wide channels) and solvent-derived local pores (50100 mm wide voids or 510 mm length plates). Indirect fabrication using casting in SFF moulds provided enhanced control over scaffold shape, porosity and pore architecture, including size, geometry, orientation, branching and interconnectivity. A shortcoming of this route is increased scaffold fabrication time compared with direct methods, as a temporary mould must be made rst.

4.4. Microsphere sintering In this process, microspheres of a ceramic and polymer composite are synthesized rst, using emulsion/solvent evaporation technique. Sintering the composite microspheres yields a 3D, porous scaffold [28,155]. 3D composites of degradable polymers and bioactive glass have been produced by sintering composite microspheres by Lu et al. [148]. Starting materials were PLAGA-Bioglasss composite microspheres obtained through a wateroilwater emulsion technique. Sintering of the microspheres into cylindrical shapes resulted in a well-integrated interconnected porous structure, with the microspheres joined at the contact necks. Average porosity was 40% with pore diameters of 90 mm, and mechanical properties close to cancellous bone. The composites were shown to be bioactive as a calcium phosphate layer formed on the surface of the composite on immersion in SBF for 7 days. Moreover, Bioglasss reinforcement gave a two-fold increase in compressive strength. The scaffolds were shown to support the adhesion, growth and mineralization of human osteoblast-like cells in vitro. Over a 3-week period, cultures with PLAGA/Bioglasss maintained pH variations within physiological ranges. More recently, Yao et al. [19] synthesized PLGA/bioactive glass microspheres by emulsication and heated them in moulds to fabricate porous 3D scaffolds. They demonstrated the bioactivity of the composites and their ability to promote osteogenesis of marrow stromal cells. A typical microsphere-sintered 3D structure fabricated by Lu et al. [148] is given by Fig. 2d.

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4.5. Coated scaffolds Bioceramic-coated porous scaffolds have been produced either as foams [135], brous bodies [171] or meshes [86,158] by slurry dipping or electrophoretic deposition (EPD). Roether et al. [135] were the rst to develop composites of macroporous PDLLA foams coated with Bioglasss particles (grade 45S5 with particle size o5 mm) by slurry dipping. A stable and homogeneous coating on the foam surface and inltration of Bioglasss particles throughout the porous network were achieved. A stable slurry of 42 wt% Bioglasss in deionized water gave relatively dense and uniform adherent coatings. EPD was investigated as an alternative route whereby charged Bioglasss particles in aqueous suspension inltrated the foam with its tubular macropores oriented perpendicularly to the larger dimension of the electrodes [135]. The slurry dipping technique was found to be more suitable than the EPD route; the latter caused frequently sealing of the interconnected pores by Bioglasss particles. EPD, however, is an attractive method to incorporate nanoparticles into porous structures with potential use as tissue engineering scaffolds [172]. Composites tested in vitro in acellular SBF exhibited increasing formation of HA (layers of 10 mm were formed after 28 days). In addition, changes in pore morphology as a result of polymer degradation with increasing immersion time were observed. The investigation of the in vitro behavior of osteoblast-like cells demonstrated that cells were able to migrate through the porous network and colonize the inner sections of the foams. Also, after 24 h a higher cell density was observed in the Bioglasss coated foams compared to the pure PDLLA foams [90]. A related approach, but using PHA woven meshes as substrate, was followed by Olsen et al. [173]. In this case, a 45S5 Bioglasss aqueous slurry was used to deposit micrometer-sized Bioglasss particles on the surfaces of PHA bers. The process was optimized to coat individual bers maintaining the pore structure of the woven mesh. There are however no published results on the in vivo behavior of these Bioglasss-PHA composites. Another promising method to coat polymer surfaces with bone like calcium phosphates is using a biomimetic approach as demonstrated in the literature [174176]. Moreover, sodium silicate gel [174] has been used to nucleate CaP coatings on polymer surfaces that were immersed in SBF. A review on biomimetic formation of calcium phosphate coatings has been published recently [177]. 5. Challenges and opportunities 5.1. Mechanical integrity of porous scaffolds Comparison of the mechanical properties of todays available porous scaffolds with relevant properties of bone reveals the insufcient mechanical integrity of the manmade scaffolds. In Fig. 3 the elastic modulus and the compressive strength of dense bioactive ceramic, biode-

Fig. 3. Elastic modulus vs. compressive strength of biodegradable polymers, bioactive ceramics and composites reviewed in this paper. Porosities of the porous scaffolds are 475% and mostly interconnected.

gradable polymers, cancellous and cortical bone are compared with porous monophasic scaffolds and composites thereof. Mechanical data for porous bioactive ceramics and for polymer foams were taken from Refs. [105,106,132]. It can be seen that some dense polymers match cancellous bone properties and approach cortical bone properties. Moreover, the bioactive ceramics region is close to the properties of cortical bone as well. Porous scaffolds however are at least one order of magnitude weaker than cancellous bone and orders of magnitude weaker than cortical bone. Interestingly, the stiffness achieved by up-to-date fabricated porous bioactive ceramics is less than the stiffness of most porous biodegradable polymer scaffolds. By comparing the mechanical properties of the porous composites to those of porous polymer scaffolds, a slight increase of mechanical properties is revealed. But the increase in stiffness and strength is certainly below expectations; most probably this can be attributed to the lack of interfacial bonding strength between the ceramic phase and the polymer matrix, which has been neglected in most studies. The ceramic phases are in general very hydrophilic whereas the polymers are hydrophobic. The increase of bonding at the interface is a challenge and might be achieved by using surfactants chemisorbed on the particle surface prior to composite processing. Using surface functionalized particles in the nanosize rangefeaturing a higher specic surface and thus a higher interface areamight even increase the interfacial bonding strength, and thus the overall mechanical properties of the composite scaffold could be effectively enhanced. However, the increase of interfacial bonding and introduction of surfactants are likely to have an impact on degradation kinetics and cytotoxicity of the composite. These effects are largely unknown and remain to be investigated. 5.2. Incorporation of biomolecules There is a signicant scope in the application of surface modications, through the use of protein adsorption or

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plasma treatment, to provide more cues to cell attachment and response [3,178]. The possibility of incorporating growth factors into composites formed by biodegradable polymers and bioactive glasses or HA inclusions has started to be explored [179182]. Integrins, laminin and RGD proteins were shown to be essential for cell attachment to materials surfaces [183186]. The immobilization of these proteins should not only promote cell adhesion and proliferation but also increase wettability of hydrophobic polymers such as PDLLA. To control protein adhesion and release kinetics, different protein immobilization routes can be used as demonstrated for polymer surfaces [187] and ceramic surfaces [188,189]. Certain growth factors were shown in in vivo studies to be osteoinductive. Possible growth factors include bone morphogenetic proteins, transforming growth factor beta, VEGF, and insulin-like growth factor as reviewed in Refs. [190192]. Immobilizing these growth factors on the scaffold surface might signicantly shorten the bone healing process and reduce patient recovery time. The incorporation of biomolecules does not allow extreme temperature ranges (470 1C) or extremely aggressive chemical conditions during processing, being a challenge to the scaffold fabrication process. Soft material routes like solgel processing might be a strategy to incorporate biomolecules during scaffold fabrication. To the authors knowledge, however, solgel-derived bioactive organic/inorganic hybrids have not yet been formed into highly interconnected porous structures, which is essential for application of these composites as scaffolds. Another related challenge is the elucidation of the local impact of growth factors on the cell and tissue systems, including long-term effects.

mandatory in order to be able to comprehensively assess the degradation of these systems with respect to pore structure, scaffolds geometry, uid ow and the inuence of the bioactive additions. Here, the use of X-ray microtomography as a reliable tool for 3D pore structure quantication is likely to gain increased impetus allowing resolutions down to 1 mm [193]. Combining image analysis and impedance spectroscopy is another possible approach to characterize pore interconnectivities of scaffolds as shown recently, being less straightforward however [164]. 5.4. In vitro and in vivo characterization While a good number of in vitro and in vivo studies exist for biodegradable polymers and bioactive ceramics alone, in vitro studies for polymer/ceramic composites have just started [15,19,86,134,136,137,148,194,195]. Table 5 gives examples of the types of composite scaffolds investigated in vitro and the applied cell cultures. Very few composite systems have been investigated in vivo up to date. More research needs also to be directed at assessing the suitability of the reviewed bioactive composite scaffolds in soft-tissue engineering strategies, including further investigations of the effect of dissolution products from the bioactive phase on vascularization and in vivo new tissue growth. 6. Summary of current status and future trends 6.1. Summary The synthetic and biodegradable, polymer/inorganic bioactive phase composites reviewed in this article are particularly attractive as tissue engineering scaffolds due to their shapability, bioactive behavior and adjustable biodegradation kinetics. Conventional materials processing methods have been adapted and extended for incorporation of inorganic bioactive phases into porous and interconnected 3D polymer networks. From the materials science perspective, the present challenge in tissue engineering is to design and fabricate reproducible bioactive and bioresorbable 3D scaffolds of tailored porosity and pore structure, which are able to maintain their structure and integrity for predictable times, even under load-bearing conditions. As reviewed here, the

5.3. Long-term characterization of porous composite scaffolds There is a lack of current understanding in the literature regarding the long-term in vitro and in vivo characterization of the porous 3D scaffold composites discussed here, specically regarding the long-term effect of the incorporation of inorganic bioactive phases on the degradation and ion release kinetics of these highly porous systems. In this regard, the development of appropriate characterization techniques coupled with predictive analytical models is

Table 5 Overview of in vitro investigated biodegradable polymer/inorganic phase composites available in the literature Composite Cell culture PLGA/45S5 Bioglasss Mouse broblasts (L929) [15], marrow stromal cells [19], mouse broblasts (208F) [86], human osteosarcoma cells (SaOS-2) [148] PDLLA/45S5 Bioglasss Mouse broblasts (208F) [137], human osteosarcoma cell line (MG-63) [134], human lung carcinoma (A549) [134,136] PLGA/HA Rat calvarial osteoblasts [194] PCL/HA Human osteosarcoma cells (SaOS-2), osteoblasts from human trabecular bone [195]

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mechanical integrity of man-made composite scaffolds is still at least one order of magnitude lower than that of cancellous or cortical bone. Achieving the mechanical properties of bone might also allow replacing bigger parts of damaged bone tissue than what is possible today. The incorporation of biomolecules such as growth factors with the aim to accelerate local bone healing is promising and currently under extensive research. Incorporating biomolecules during scaffold processing however is not simple as biomolecules are sensitive to elevated temperatures and extreme chemical conditions. A promising strategy is the immobilization of proteins and growth factors in the post-processing phase via surface functionalization of the scaffold. Hardly any in vitro and particularly in vivo studies exist for the composite scaffolds reviewed in this article. However, in order to target clinical applications, in vitro and in vivo studies are inevitable and the need for more studies in biological systems is imperative. 6.2. Future trend: stem cells and ideal biomaterials The application of biomaterials in hard tissue repair started with bioinert approaches, which involved the development and application of bioinert materials. These materials are applied in most permanent bioimplants in todays clinical use such as, for example, hipjoint replacements. The subsequent development of biomaterials focused on bone-bonding properties of bioactive glasses and ceramics [81]. This period was soon followed by the development of biodegradable materials for bone tissue engineering scaffolds that can stimulate specic cellular responses at the molecular level [23]. The composite scaffolds reviewed in this article combine the features of the biomaterials in the second and third periods: they possess bioactivity, degradability and the possibility of biomolecule incorporation. Over the years, the developed biomaterials have addressed biological aspects of increased complexity, starting on the level of ion interactions and moving then to growth factor incorporation. The biomaterials were extended from purely synthetic materials to material/biologic hybrids, engineering at the same time bioactivity and biodegradability. While current research is still focused on the interaction between stromal cells and biomaterials, the fundaments for biomaterials seem to originate from introducing stem cells. Scaffolds seeded with stem cells allow local cell function adaptation by differentiation of stem cells as demonstrated by Levenberg et al. in 2003 [8,196]. This new approach enables the scaffold surface to mimic complex local biological functions and may lead in near future to in vitro and in vivo growth of tissues and organs. The interface of stem cells and scaffolds are at the moment in the center of attention, issuing growth factor incorporation and cell adhesion [8]. In this approach, we anticipate that engineered composite scaffolds made by biodegradable polymer matrices with bioactive inorganic phases, as

reviewed here, will play a vital role and perhaps they will be the scaffolds of choice in combination with stem cell seeding. Acknowledgment Stimulating discussions with Prof. L.L. Hench, Prof. J. Polak and Dr. A. Bishop (TERM-Centre, Imperial College London) and with Dr. F. Filser (ETH Zurich) are acknowledged. References
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