TCP

Materials Science and Engineering C 71 (2017) 1293–1312

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Review

Biphasic calcium phosphates bioceramics (HA/TCP): Concept, physicochemical properties and the impact of standardization of study protocols in biomaterials research Mehdi Ebrahimi a,⁎, Michael G. Botelho a, Sergey V. Dorozhkin b a b

Oral Rehabilitation, Faculty of Dentistry, The University of Hong Kong, Hong Kong Kudrinskaja sq. 1–155, Moscow 123242, Russia

a r t i c l e

i n f o

Article history: Received 9 October 2016 Received in revised form 6 November 2016 Accepted 10 November 2016 Available online 12 November 2016 Keywords: Biphasic calcium phosphates Bioceramics Bone tissue engineering Bone substitutes Pre-clinical study Study protocol

a b s t r a c t Biphasic calcium phosphates (BCP) bioceramics have become the materials of choice in various orthopedic and maxillofacial bone repair procedures. One of their main advantages is their biodegradation rate that can be modified by changing the proportional ratio of the composition phases. For enhanced bone tissue regeneration, the bioactivity of BCP should be increased by optimizing their physicochemical properties. To date, the ideal physicochemical properties of BCP for bone applications have not been defined. This is mostly related to lack of standard study protocols in biomaterial science especially with regards to their characterizations and clinical applications. In this paper we provided a review on BCP and their physicochemical properties relevant to clinical applications. In addition, we summarized the available literature on their use in animal models and evaluated the influences of different composition ratios on bone healing. Controversies in literature with regards to ideal composition ratio of BCP have also been discussed in detail. We illustrated the discrepancies in study protocols among researchers in animal studies and emphasized the need to develop and follow a set of generally accepted standardized guidelines. Finally; we provided general recommendations for future pre-clinical studies that allow better standardization of study protocols. This will allow better comparison and contrast of newly developed bone substitute biomaterials that help further progress in the field of biomaterial science. © 2016 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Biphasic calcium phosphate . . . . . . . . . . . . . . . . . . . . . Review of literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Search strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Inclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . BCP bioceramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Concept and history . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. BCP synthesis and preparation . . . . . . . . . . . . . . . . . . . . 3.3. Physicochemical properties and characterization . . . . . . . . . . . . 3.4. The optimal composition ratio . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The controversies on composition ratio of BCP . . . . . . . . . . . . . 4.2. Recommendations for characterization and documentation of biomaterials 4.3. Rationale and duration of pre-clinical animal study . . . . . . . . . . . 4.4. Sample size calculation; importance and impact . . . . . . . . . . . . 4.5. Critical size defect; a clarification . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Oral Rehabilitation, Faculty of Dentistry, Prince Philip Dental Hospital, The University of Hong Kong, 34 Hospital Road, Hong Kong. E-mail address: [email protected] (M. Ebrahimi).

http://dx.doi.org/10.1016/j.msec.2016.11.039 0928-4931/© 2016 Elsevier B.V. All rights reserved.

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M. Ebrahimi et al. / Materials Science and Engineering C 71 (2017) 1293–1312

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Background The application of biomedical materials in reconstructive surgery for repair of surgical or traumatic defects has involved significant research in the field of biomaterial science with the aim to achieve faster and better biological healing outcomes. Currently, autograft and allograft remain the gold standard for bone replacement therapy. However; disadvantages such as limited supply of donor bone graft and a secondary trauma for autograft as well as the issue of immune response of the allograft challenge their clinical applications. This has stimulated interest in the development of synthetic materials for bone replacement and more recently novel biomaterials with similar properties to native bone [1]. A major advance in biomaterial science has been the development of bioceramics as bone substitutes. The most common bioceramics in use are calcium phosphate-based (CaP) biomaterials which include: hydroxyapatite (HA), α- and β-tricalcium phosphates (α-TCP, β-TCP), octacalcium phosphate (OCP), amorphous calcium phosphate (ACP) and biphasic calcium phosphates (BCP) which is a combination of two different CaP phases [2–4]. All types of CaP biomaterials can be manufactured in both porous and dense forms as bulk, granules, and powders or in the form of coatings. Their biocompatibility, safety, availability, low morbidity and cost effectiveness are important advantages over autografts and allografts. CaP bioceramics are now in common use for different medical and dental applications such as treatment of bone defects and fracture, total joint replacement, spinal surgery, dental implants, periodontal therapy and cranio-maxillofacial reconstruction [5,6]. The majority of research on CaP based biomaterials has focused on HA (Ca5(PO4)3OH) and β-TCP (Ca3(PO4)2) [7]. CaP based biomaterials are bioactive and have a composition and structure similar to the mineral phase of bone and can be processed to have osteoconductive properties [8]. Furthermore, they have a high affinity for protein adsorption and growth factors [9] that in turn influence osteoinductivity. The osteoinductive properties can be achieved in two ways; intrinsic and extrinsic. Intrinsic is by structural or chemical optimization of the biomaterials themselves and the extrinsic is by the addition of osteoinductive signal molecules, such as bone morphogenetic proteins (BMP) or osteogenic cells [10,11]. Although the CaP bioceramics have many advantages, they also suffer from disadvantages such as; poor mechanical strength, lack of organic phase (i.e. collagen), presence of impurities, micro-scale grain size and non-homogenous particle size and shape. Furthermore, the processing techniques for preparation of bioceramics suffer from prolonged fabrication time, low-yield final product and difficult porosity control [2]. However, over recent years, several modifications of fabrication parameters such as sintering temperature, sintering soaking time, pH and purity of the initial materials have given rise to biomaterials with improved physicochemical properties such as specific surface areas, surface energy, surface charge, surface topography and roughness, grain size and porosity [12]. Another area of recent interest in the literature is to produce bioceramics at the nanoscale level. This is because the bone matrix is also a precise composition of two major phases at the nanoscale level namely, the organic phase (proteins) and inorganic phase (minerals, mainly CaP nanocrystals). The conventional synthetic CaP ceramics are composed of large grain size at microscale level, which seems to possess

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different biological behaviors of bioactivity, biodegradability and mechanical properties than nanoscale alternatives. Therefore, fabrication of nanoscale bioceramics may improve the biological behavior of CaP bioceramics [13]. In addition, it has been shown that nano-HA promotes osteoblast cells adhesion, differentiation, and proliferation thereby enhancing osseointegration and deposition of calcium containing minerals on its surface better than microcrystalline HA [14]. Moreover, the superiority of biological calcified tissues (e.g., bones) are also due to the presence of biopolymers (mainly, collagen type I fibers) which confer strength and partial elasticity. Therefore, one of the most promising ideas is to apply biomaterials with similar composition and nanostructure to that of natural bone tissue. In this regard, development of composite organic–inorganic biomaterials may provide better opportunities for optimizing the conventional bone substitutes [13]. 1.2. Biphasic calcium phosphate Another major area of research on CaP based biomaterials has focused on BCP [15]. According to the definition, BCP consists of two individual CaP phases: most commonly from a more stable phase (HA) and more soluble phase (β-TCP) in different proportions. This combination has presented significant advantages over other types of CaP bioceramics by allowing a better control over bioactivity and biodegradation which guarantees the stability of the biomaterial while promoting bone ingrowth. BCP ceramics are osteoconductive with the possibility of acquiring osteoinductive properties [16,17]. Different researchers have attempted to achieve desirable biological responses by modification of different parameters during biomaterial fabrication or by incorporation of different biocompatible polymers. The biological response to BCP ceramics varies according to their chemical compositions and physical properties which can give rise to different rates and patterns of bone regeneration. To the best of our knowledge, a consensus of the ideal physicochemical properties of BCP scaffolds such as; composition ratio, pore size, total porosity and interconnected porosity has yet to be determined. In addition, there are no generally accepted standards for in vitro and in vivo studies with regards to experimental protocol and interpretation of results; this most likely appears to contribute to the variation of results and findings on the properties and effectiveness of BCP. For the purpose of this review, it is divided into two main sections. First, Sections 3.1–3.4 provide a general overview on BCP (HA/β-TCP) elaborating the concept, synthesis and importance of physicochemical properties and characterization. This section also includes a systematic review of animal studies comparing application of different composition ratios of BCP (Section 3.4). Second, Sections 4.1–4.5. provide discussion regarding controversies on BCP composition ratio followed by general guidelines for characterization and documentation of BCP. This section also presents recommendations to help in reducing the inconsistencies in protocols and reporting strategies of future studies. Of course, the provided guidelines and recommendations can also be applied to study of other biomaterials in the field of bone tissue engineering. 2. Review of literature It is well accepted that the best representative model of biological condition are animal studies. Therefore, for systematic review on application of different composition ratios of BCP, we restricted our search to animal studies as the best models that represent the real biological


condition with less diversity compared to in vitro models. As long as the human trials of BCP are limited to only few composition ratios, we decided to exclude human studies from our systematic review. 2.1. Search strategy An electronic search was performed from 1989 to 2015, using MEDLINE (National Library of Medicine) and PubMed following the criteria as below: 2.2. Inclusion criteria 1. articles in English, 2. use of biphasic calcium phosphate (BCP, HA/ β-TCP) ceramics, 3. animal studies, 4. the keyword “bone substitutes”, 5. use of BCP for intraosseous implantation, 6. use of pure BCP (only inorganic phases). 2.3. Exclusion criteria 1. literature review, 2. use of single composition ratio of BCP, 3. use of BCP in extraosseous implantation sites (subcutaneous or intramuscular), 4. presence of other polymeric phase within BCP, 5. in vitro studies, 6. human trials, 7. non-English articles. After the review process, the available papers were studied carefully to determine the following data: 1. author/year, 2. studied HA/β-TCP ratios, 3. animal type and number, 4. site of implantation of BCP, 5. osseous defect dimension, 6. characterization of BCP (including particle/granule size, pore size and porosity), 7. follow up time and analysis intervals, 8. type of investigations and 9. main findings. All identified studies were assessed independently and in detail to ensure meeting the review's inclusion criteria. A total of 15 articles (out of 101) meet the inclusion criteria and underwent data extraction independently and in duplicate. Data were registered in a table and arranged by year chronologically. 3. BCP bioceramics 3.1. Concept and history The majority of biomedical materials usually consist of a single phase referred to as a monophasic system e.g. HA bioceramics. However, the term multiphasic is referred to the mixture of two or more individual phases with almost similar physical properties, for example a combination of HA and TCP will be called biphasic as it consists of only two phases. The distinction should be made from the term “composite” that is made from two or more constituent materials with significantly different physicochemical properties [18]. Nery et al. [19] in 1975 were the first to report on clinical application of a “tricalcium phosphate” preparation as bone substitutes in surgically created “infrabony” periodontal defects in animals. This preparation was subsequently analyzed by X-ray diffraction (XRD) and found to be actually a mixture of 20% HA and 80% β-TCP [20]. Later, the term “biphasic calcium phosphate” (BCP) was first introduced by Moore et al. [21] and Anuta et al. [22] in 1985 presentations at the 11th annual meeting of the society for biomaterials and later on, in 1986 by Ellinger et al.

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[23] who reported its application in periodontal osseous defects in a case report. LeGeros [24] (1986) and later on Daculsi [16] (1989) started basic research on preparation and clinical application of BCP. BCP formulations are of two major types; 1) BCP consisting of CaP phases with similar molar Ca/P ratio (e.g. α-TCP and β-TCP, Ca/P = 1.5 for both); 2) BCP consisting of CaP phases with different molar ratio (e.g. β-TCP and HA, Ca/P = 1.5 and 1.67, respectively). Table 1 summarizes the different composition phases of BCP and their properties. BCP (HA/β-TCP) appear to be the focus of many research papers in the field of bioceramics and therefore they are investigated extensively [25,26]. Selection of appropriate phases is of great importance to overcome the shortcomings of a single phase formulation. HA is used because of its similarity to the mineral phase of bone and a better mechanical properties compared to α- and β-TCP. However, to overcome its poor biodegradation rate, HA is combined with other more biodegradable bone phases at an appropriate ratio. For the second phase, usually β-TCP is selected because of its higher chemical stability (compared to α-TCP) and more favorable biodegradation rate. The BCP scaffolds proved to be highly biocompatible and could support attachment, proliferation and differentiation of osteoblast cells [12,27]. The main concept behind the use of BCP is to improve the biological properties of the bioceramics such as bioactivity, bioresorbability, osteoconductivity and osteoinductivity so as to enhance bone tissue formation [26]. It has to be highlighted that a delayed or fast biodegradation rate may interfere with rate and pattern of new bone formation. Therefore, the main advantage of BCP is the possibility to manipulate the composition ratio of more stable phase and more biodegradable phase to optimize biodegradation rate and enhance bone repair process for specific application. This is achieved by selection of an optimum composition ratio of the HA and the α- or β-TCP, where increasing the ratio of latter, improves the bioactivity and biodegradability of BCP [16]. The biodegradation process initiates after BCP implantation by dissolution of CaP crystals and precipitation of ion-substituted carbonated calcium deficient-HA (CDHA) which will be replaced by new bone ingrowth during bone healing [28]. Greater biodegradation of TCP and partial dissolution of the HA facilitate an increase in the supersaturation 3− of calcium (Ca2+) and phosphate (HPO2− 4 , PO4 ) ions concentration in the local microenvironment. This results in precipitation of released ions and formation of CDHA microcrystals [27], which incorporates 2+ , Na+) present in the biological fluid. This proother ions (CO2− 3 , Mg cess results in subsequent mineralization of extracellular matrix (ECM) and incorporation of CDHA in the newly generated bone, forming a strong interface through direct chemical bonds [25]. The biodegradation process of BCP depends on several factors such as; chemical composition, particle sizes, crystallinity, specific surface area and porosity. It is known that both in vitro and in vivo biodegradation process of BCP follow a common pattern. A general accepted pattern of biodegradation of different phases of BCP in increasing order is as follow: HA b β-TCP b α-TCP; where α-TCP manifests the fastest biodegradation rate [12,27,29,30]. The biodegradation kinetics of BCP with similar particle size and porosity depends on types of available chemical phases (HA/TCP) and their percentage ratio, where the higher the ratio of TCP, the higher the biodegradation rate of BCP. However, the biodegradation process is also influenced by other factors, for example, a lower

Table 1 BCP composition phases (HA and TCP) and their major properties. Material

Hydroxyapatite (HA) Beta-tricalcium phosphate (β-TCP) Alpha-tricalcium phosphate (α, α′-TCP) Compiled from references [2–4].

Stoichiometry

Ca10(PO4)6(OH)2 β-Ca3(PO4)2 α-Ca3(PO4)2 α′-Ca3(PO4)2

Crystallography

Hexagonal Rhombohedral α = monoclinic α′ = hexagonal

Molar ratio (Ca/P)

1.67 1.5 1.5

Solubility at 25 °C −log Kps

mg L−1

116.8 28.9 25.5

0.00010 0.20 0.97

Molar mass g/mol

Density g/cm3

988.62 310.17 310.17

3.156 3.066 α = 2.866 α′ = 2.702

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porosity and surface area or a higher crystallinity and larger particle sizes are associated with a lower rate of biodegradation process [31]. The biodegradation process of BCP is characterized in vivo by decrease in crystal size and increase in total porosity [27]. Currently, there are over 30 commercially available BCP bone substitute products for various orthopedic and maxillofacial applications. Table 2 summarizes the available BCP products based on composition ratio of HA and TCP. Furthermore, BCP has been used as carrier or delivery system for therapeutic drugs, antibiotics, hormones and growth factors to enhance bone tissue engineering [16,17]. 3.2. BCP synthesis and preparation For bone tissue engineering, the fabricated scaffolds should simulate the physicochemical properties of the bone ECM. BCP bioceramics best represent the inorganic phase of the bone ECM with mineral composition similar to that of natural bone. More stable HA phase could function as a structural framework that support the scaffold and newly formed bone, while the less stable TCP phase create space for new bone ingrowth during biodegradation process. Various types of CaP bioceramics have been used in medicine as bone replacement materials due to their chemical similarity to the inorganic mineral phase of natural bones. Therefore, several synthetic routes have been developed to prepare BCP bioceramics of variable HA/β-TCP ratios simulating the physical and biological properties of natural bones. Nevertheless, up to now, there has been only a relative success in the fabrication of bone substitute materials similar to natural bone; this emphasizes the complexity of the natural structures. Among the available BCP preparation techniques, the most usual one consists of

Table 2 The commercially produced BCP and percentage ratio of composition phases. HA %

TCP %

Brand name

N96 80 75 70

b4 20 25 30

65

35

60

40

55

45

30–50 20

50–70 80

Calciresorb (Ceraver, France) Osteosynt (Einco, Brazil) TCH (Kasios, France) Ceratite (NGK Spark Plug, Japan) OrthoCer HA TCP (Baumer, Brazil) CuriOs (Progentix Orthobiology BV, Netherlands) Ceraform (Teknimed, France) Calcicoat (Zimmer, IN) BCP (Depuy Bioland, France) BCP BiCalPhos (Medtronic, MN) BonaGraft (Biotech One, Taiwan) BoneMedik-DM(Meta Biomed, Korea) CellCeram (Scaffdex Oy, Finland) GenPhos HA TCP (Baumer, Brazil) Graftys BCP (Graftys, France) Hatric (Arthrex, US) Hydros (Biomatlante SA, France) Kainos (Signus, Germany) MasterGraft Granules (Medtronic Sofamor Danek, US) MBCP (Biomatlante SA, France) OpteMx (Exactech, FL) Ossceram nano (Bredent Medical, Germany) Osteosynt (Einco, Brazil) Ostilit (Stryker Orthopaedics, NJ) SBS (ExpanScience, France) TriOsite (Zimmer, IN) 4Bone (MIS, Israel) CuriOs (Progentix Orthobiology BV, Netherlands) Eurocer (FH, France) Indost (Polystom, Russia) BoneCeramic (Straumann, Switzerland) BoneSave (Stryker Orthopaedics, NJ) Kainos (Signus, Germany) MBCP+ (Biomatlante SA, France) OsSatura BCP (Integra Orthobiologics, CA) Osteosynt (Einco, Brazil) ReproBone (Ceramisys, UK) Tribone 80 (Stryker, Europe)

sintering of non-stoichiometric CaP, such as ACP and CDHA, at temperatures above ~ 750 °C [32,33]. However, commonly, this procedure is made at temperatures exceeding ~1000 °C [34–39]. The process is simple, more economic and bypasses the time-consuming purification process compared to other preparation techniques. Various modifications of sintering, such as a two-step sintering [40] and a microwave heat processing [41–44] of both ACP and CDHA have been applied as well. Namely, the chemical reaction of a thermal decomposition of “Ca10 − x(HPO4)x(PO4)6 − x(OH)2 − x (CDHA)” with x = 0.5 is represented as: 2Ca9:5 ðHPO4 Þ0:5 ðPO4 Þ5:5 ðOHÞ1:5 ¼ Ca10 ðPO4 Þ6 ðOHÞ2 þ3Ca3 ðPO4 Þ2 þH2 O ð1Þ This reaction results in to formation of a BCP, consisting of HA and βTCP. For this decomposition, a reasonable solid-state transformation mechanism based on the diffusion of OH− and Ca2+ ions was proposed in 2002 [45,46]. In 2015, that mechanism was further itemized by discovering a fully separated growth of microscopic HA and β-TCP crystals, which could be adjacent, forming particles but without mutual intergrowth [47]. It is important to notice, that under strictly equal conditions, the numerical value of the Ca/P ratio of the initial CDHA influences the grain sizes of the sintered BCP, where the average grain size decreases with increasing Ca/P ratio [48]. Another preparation approach is based on solid-state reactions between two solid compounds, performed at elevated temperatures. The examples comprise solid state reactions between calcium hydrogenphosphate dihydrate (brushite, CaHPO4·2H2O) and calcium carbonate (calcite, CaCO3) [49,50], TCP and Ca(OH)2 [51], monocalcium phosphate monohydrate (Ca(H2PO4)2·H2O) and CaCO3 [52,53]. Thus, to prepare BCP, initially, the chosen Ca- and P-containing compounds are mixed at known proportions to get the desired Ca/P ratio (1.50–1.67) followed by heating and sintering. When milling of the initial reagents was applied, the proportion of HA and β-TCP phases in the prepared BCP was found to depend on the milling time [50]. Furthermore, BCP could be prepared by other methods, such as a flame spray pyrolysis [54], a liquid mix [55] and a sol-gel [56,57] techniques; both latter processes must be followed by sintering [55–57]. Regarding the initial raw materials, BCP could also be manufactured from the natural CaP containing recourses, such as bovine [58–60] and cuttlefish [61] bones, corals [62] and algae [63]. The various processing techniques yield diverse compositions and properties of the final BCP formulations, which is crucial for further applications. In addition, a mechanical blending of the desired amounts of either the individual powders of HA and β-TCP [64–69] or their precursors might also be used to produce BCP. For instance, one can mix two types of CDHA powders, such as one with x = 0.1 (almost HA) and another one with x = 0.9 (almost TCP) and sinter the mixture. In this case, the first CDHA will decompose to HA with a small admixture of β-TCP, while the second CDHA will decompose to β-TCP with a small admixture of HA, resulting in BCP (HA + β-TCP). Synthesis conditions of the precursive non-stoichiometric CaP were found to influence the phase ratio in the final BCP [70]. However, due to inability to get homogenous crystal distributions of the mixed phases, a blending of both individual CaP and their precursors is not suggested for production of BCP formulations with reproducible structure and composition. Furthermore, such types of mechanically blended BCP bioceramics were found to exhibit both an elevated extent of dissolution [71] and an inferior sintering performance [72] if compared to those manufactured by a thermal decomposition of a CDHA powder of the equal Ca/P ratio. On the other hand, when soaking in simulated body fluid, the weight increase for the mechanically blended BCP was found to be greater than that for precipitated BCP [64]. Besides, in mechanical blends with β-TCP, HA could be partly decomposed at sintering, changing the HA/β-TCP ratio in the final BCP [67]. Moreover, if sintering is performed at temperatures exceeding ~1250 °C, formation of α-TCP phase becomes possible, which


results in transformation of the initially biphasic blends of HA + β-TCP into triphasic (HA + β-TCP + α-TCP) ones [73,74]. The latter process is reversible because dwelling of the HA + β-TCP + α-TCP formulations for several hours at ~900 °C results in a reverse change of α-TCP to βTCP, and, thus, transformation of the triphasic formulations into BCP (HA + β-TCP) again [73]. Furthermore, in the presence of dopants, various ion-substituted forms of BCP might be synthesized [75–78]. Depending on the nature of dopants, in such formulations the doping elements could be present in either phase. For example, F [79], Cl [80], Si [81] and carbonates [82, 83] appeared to enter more readily into the HA phase, Zn [84] and Sr [85,86] were found to enter into both phases, while Na [77], K [87], Mg [88–91], Mn [92–94] and Nb [95] entered preferably into the βTCP phase [96]. Furthermore, addition of MgO to BCP was found to suppress a phase transition from β-TCP to α-TCP [88]. 3.3. Physicochemical properties and characterization BCP chemical formulation and phase compositions should be stable to fit the purpose of clinical applications. BCP is vulnerable to moisture contamination that could transform it to single phase products such as CDHA. For this reason they should be stored in a clean and dry condition away from heat to maintain its chemical nature prior to clinical application [18]. The major physicochemical properties of BCP are similar to the single phase bioceramics (HA and β-TCP) and depending on the composition ratio of each phase their properties may differ. However, the critical issue is their in vivo differences such as biodegradation, precipitation of apatite crystal on their surface, protein adsorption and cellular behavior [25,97,98]. The most common method for characterization of BCP is X-ray diffraction (XRD) technique. XRD is used for crystallography and phase

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identification and quantification of bioceramics materials using X-ray diffractometer machine (with Cu-Kα radiation). For BCP the usual XRD scan range is from 20° to 60° because most of the CaP phases have the strongest peaks at this range [99]. XRD analysis is the only technique that can provide a mean of approximating the HA/β-TCP ratio in the BCP. This ratio is determined using the ratio of intensities of the most intense diffraction peaks of the HA phase to those of the most intense diffraction peaks of β-TCP phase compared with the ratios obtained from calibrated standard mixtures of pure HA and β-TCP [27] (standard XRD card, JCPDS Card 09–0432 for HA and 09-0169 for βTCP). XRD pattern of BCP (HA/β-TCP) indexes main peaks corresponding to HA (JCPDS no. 09-0432) and β- β-TCP (JCPDS no. 09-169) in accordance to ICDD standard (The International Centre for Diffraction Data). Fig. 1 shows the main characteristic peaks of HA at 2-Theta diffraction angles and the absolute intensity (a.u.) relevant to each peak as follow (°/a.u.); 25.9°/628, 31.8°/1469, 32.2°/816, 33°/900, 34.1°/425, 46.8°/442 and 49.6°/519. For β-TCP, the main peaks are indexed at; 25.8°/315, 26.7°/495, 29.7°/380, 31.1°/887, 32.6°/350 and 34.5°/639 (Fig. 1) [100]. The presence of other secondary phases (i.e. α-TCP or calcium oxide) can also be evaluated to determine phase decomposition or transformation during biomaterials processing such as sintering process [12,27,99,100]. It is obvious that by decrease of β-TCP ratio in BCP, a lower intensity of β-TCP and a higher intensity of HA peaks are indexed which is relevant to each HA/β-TCP ratio (Fig. 1). The Reference Intensity Ratio (RIR) is a general method for quantitative phase analysis by scaling all diffraction data to the diffraction of the standard reference materials [101]. Software programs such as “ICDD's Sleve” allow identification of materials and calculation of peak intensity and concentration of each phase by RIR method. For complete structural description of a material, use of Rietveld structural refinement techniques and powder pattern indexing are complementary. These

Fig. 1. XRD pattern of sintered BCP scaffolds with different HA/β-TCP composition ratios.

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methods allow accurate phase identification by lattice matching techniques [102]. Fourier transform infrared spectroscopy (FTIR) is useful for chemical characterization of major functional groups. This technique can be used for chemical analysis of BCP and other organic components if present (Fig. 2). FTIR is a very sensitive technique for determining phase composition and transformation of one phase to another [99,103]. Analyzing the chemical structure using FTIR reveals that both HA and β-TCP group at approxishow the absorption bands corresponding to PO3− 4 mately 1090–1120 (ν3), 1040–1050 (ν3), 945–970 (ν1), 600–610 (ν4) and 550–570 (ν4) cm− 1, and bands corresponding to CO2– 3 group at 870–880 (ν2) and 1445–1455 (ν3) cm−1 (Fig. 2) [100]. Furthermore, for HA, additional absorption band corresponding to OH− group can also be detected at 630 and 3572 cm−1 due to stretching vibration of OH− ions. However, this peak cannot be seen in TCP since chemical structure of TCP lack OH− group. It is worth mentioning that adsorbed H2O molecules may express extra broads at 3433 and 1642 cm− 1 [104]. Therefore, in FTIR pattern of BCP, in addition to peaks of PO3− 4 − and CO2− groups can also be detected relevant to 3 , the peaks of OH the ratio of HA. Other available techniques such as X-ray fluorescence (XRF) and inductively coupled plasma-optical emission spectrometry analysis (ICPOES) can also be used for analyzing the calcium phosphate molar ratio [105]. Physical characterization of bioceramics including BCP can be performed to determine the shape, size and distribution pattern of bioceramics particles using transmission electron microscopy (TEM), laser diffraction particle size analyzer (PSA) and scanning electron microscopy (SEM). 3.4. The optimal composition ratio The abundance of CDHA crystals on the surface of BCP implant appears to be inversely related to HA/β-TCP ratio. The lower the ratio, the greater the abundance of CDHA crystals on the surface [106]. This in turn influences the bioactivity of BCP therefore, the control of the ratio of the BCP is crucial to achieve desired biological outcomes. Various HA/β-TCP ratios have been tested in order to improve BCP properties. An ideal balance between these two phases may improve mechanical strength and enhance the biological behaviors of BCP

scaffolds [27]. The first studies of BCP with multiple ratios of HA/β-TCP reported by Daculsi et al. (1989) demonstrated that manipulating the HA/β-TCP ratio could control the bioactivity of these bioceramics [16]. However, similar physicobiological properties of BCP may not be the distinctive feature of a single ratio of HA/β-TCP but rather applies to a range of close ratios. For example, in comparison to HA/β-TCP ratio of 20/80, similar results were also found with HA/β-TCP ratios of 15/85 [107], 25/75 [108] and 30/70 [109]. Furthermore, some studies reported that formulations containing HA/β-TCP ratio of 50/50 [110] and 60/40 [111] enhanced cell proliferation to a higher extent. Therefore, the relationship between the composition ratio of BCP and cellular behavior is complicated and requires further development of standardized protocols for material preparation, characterizations, and analysis of biological behaviors. The literature is not clear with regards to the effect of each particular ratio on bone tissue regeneration, particularly in animal studies. The findings in the literature on biological in vivo performance of different ratios of BCP can be categorized into three groups; 1) those who reported the beneficial effects of a particular ratio compared to other ratios in enhancing bone regeneration, 2) those who did not find any differences between various studied ratios, and 3) those who reported detrimental effects of BCP on healing and bone regeneration in comparison to autograft and control groups (no graft). Table 3 summarizes the comparison of different parameters among animal studies, including all the available and reported data by authors. An in vivo study reported a greater gain in probing attachment levels and bone regeneration in periodontal defects when lower ratios of TCP were used (HA/β-TCP ratios of 85/15 and 65/35 versus 0/100 and 50/ 50) [107]. On the other hand, it has been reported that faster and greater quantity of bone is formed in 15/85 ratio due to the greater osteoinductive potential compared to 85/15 ratio [112]. JW Park et al. [113]. reported that 60/40 ratio exhibited a significantly greater percentage of newly formed bone when compared with 0/100 ratio at both 4 and 8 weeks with direct ingrowth of bone tissue and blood vessels. However, other researchers have reported that 30/70 ratio (compared to 20/80) showed a faster bone resorption and more favorable result in new bone formation and space maintenance, particularly at 8 weeks [114]. In contrast to previous findings, some studies reported no statistical significant differences (p N 0.05) between ratios in terms of new bone

Fig. 2. FTIR pattern of HA and β-TCP showing major corresponding peaks of PO3− and CO2− 4 3 .


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Table 3 Summary of literature on animal studies used different composition ratios of BCP (HA/β-TCP), their investigated parameters and main findings. Authors/year/reference HA/TCP Animal/no. % age. wt.

Site of implant

Defect dimension

BCP characterization

Investigation times and follow up period

Type of investigations

Daculsi G et al. 1989 [16]

85/15 65/35 15/85

Beagle dogs no: NR age: NR wt: NR

Surgically created periodontal defects?

NR

Macroporosity 150-200 μm, porosity: NR mechanical property: NR

6 & 12 months

Histologic, TEM, electron microdiffraction, microanalysis X-ray

Nery EB et al. 1992 [107]

100/0 85/15 65/35 50/50 35/65 15/85 0/100

Adult beagle dog, no:21 age: NR wt: NR

Mesial to maxillary & mandibular first molars and canines

NR 3-wall intrabony defects?

NR

6-months

Histologic

Schopper C et al. 2005 [115]

30/70 50/50 100/0

Female sheep, no: 6 24–48 months 58.5 kg

Rib 5 × 5 mm corticocancellous defects 6 holes/rib

Granulates, particle sizes 0.5–1 mm, porosity: NR mechanical property: NR

6 & 12 months

Histologic, histomorphometric, backscattered SEM

Main findings

⁎ Bone formation is associated with each BCP regardless the ratio. ⁎ After implantation the average grain size decreased and the size of microporosity increased. ⁎ Resorbability of BCP can be controlled by varying its HA/β-TCP ratio. ⁎ The higher the β-TCP ratio, the greater the resorbability and microcrystals formation. ⁎ Compared to other ratios, the higher HA ratio (85/15 & 65/35) had significantly greater gain in probing attachment levels and bone regeneration in periodontal bony defect. ⁎ Higher HA ratio (but not 100% HA) showed accelerated new bone formation and new attachment levels. ⁎ Based on histological results, the 85/15showed greatest gain in attachment level and bone regeneration. ⁎ As the proportion of β-TCP increases, osteoclastic activity also increases. ⁎ No statistical differences between 30/70 & 50/50 in degree of bone formation. ⁎ 30/70 scaffolds were better integrated into physiological bone remodeling. ⁎ 100/0 produced significantly lower bone values than other ratios. ⁎ Bone formation did not significantly increase from 6 to 12 months. ⁎ Remodeling activity and biomaterial replacement increased from 6 to 12 months and were more pronounced in 50/50 and 30/70 than 0/ 100. (continued on next page)

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Table 3 (continued) Authors/year/reference HA/TCP Animal/no. % age. wt.

Site of implant

Defect dimension

Bodde EWH et al. 2007 [132]

75/25 0/100

Female sheep, No: 9 24–36 months 50 kg

Trabecular bone of the femoral medial condyles, bilaterally 2 holes/side

Ø 6 × 9 mm 0/100 (Conduit™); 3, 12, 26 weeks Histologic, depth irregular granules, histomorphometric, Ø 1.5–3 mm. radiologic Interconnectivity 70%, pores 1– 600 μm. 75/25 (Biosel®); 3 mm cube-shaped particles, interconnectivity 70%, pore 200– 500 μm. Mechanical property: NR

Jensen SS et al. 2007 [119]

60/40 100/0 0/100

Adult Göttingen minipig no: 16 age: NR 55 kg

lateral Ø 7 × 4 mm Particle size of mandibular body depth 0.5–1 mm, macropore 100– and ramus 500 μm, total 4 holes/side porosity of 90%, crystallinity 100%. Mechanical property: NR

2, 4, 8, 24 weeks

10 mm long segment

4, 6, 8, 12, 18 weeks

Balçik C et al. 2007 [117]

60/40 100/0

White rabbit no: 38 4 months 1.94 kg

Central third of the right and left tibia


Macropore 150–200 μm, 2nd pores 20–100 μm. Microporosity 1–2 μm, compressive strength 4.9–5.3 MPa, elastic modulus



Histologic, histomorphometric, radiologic

Histologic, radiologic, DXA, Q-CT scan (quantitative), 3-point bending test

Main findings

⁎ The amount of newly formed bone did not differ significantly between 75/25 & 0/100. ⁎ 0/100 showed more degradation and less cellular intervention as compared to 75/25. ⁎ 0/100 degraded significantly during time and only 36% of the material was left after 26 weeks. ⁎ 75/25 showed more multinucleated cells at its surface, but were not able to degrade the ceramic. ⁎ There was no significant difference in distribution of newly formed bone over the defect except after 26 weeks where 75/25 showed more bone formation in outer region. ⁎ At 8 weeks: more bone formation in defects with autograft and 0/ 100 N 60/40 N 100/0. ⁎ Bone formation was faster with 60/ 40, so the TCP ratio had some effect on the healing events. ⁎ No difference in bone formation between the groups at 24 weeks. ⁎ 100/0 and 60/40 exhibited limited and similar resorption patterns over 24 weeks, whereas autograft and 0/ 100 showed fast degradation and substitution by newly formed bone in 8 weeks. ⁎ BCP with slow substitution (higher HA ratio) may be suitable for volume expansion. ⁎ No statistical difference in mean radiological grade of healing and bonding to bone between ratios. ⁎ Both ceramics have a limited application in the treatment of


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Site of implant

Defect dimension




Main findings

3.1–3.5 GPa, porosity: NR ⁎

⁎

Fariña NM et al. 2008 [112]

85/15 15/85

Beagle dog no: 6 24 months 20 kg

Right and left mandibular bone, 4 holes/side

Cylindrical 2 × 6 mm

Particle size 5 μm porosity: NR mechanical property: NR

4,12, 26 weeks

Histologic, histomorphometric, SEM, X-ray microanalysis

⁎

⁎

⁎

⁎

Chissov VI et al. 2008 [133]

100/0 80/20 0/100

Wistar rat no: 108 age: NR wt: NR

Fenestral defect in rat shin bone

5–7 mm fenestral defect in shin bone

Granule size 300–600 μm, Pore 10–30 μm, porosity: NR mechanical property: NR

3,6,9,12 weeks, Histologic & 6, 9 months

⁎

⁎

⁎

⁎

Jensen SS et al. 2008 [120]

80/20 60/40 20/80

Göttingen Minipig no: 24 age: NR 59.2 kg

Mandibular body, 3 holes/side

Ø 9 × 4 mm Macropores depth 100–500 μm, total porosity 90%, crystallinity 100%, crystal size b5 μm, particle sizes 0.5–1 mm, mechanical property: NR

4, 13, 26, 52 weeks

Histologic, histomorphometric

⁎

⁎

⁎

load-bearing segmental bone defects. DXA and QCT showed a time-dependent increase in ceramics density due to the healing process in these ceramics, while densities at the bone–ceramic interface decreased. Flexural resonant frequencies and 3-point bending strength increased with time in both ratio, revealing an increase in mechanical stability The percentage of bone formation of both ceramics was significantly higher at 26 weeks. An earlier and more quantity of bone formed in 15/ 85 due to higher osteoinductive potential. Faster resorption and subsequent migration of bone in 15/85 than 85/ 15 at 26 weeks. Osteoinductive potential of BCP can be improved by varying HA/β-TCP ratio. The rate of resorption was: 100/0 b 80/200/100. Rate of resorption of 0/100 lagged behind reparative bone capacity. Reparative ossification and bone tissue mineralization completed by end of 3 month. All ratios were biocompatible and bioactive in replacement of bone defects. Autograft and 20/80 had a higher degradation rate and bone formation over 52 weeks. 80/20 and 60/40 had a lower degradation rate and bone formation over 52 weeks. 80/20 and 60/40 had significantly

(continued on next page)

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Site of implant

Defect dimension




Park JW et al. 2010 [113]

60/40 0/100

Adult male New Zealand white rabbit no: 30 age: NR 3.7 kg

Transosseous defect in the mid-portion of parietal bone

Ø 8 mm?

Hung C-L et al. 2011 [122]

70/30 60/40 0/100

Mature beagle dog no: 4 24–36 months 10 kg

All Premolars extraction site max. 2 holes man. 4 holes

Cylindrical Granules, particle Ø 3 × 6 mm size 0.5–1 mm, length macropore 400– 600 μm, porosity: NR, mechanical property: NR

2, 4, 6, 8 weeks Histologic, histomorphometric

Yun P-Y et al. 2014 [114]

30/70 20/80

Pilot 1: rabbit age: NR 2.8 kg no: 12 pilot 2: beagle dog no: 6 5–6 months 9 kg

Pilot 1: calvarial defects 4 holes/rabbit pilot 2: mandibular sockets of extracted premolars

Pilot 1: Ø 7 × 7 mm pilot 2:

Macropore 250-400 μm, macroporosity 70–75%, mechanical property: NR

⁎ 30/70 showed faster rate of bone resorption than 20/80. ⁎ 30/70 showed more new bone formation and space maintenance than

60/40A: donut shaped, 300–400 μm central pore, particle size 0.8 mm. 60/40B: rod shaped, no central pore, particle size 0.6 × 2 mm. Micropore 20–60 μm, small micropore 1–8 μm, grain size 300–400 nm, porosity: NR, mechanical property: NR

6 × 6 × 3 mm

4, 8 weeks


2, 4, 8 weeks

Main findings

lower bone % occupying the defect volume compared to autograft. ⁎ The % of TCP in the BCP has a direct impact on the amount of bone formation. ⁎ In all groups, % of new bone formation increased over time from 4 to 8 weeks. ⁎ 60/40A showed optimum biodegradation and higher % of new bone formation at entire defect area when compared with the other groups at 4 and 8 weeks with direct ingrowth of bone tissue and blood vessels into central pores. ⁎ Decreased interparticle space and lack of a central pore in 60/40B contributed to the lower % of new bone formation. ⁎ 0/100 showed no significant signs of graft degradation after 8 weeks. ⁎ All ratios showed significantly greater new bone formation than the empty control group. ⁎ 70/30, 60/40 had greater new bone regeneration and material replacement than 0/100. ⁎ Blood-vessel-like cavities were observed more often in 70/30, 60/40 than 0/100. Histologic, histomorphometric, micro-CT


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20/80, especially at the 8 weeks. ⁎ There was no difference among groups in the volume of residual bone graft material at 2,4,& 8 weeks. Hong JY et al. 2014 [121]

Site of implant

Defect dimension




100/0 60/40 0/100

Male beagle dog no: 8 18 months 15 kg

Extraction socket -NR of all third premolars of maxilla & mandible. 4 exo/jaw

100/0 (Calcitite® 2, 4, 8 weeks 2040); particle size 420–840 μm. 0/100 (Cerasorb®); particle size 0.5–1 mm. BCP (BoneMedik-DM®) particle size 0.5–1 mm. Porosity: NR mechanical property: NR


Kunert-Keil C et al. 2015 [116]

60/40 0/100

Lewis rat no: 24 2 months 300 g

Parietal region of the cranium

Ø 5 mm?

Round granules, total porosity 70%, mechanical property: NR

4 weeks

Histologic, immunohistochemistry, RT-PCR

Lim H-C et al. 2015 [118]

70/30 30/70

Adult New Zealand white rabbit no: 8 age: NR 3 kg

Bilateral sinus lift Ø 6 mm?

Porosity 77%, pore size; BCP 70/30: 300–500 μm, BCP 30/70: 250 μm. Mechanical property: NR

2, 8 weeks

Histologic, histomorphometric, micro-CT

Main findings

⁎ % new bone was significantly higher in control group (no graft) compared with the grafted groups at all healing periods. ⁎ Bone formation was delayed in the sockets grafted with BCP compared to control group. ⁎ % new bone volume at 8 weeks was greater in 0/ 100 than other ratios. (0/ 100 N 60/40 = 100/0) ⁎ % residual bone of 0/100 decreased with time and it was less than other ratios at all healing periods. ⁎ More number of multinucleated cells in 60/40 and 0/100, followed by 100/0 and smallest in control group. ⁎ The amount of newly formed bone in the defect site did not differ between the two ratios. ⁎ 0/100 group showed significantly higher bone resorption and osteogenic differentiation marker. ⁎ 60/40 can be used as osteoconductive bone substitute that remains stable in the implantation bed. ⁎ The amount of new bone increased significantly between 2 and 8 weeks of healing in both groups. ⁎ The bone volume, volume stability and osteoconductive capacity did not differ between two ratios. ⁎ High β-TCP ratio did not impair (continued on next page)

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Site of implant

Defect dimension




Main findings

volume stability. ⁎ The residual material was significantly more resorbed in 30/70 than in 70/30 at both 2 and 8 weeks along with the presence of greater number of multinucleated giant cells. Abbreviations and symbols: DXA: Dual-energy X-ray absorbtiometry, Exo: extracted tooth, NR: not reported, RT-PCR: reverse transcription polymerase chain reaction, TEM: transmission electron microscopy, wt: weight, Ø: diameter, ?: not fully reported.

formation, bone volume, volume stability, bone mineral density and osteoconductive capacity in the defect site (HA/β-TCP ratios of 30/70 vs. 50/50 [115]; 60/40 vs. 0/100 [116], 60/40 vs. 100/0 [117] and 70/ 30 vs. 30/70 [118]). However, a high β-TCP ratio did not impair the volume stability of an implant biomaterial before bone ingrowth. In comparing different BCP ratios with autograft at 8 weeks, Jensen et al. [119]. reported the greatest bone formation in defects with autograft. This was then observed to be reduced over the different BCP with decreasing TCP ratios - 0/100 N 60/40 N 100/0, respectively. However, at the end of the study (24 weeks), they failed to detect any difference in bone formation between the study groups. HA/β-TCP ratios of 100/0 and 60/40 demonstrated similar resorption patterns which was at a slower rate, whereas the autograft and 0/100 ratio showed faster biodegradation and substitution with newly formed bone. In a later report by same group [120] they revealed that 20/80 ratio showed bone formation and biodegradation similar to autografts whereas 60/40 and 80/20 ratios were similar to DBBM (deproteinized bovine bone mineral). They concluded that the amount of bone formation and biodegradation of BCP is inversely proportional to the HA/TCP ratio. The majority of studies have reported that regardless of the ratio, BCP in general enhances the rate and quality of bone regeneration when compared to empty control groups. However, Hong et al. [121]. reported that bone formation in extraction sockets was delayed in the sockets grafted with BCP and showed different healing process according to the biodegradation patterns. In addition, the percentage of new bone was significantly higher in the control group (no graft) compared with the grafted (BCP) groups at all healing periods (2, 4, and 8 weeks). Furthermore, they reported higher numbers of multinucleated cells in 60/40 and 0/100 ratios, followed by 100/0 ratio and smallest number in the control group. This was contributed to a lower percentage of residual bone of 0/100 ratio than 100/0 and 60/40 ratios at all healing periods. Contrasting to these results, in a similar study of dental extraction sites of beagle dogs, Hung et al. [122] reported that ratios of 70/30 and 60/40 provided better new bone regeneration rate than 0/100 ratio and empty control groups without any toxic or inflammatory reactions. In general, BCP scaffolds are osteoconductive, but as mentioned earlier, some studies reported their possible osteoinductive property. This is due to the evidence of ectopic bone formation upon implantation in non-osseous sites (e.g. subcutaneously or in intramuscular sites). It has been shown in ectopic sites that an optimal balance between the ratio of the HA and β-TCP can induce mesenchymal stem cells (MSC) for enhanced rate of new bone tissue formation. HA/β-TCP 20/80 scaffolds seeded with human MSC have been shown to have a highest rate of bone formation compared to other HA/β-TCP ratios (76/24, 63/ 37, 56/44), pure HA and pure β-TCP [123]. However, osteoinductive property may not be inherent in nature but could be attributed to the critical geometry, topography and ratio which allow local concentration

of endogenous BMPs or growth factors on materials' surface to induce osteoinductivity [124,125]. Currently, there is no general agreement on an ideal ratio of each phase of BCP for clinical applications. Various HA/β-TCP ratios have been evaluated in the literature in order to determine the best ratio for optimum bone regeneration, however, only BCP ratios of HA/β-TCP 65/35, 60/40 and 50/50 have been applied successfully in human clinical trials [126–130]. Interestingly, two ratios of HA/β-TCP; 30/70 and 20/80 have also been reported to reveal some osteoinductive properties [2,29,131]. 4. Discussion 4.1. The controversies on composition ratio of BCP It is accepted that the higher the ratio of β-TCP compared to HA, the greater the biodegradation rate of BCP. Subsequently, the higher ionic dissolution results in formation of microcrystals in the supersaturated microenvironment [25,27]. In addition, a higher number of multinucleated cells were detected at the surface of BCP with a higher β-TCP ratio [132]. The resorption rate can be modified by controlling the percentage ratio of β-TCP [16], despite this the rate of resorption of β-TCP is still slower than the reparative capacity of bone [133]. This review of literature has revealed that based on biological outcomes the optimum ratio of two phases of BCP (HA and β-TCP) has not been consistent between studies, therefore, the relative clinical importance of each ratio is uncertain (Table 3). Furthermore, the particular biological effects and physicochemical features of each BCP ratio are not clear. It should be noted that the differences in physicochemical properties of a composition ratio of BCP such as; preparation techniques, crystallinity, surface topography and porosity are the main factors that contribute to inconsistencies in study outcomes. Particularly, it is advised to report the accurate method of BCP ratio calculation and preparation in volume or weight percentage. Other factors may be related to clinical experimental heterogeneity in the study designs, outcome measures, measurement methods, definition of variables, subjects, and duration of study which may all contribute to biased outcomes and inconsistencies in findings. For example, with regards to implantation procedures of biomaterials, the differences in surgical technique, amount of surgical trauma and duration of surgical procedure may influence the resorption rate and other biological responses. In conclusion, considering these variables, it should be noted that, it is possible for similar composition ratios to exhibit different behaviors at a particular healing time in animal models. Therefore, it is recommended to follow available standardized protocols and report all study variables in appropriate details (Table 4). In this way, one can come up with a more informed judgment about

M. Ebrahimi et al. / Materials Science and Engineering C 71 (2017) 1293–1312 Table 4 Investigated parameters in the study of BCP biomaterials and recommendations for future animal studies. Parameters

Findings in literature review

Type of animal

The most common animal model studied were dogs (6 studies, 40%) and rabbit (4 studies, 27%).

Table 4 (continued) Parameters

Recommendations

Larger animal model may express closer similarity to human clinical application. Full detail of species, sex, age and weight should be reported Sample size There was significant The objectives and difference among studies in the justifications for sample size number of animals. selection should be stated There were no standard considering both ethical and protocols and justifications for economic factors. the number of animals in the Minimum number of animals studies. in a study should be determined that allow statistical significant testing and assure sample mean calculation. Anatomical The most common study site Exact anatomical sites and the site was the mandibular (8 studies, rationale of their selection 53%) defects (extraction should be stated considering sockets 20%, body critical the amount of functional load defects 20%, periodontal defect subjected to implant materials. 13%) followed by maxilla (4 studies, 27%) and cranium defects (3 studies, 20%) Most papers fail to report this Full details of housing and Housing and husbandry in full detail. husbandry conditions should conditions be reported to address bias of confounding factors in findings. Experimental Almost all of the paper Full detail of surgical surgical reported this in adequate procedure, including time, date procedure detail. and location is necessary. Report of materials and instruments used and techniques for creating standard reproducible CSD is required. Defect The defects created were There should be clear definition dimension ranging from 2 to 10 mm. of CSD based on type of animal The exact defect dimension is model and nature of the study. not reported in majority of The defect dimension should cases. be reported in full detail (3D) in SI standard. HA/β-TCP ratio Almost all ratios were included Ratio calculation methods for animal studies with the should be clearly defined most common 60/40 ratio (V/V%, W/V %). (47%) followed by 30/70 ratios Full crystallography is required (20%). based on XRD studies. Additional investigations (i.e. SEM) support the evidence. Phase purity Almost no study investigated Additional characterization the possible presence of other (FTIR) is required to roll out the phases' impurity or phase presence of impurities and transformation. other phases that may trigger certain biological response. Sintering Majority of studies failed to Detail of full sintering program program report the full details of should be reported i.e. sintering program for BCP sintering temperature, heating production. rate, soaking time, cooling rate. Macropore The size of macropores ranged Pore size and the technique of from 100 to 850 μm, however, measurement should be the majority of macropores reported. were b500 μm. Rationale for selection of pore Micropore The size of micropores ranged size should be stated. from 1 to 60 μm. There should be a clear distinction in use of these terminologies. This important parameter need Interconnected There was almost no report of porosity interconnected porosity with to be characterized as it has exception of one study. major effect on biological responses by controlling rate and pattern of bone ingrowth. Total porosity Total porosity ranged from 70 Total porosity and the to 90%. Almost all of the studies technique of calculation should failed to report the effects of be reported.

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Findings in literature review

Recommendations

porosity on mechanical properties of BCP.

The relationship between total porosity and mechanical behaviors should be reported. Particle/grain size and shape should be determined and reported. Full mechanical properties should be reported i.e. mechanical strength, toughness, young's modulus (wet/dry condition). Sample preparation, size, type of the test and test parameters should to be stated. Use of proper sterilization techniques, short and optimum storage condition and characterization of biomaterials just before the implantation is recommended. Rationale for selection of analysis time frame should be reported. There is a clear need for standardization. Rationale should to be clarified for short and long study period. There is a clear need for standardization in selection of study period. Short term study (2–3 months) can be used for screening and optimization, but long term study (N6 months) is required to analyze detailed effects on biochemical composition and remodeling of bone. In addition to histological and histomorphometrical analysis, radiograph should be used to assure implant insertion and exclude displacement. It will help in evaluation of amount and quality of newly formed bone. Radiograph scoring system should be reported as well. CT-scan can further support the findings and allow 3D monitoring of newly formed bone.

Particle size/shape

The particle size ranged from 0.3 μm to 3000 μm

Mechanical properties

Mechanical properties of BCP were not reported in majority of studies.

Sterilization and storage condition

Few papers reported the full detail of biomaterials sterilization and storage conditions.

Analysis time

Analysis intervals varied significantly and ranged from 2 weeks to 52 weeks.

Total study period

Total study period ranged from 4 weeks to 12 months among studies. Majority of study period was 8 weeks (36%).

Investigation types

Majority of investigations included histology (15 studies, 100%) and histomorphometry (11 studies, 73%) and Radiographic reports (40%)

clinical effects of each particular BCP composition. This perhaps provides the researcher with a better prospective and allows more reproducible techniques for future applications. Finally, we believe that the debate of optimum ratio should not focus on finding a particular ratio as a magic ratio that fit all purposes. Rather, there may be a need to develop a set of ideal composition ratios for different applications based on clinical requirements. Therefore, BCP bone substitute needs to be engineered for the customized use in different anatomical sites for various functional and clinical needs; i.e. in elderly patients with lower bone remodeling and lower density, in patients with bony diseases (i.e. osteoporosis and tumor) and in trauma patients. 4.2. Recommendations for characterization and documentation of biomaterials It is recommended that for in vivo as well as in vitro studies, there should be a full disclosure of techniques utilized for BCP preparation as well as the details of sintering parameters. Unfortunately, these details seem to be frequently disregarded in the literature particularly in animal studies. Although, the produced final BCP products may bear

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comparable physicochemical properties, this will not guarantee their similar in vivo behaviors as well [107,112–114]. This may be contributed to minute differences in surface specific energy, surface charges, minerals spatial orientation and detailed topography which are not normally reported on. Furthermore, the BCP of the same chemical composition processed under different sintering conditions could produce different physicochemical and biological behaviors [134]. In addition, our review of literature has revealed the lack of proper characterization of bioceramics before their applications in animal studies. In general, XRD analysis should be an integral part of material characterization in all types of BCP studies. In addition to porosimetry and analysis of mechanical properties, other investigations such as SEM and TEM are also recommended (Fig. 3). A report of appropriate details including exact scaffolds' dimension, particle size, grain size, crystal size and shape provides a clear view of their impact on physicobiological properties [99]. A review of the related literature has also revealed that the majority of papers failed to report the complete porosity details of investigated BCP. A detailed report stating method of pore size analysis and technique of measurement of total porosity (i.e. macroporosity, microporosity, and interconnected porosity) should be clearly provided. The rationale for selection of total porosity and a particular range of pore sizes should be also stated, as this influences both the mechanical behaviors of biomaterials and the biological responses. Furthermore, distinction between terminologies such as nano, micro, meso and submicron should also be defined for any report of porosity. Unfortunately, the literature is not consistent with the use of these terms and there are no common definition criteria. According to the technical report by IUPAC (International Union of Pure and Applied Chemistry), the pore sizes or pore widths of nanoporous materials are subdivided into 3 categories as; micropores (b2 nm), mesopores (2–50 nm) and macropores (N 50 nm) [135]. However, the pore sizes have also been defined as macropores (N 100 μm), micropores (1–10 μm), and submicron pores (b1 μm) [136–138] while others simply classified pore sizes as macropores (N50 μm) and micropores (b50 μm) [139]. Nevertheless, the term “nano” was defined as being of the order of 100 nm or less (b 0.1 μm) by European Commission (SCENIHR) [140]. Therefore, there is a need to develop a generally accepted definition criteria for such terminologies to prevent confusion of the literature and avoid their misuse in scientific reports. In addition to the abovementioned biomaterials characterizations, there are other important factors that need to be reported in appropriate detail. It is necessary to consider the impact of functional load that the biomaterial is subjected to, which affects the process of bone repair as well. This impact varies depending on type of animal, anatomical site and dimension of the defect (implanted scaffold). For example, various anatomical sites may express different influences on the implant responses such as biodegradability and bioactivity, and result in dissimilar mechanical behaviors. Therefore, for more accurate reporting, the detail of the anatomical site of the implant, the nature, extent and duration of mechanical loads should also be provided. Furthermore, the cambium

layer of the periosteum has been confirmed to be a source of progenitor cells that develop into osteoblast cells. It should be reported clearly whether the periosteum is kept or removed after surgical defects preparation as this affects the bone healing process [141,142]. Other factors that have not been considered before are the remodeling capacity and type of the recipient bone (D1-D4) that could also affect the process of bone healing. All produced bioceramics should pass cytotoxicity and biocompatibility tests before in vivo trials. Furthermore, each lot of implant materials should be sterilized before application, however, it has been reported that some sterilization techniques may change the physicochemical properties of bioceramics [27,143]. Therefore, detail of sterilization techniques should be included in the reports of biomaterials studies. For a complete review, the interested readers can refer to CDC (Centers for Disease Control and Prevention) guidelines for disinfection and sterilization in healthcare facilities [144,145]. It is recommended that physicochemical characterizations be performed after bioceramics implant sterilization and shortly before implantation to address the possible changes in bioceramics nature. In this way, one can assure the similarity and consistency of all bioceramics materials before implantation and analysis. Another important issue is the possible effect of storage time and contamination on bioceramics properties. Therefore, it is highly recommended to include these data in clinical reports. Significant effort should be made to reduce the storage time and avoid contamination of implant materials before application. All of the abovementioned factors could contribute to the process of biomaterials integration after implantation and can influence the rate and pattern of bone healing. A clear statement of these contributing factors provides insight for comparison of the available literature so as to support better planning and execution of clinical research. Table 4 provides general recommendations for conducting and reporting animal studies analyzing biomaterials as bone substitutes. 4.3. Rationale and duration of pre-clinical animal study There is a significant gap between the outcomes of in vitro studies and intended clinical applications in patients due to absence of 3D simulation, circulating hormones and growth factors. Furthermore, the results of in vitro studies are very diverse; hence, these results cannot be applied directly or comprehensively to the biological models. Therefore, animal models are of great importance for examining the complexity of biological environment and study of disease pathogenesis, therapeutic materials, drug safety and efficacy [146]. Over recent years the number of animal studies appear to have decreased mainly due to ethical and economic considerations [147]. However, many of new materials and techniques still require testing on animal models and for the majority of medical implants, the number of animal studies has actually increased considerably in recent years [148]. Research using animal models should be conducted as per governmental and institutional regulations and guidelines. Furthermore, a comprehensive review and consultation should be made for

Fig. 3. SEM view of sintered BCP scaffold (HA/β-TCP, 60/40) at different magnifications. The presence of large number of pores at different scales significantly increases the surface roughness and total porosity.


development of study protocols before initiation of animal research. Appropriate species and anatomical sites should be selected according to the type of biomaterial implants. Different animals express different levels of endogenous signaling molecules such as circulating hormones, and growth factors. These factors may account for similar types of BCP expressing different biological behaviors in different animal models. It is presumed that larger animal models express closer similarity to human clinical trials. Many animal species have been used to investigate the efficacy of biomaterials in bony defects, including mice, rats, rabbits, dogs, pigs, sheep and goats. However, approximately 95% of all lab animals are rodent models as the animal model of choice for research because of reproducibility, throughput and economic considerations [149]. Small animal models (rats, rabbits) provide good choice for initial evaluation of different biomaterials effect on bone tissue regeneration before investigation of larger animal models (goats, sheep, dogs, pigs) [150]. The ideal animal models should have the following characteristics: similarity to human genome, high reproducibility, low morbidity and mortality rate, easy husbandry, high throughput, low cost, allow multiple investigations and offer adequate area of interest for study [151]. Anatomically, various areas of the animal skeleton have been examined as recipient sites for implant materials, including the femora, tibia, spine, mandible, maxilla and calvarium. The duration of the animal study depends on many factors such as, the purpose of study, type of animal, size of defect and type of implant. For small animal model a study period of 2 to 3 months provides enough information on type of repair, however, for larger animals this period may not be sufficient and information provided may be limited to the initial cellular behavior and biocompatibility. According to ASTM standard guidelines (F2721-09), usually a study period of N3 months is considered compulsory to confirm the histological extent of bone regeneration [142]. In general, many factors may contribute to an increase in the variability of outcome measures and should be considered in the research protocols and reports of animal studies. These include; the sex and age of animal, unilateral/bilateral defects, quality of animal handling, weight/non-weight bearing model, amount of mechanical force on implant, animal activity, type of diet, and duration of study. All of these factors can influence the bone healing process and need to be controlled and fully documented and reported. For example, improper maintenance of standard husbandry conditions or animal malnutrition may negatively influence the physiologic bone healing process in response to biomaterial implants. Therefore, a well-planned experimental design should consider all possible study variables (primary and secondary) and takes into account the influences of potential confounding (tertiary) variables. A failure to avoid or control these confounding variables affects the internal validity of the experimental animal studies. The ultimate goal of pre-clinical animal studies is to apply the outcome findings to clinical trials in human models. A general review of research studies that have extrapolated the protocols of animal studies to human trials reveals that many researchers have significantly changed their study protocols and investigation parameters to fit the human model [152]. This potentially calls into question the reproducibility and generalizability of the animal study results. However, due to nature of the human model, some changes in variables are considered acceptable during the transition from pre-clinical studies to clinical applications. Researchers should therefore adhere to available standard guidelines and procedures that tend to minimize the risk of bias in conducting and reporting the studies. This will also help in the smooth transition from animal models to human trials by improving the efficacy of all phases of the study process. It is important to notice that the findings of animal studies are not necessarily predictive of human conditions and as such these findings should be cautiously interpreted before potential human applications [142]. In conclusion, there is a significant need in improving the quality of research papers on both basic and animal studies to avoid the reporting

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of inaccurate and inconsistent findings. Such inconsistencies would probably interfere with the progress of future clinical trials and delay potential translation to human applications. For randomized controlled trial (RCT), the Cochrane guidelines provide general recommendations to reduce the risk of bias [153]. Furthermore, for conducting animal research, the ARRIVE guidelines are prepared to improve the quality of bioscience research reporting [154]. The ARRIVE guidelines consist of 20 items that promote reproducible, concise and comprehensive reporting of animal experiments, and improve the communication of research findings. 4.4. Sample size calculation; importance and impact Calculation of accurate sample size is important for both scientific validity and ethical approval of related studies. Appropriate sample size improves the reliability of study and precision of measurements in animal experiments [155]. A pre-study power determination (the probability of rejecting a null hypothesis when it is false) is required to select an appropriate sample size for correct detection of a clinically meaningful difference at a given level of significance [155,156]. This is important to avoid type II errors (failure to reject a false null hypothesis) that occur as a result of insufficient study power to detect a difference between interventions [157,158]. Typically the power is set to 80–90%, further we need to set the significance level (i.e. type I error rate; the probability of incorrect rejection of true null hypothesis) usually to 1% or 5%, the expected mean difference between control and study groups, and the expected variation as standard deviation (SD). For sample size calculation the mean differences and the SD can be estimated from pilot studies or previous publications of a similar model. This method is based on assumption of data with normal distribution [159]. The maximum accepted level of significance is set at 5% with 80% power to get the minimum possible number of animals required for the study. Although, some argue that the optimum number of animals is attained at 90% power, however, as the power increases and level of significance decreases, the sample size increases accordingly [159]. An appropriate sample size supports the statistical testing by allowing more confident calculation of the sample mean. Typically the minimum and maximum number of animals recruited in a study may vary at different animal research labs. Generally, the minimum number of animals used in experimental study is usually set at 3, while the maximum number of animals likely to be accepted per group may vary (n = 15–20). Recruitment of greater number of animals necessitates a strong justification that should be provided [160]. In general, sample size depends on many factors that should be considered before recruitment of animal models. The factors such as; objective and type of the study, number of study and control groups, number of investigations per group, nature of animal model, intrinsic variation among the animal model, type of treatment, consistency of performed surgical techniques, validity and reliability of analysis techniques and measurements, attrition rate, statistical power and statistical test, all influence the number of required animals in the study [155,159]. The majority of sample preparation techniques for assigned investigations results in the destruction of samples that is usually discarded and cannot be reused for a new set of investigations. The advantages of large animal models are that they provide larger samples sites and allow multiple biopsies within a specified sample of bone that in turn help in reducing the number of total animal required. Furthermore, they allow non-invasive monitoring and biopsy collection without the need to sacrifice the animals. However, large animals suffer from more genetic variation, require higher housing facilities and cost per animal compare to rodents model [161]. Although the importance of sample size calculation has been emphasized strongly in the literature, evidence suggests that it has been frequently disregarded in animal studies as apparent in the available literature [159]. Inappropriate sample size will affect the validity of study results and may interfere with the progress of further clinical studies.

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However, increasing the number of animals in a study is not always the proper method [155]. The challenges of increasing the sample size are the unavoidable higher cost and ethical justification for maintaining such large colonies of animals. Conducting multicenter animal studies may help to bypass these challenges by increasing the total sample size and distributing the cost related issue between different study centers. In conclusion, it is suggested that researchers include a detailed statement of sample size calculation and justification. Additional reports of effect size, SD, type I error, power, direction of effect (one or two tailed), attrition rate, and statistical techniques are also recommended to provide a full view and add strength to the paper [155,162]. 4.5. Critical size defect; a clarification The critical size defect (CSD) model is one of the main experimental methods for in vivo investigation of new biomaterials in tissue engineering. Classically, Schmitz et al.. [163] defined the CSD as the smallest size tissue defect that will not heal completely over the natural lifetime of an animal. In bony tissue, a CSD refers to a non-pathological artificial or naturally occurring defect that will not undergo spontaneous healing. According to the ASTM standard guidelines (F2721-09), CSD is defined as “a defect that will not heal without intervention”. However, clinically, CSD applies when the healing phase exceeds a period of 6 months in healthy adult patient [142]. On the other hand, some researchers argue about the abandonment of term “CSD” and use of “non-healing” as an alternative due to deviation from the original definition [164]. The typical dimension of CSDs has been defined by the ASTM standard guideline (F2721–09) for some animal models such as rat (0.5– 1.0 cm), rabbit (2 cm), dog (2.1–2.3 cm), goat (2.6–3.5 cm) and sheep (2.2–5.0 cm) [142]. However, this guideline is limited to mid-diaphyseal segmental defects in long bones. The detail about CSD in other bones such as calvaria is variable and can be found elsewhere [164,165]. In addition, the site specificity of CSD in many animal models has not undergone the required analysis to determine the smallest non-healing defect size according to the definition. The exact dimension of CSD is dependent on the type of animal, weight, age and exact anatomical location. For example an 8 mm defect is accepted to be the CSD for the rat calvaria of particular age but it is not true for larger animals [151,166]. In general, the length of artificial bone defect should be 1.5 to 2 times the diameter of selected bone [167]. It is strongly recommended to have a clear justification and reference about the use of term “critical size defect” before conducting any animal study. In order to confirm that the created CSD is in accordance to the standard definition, each animal study should include an untreated empty defect as a control group. However, if the literature is clearly established on the defect size, one can use the historical data instead of a comparative control group, thereby keeping the number of animals to a minimum [142]. Furthermore, during fabrication of bioceramics implants, the size of the implants should be considered according to the dimension of CSD as it varies among different animal species. 5. Conclusion Over the past century, animal research was the foundation for the development of new biomaterials for bone tissue engineering. To date, there is no comprehensive alternative for animal models in research to fully duplicate a whole, living system. In general, there is a lack of proper knowledge regarding the quality of experiments and regenerative potential of different biomaterials in animal studies, in particular BCP bioceramics. BCP has proved to be of great benefit for biomedical application that aid in bone regeneration. They are osteoconductive with possibility of acquiring osteoinductive features by controlling their physicochemical properties. Various efforts are made through the literature to improve their properties for enhanced bone tissue

regeneration. However, to date there is no agreement on ideal biomaterials properties for clinical applications. In this detailed review, we have selected BCP as an example to shed the light on one of the main challenging issues in biomaterials studies. A review of literature has revealed that investigators of biomaterials in clinical studies and animal study models in particular, have not standardized their experimental study protocols and have applied different approaches in characterization of biomaterials and different methods in reporting their findings. Because of this, it is extremely difficult to compare and contrast different study outcomes and so comparison of different study outcomes does not allow knowledge building for the improvement of biomaterials and application; this hinders further advancement in the field. The discrepancy in findings among studies will continue unless there are standard guidelines and protocols for researchers to follow that help to reduce experimental heterogeneity and bias in reporting. Therefore, there is a need to standardize the materials and methods in biomaterials characterization and analysis and reporting of biological outcomes. This better facilitates understanding of biological behaviors of biomaterials and allows their comparison under similar conditions. Furthermore, comparison of materials should be limited to studies in similar animal models, as there will be differences among different species of animal in response to bioceramics. This review emphasizes the need for development of standard protocols that should to be followed by researchers in animal studies. For this purpose, we have provided some guidelines in order to respect the animal ethics regulations and help to standardize study protocols for future research and publications. We believe this proposed set of standard criteria will encourage a more uniform method in conducting and reporting biomaterial analysis in pre-clinical and clinical studies and will facilitate the exchange of data among researchers. These guidelines along with other relevant guidelines should be followed in all animal research protocols as well as human trials so as to increase homogeneity and to facilitate the comparison and reproducibility of the studies and investigated biomaterials. In addition, this review encourages a better control over physicochemical parameters of biomaterials that improves understanding of their impacts on biological cellbased behaviors. A complete view of cell-scaffold interactions ultimately helps in improving the properties of future biomaterials for enhanced clinical performance in bone tissue regeneration. Acknowledgment The authors sincerely thank Dr. Naruporn Monmaturapoj (National Metals and Materials Technology Centre (MTEC, NSTDA), Thailand) for her useful comments and review of the manuscript. The authors declare there is no conflict of interest. References [1] S.W.S. Laurie, L.B. Kaban, J.B. Mulliken, J.E. Murray, Donor-site morbidity after harvesting rib and iliac bone, 1984. Plast. Reconstr. Surg. 73 933–938, http://dx.doi. org/10.1097/00006534-198406000-00014. [2] S. Dorozhkin, Bioceramics of calcium orthophosphates, 2010. Biomaterials 31 1465–1485, http://dx.doi.org/10.1016/j.biomaterials.2009.11.050. [3] R.G. Carrodeguas, S. De Aza, α-Tricalcium phosphate: synthesis, properties and biomedical applications, 2011. Acta Biomater. 7 3536–3546, http://dx.doi.org/10. 1016/j.actbio.2011.06.019. [4] G. Vereecke, J. Lemaître, Calculation of the solubility diagrams in the system Ca(OH)2-H3PO4-KOH-HNO3-CO2-H2O, 1990. J. Cryst. Growth 104 820–832, http://dx.doi.org/10.1016/0022-0248(90)90108-W. [5] S.M. Best, A.E. Porter, E.S. Thian, J. Huang, Bioceramics: past, present and for the future, 2008. J. Eur. Ceram. Soc. 28 1319–1327, http://dx.doi.org/10.1016/j. jeurceramsoc.2007.12.001. [6] M. Vallet-Regí, Ceramics for medical applications, 2001. J. Chem. Soc. Dalton Trans. 97–108. http://dx.doi.org/10.1039/b007852m. [7] J. Osborn, H. Newesely, The material science of calcium phosphate ceramics, 1980. Biomaterials 1 108–111, http://dx.doi.org/10.1016/0142-9612(80)90009-5. [8] P.V. Giannoudis, H. Dinopoulos, E. Tsiridis, Bone substitutes: an update, 2005. Injury 36 S20–S27, http://dx.doi.org/10.1016/j.injury.2005.07.029.

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