Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies

Скачать 160.06 Kb.
НазваниеCurrent Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies
Дата конвертации20.04.2013
Размер160.06 Kb.
1   2   3

(6) Computer Modelling of Bone Regeneration In CaP Scaffolds

Improvements in computer capacity now enable an increased model realism and complexity (e.g. 3D calculations, complex geometries, multi-scale and multi-physics) [151]. As a consequence of this technological revolution, there has been an enormous increase in the use of mathematical models in biology and medicine. These mathematical models can propose and test possible biological mechanisms, contributing to the unravelling of the complex nature of biological systems, like bone regeneration processes inside CaP structures. Moreover, they can be used to design and test possible experimental strategies in silico before they are tested in vitro or in vivo [152-153]. Eventually, all this knowledge can be used to develop clinically relevant CaP-based bone reparative units (figure 4).

Currently, many computational models of bone formation and regeneration in general [154], or even in scaffolds specifically [11] exist. Bohner et al. propose a theoretical approach to determine the effect of geometrical factors on the resorption rate of CaP scaffolds [155]. Their theoretical model was based on five assumptions: (i) the sphericity of the pores, (ii) a face-centered cubic packing of the pores, (iii) surface-controlled resorption, (iv) the resorption requires the presence of blood vessels (50 µm in diameter) and (v) the resorption time is proportional to the net amount of material [156]. The model calculations show that, based on these assumptions, the resorption time of a macroporous block depends on the pore radius which is determined by the size of the bone substitute and interpore distance [156]. Subsequently, the model was used to optimize the pore size of CaP scaffolds and validated with experimental data.

The theoretical model mentioned above looks, however, exclusively at geometrical scaffold properties and does not include biological variables such as cell or matrix densities. Byrne et al. developed a 3D mechanoregulatory model of bone regeneration in a regular scaffold to investigate the effect of porosity, Young’s modulus and dissolution rate on bone regeneration in different loading conditions [157]. They modelled the scaffold as a poroelastic material which resorbs in a linear, load-independent fashion, i.e. the porosity will be increased by a 0%, 0.5%, 1% per iteration for low, intermediate and high dissolution rates respectively [158]. Consequently, the size of all scaffold elements decreases uniformly resulting in an overall volumetric reduction while the scaffold geometry remains unaltered. Their calculations show that as scaffold degradation progresses, the regenerating tissue must take over the mechanical function of the bone-scaffold system, which would otherwise collapse due to a lack of mechanical strength. Moreover, all three variables (i.e. porosity, Young’s modulus and dissolution rate) appear to influence the amount of bone formation in a non-intuitive way, demonstrating the need to optimize scaffolds for site-specific loading requirements. This model was improved by including blood vessel growth thereby establishing a framework to investigate the effect of vascularization on bone formation [159].

Other studies have modeled the bone regeneration process inside biodegradable polymer-based scaffolds. Stops et al. further investigated the influence of mechanical strain and perfusive fluid flow on cell differentiation and proliferation within a collagen-glycosaminoglycan scaffold [160]. Sanz-Herrera et al. presented a multi-scale model of bone regeneration inside a porous scaffold [161]. The degradation mechanism of the biodegradable polymer scaffold was modelled as a hydrolysis process, i.e. the water content in the polymer chemically reacts and breaks down the polymer molecules resulting in biomaterial bulk erosion. The mechanical properties of the polymer were assumed to relate linearly to its molecular weight. The model was used to predict the evolution of the bone formation process in a scaffold implanted in the femoral condyle of a rabbit. They found a good qualitative agreement between the obtained computational and experimental results. Although further validation is necessary, the proposed multi-scale model is a useful tool to investigate the complex phenomena that occur at different length and time scales, i.e. the bone formation and scaffold resorption at the microscopic scale and the change of mechanical properties at the macroscopic scale. Lacroix et al. reviewed the current techniques used for scaffold development: from scaffold optimization of scaffolds by mathematical models (e.g. FEM) to scaffold design using computer aided design (CAD) and scaffold characterization by computed tomography (CT) [162].

Although the above models can be used to optimize some (mechanical) properties of scaffolds, e.g. the porosity, the micro-architecture, the Young’s modulus and dissolution rate, they neglect the influence of growth factors and other biochemical signals on the bone formation process. Moreover, the dissolution process is only crudely modeled, neglecting the influence of the degradation products (e.g. Ca2+ and Pi) on the cellular activities and bone formation processes. Carlier et al. developed and implemented an experimentally informed bioregulatory model of the effect of calcium ions released from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven ectopic bone formation [163]. The dissolution kinetics of the CaP scaffold were modeled by a general empirical equation [164], assuming that the dissolution rate is proportional to the driving force (i.e. the difference between the current and the saturated calcium concentration) and that the rate constant is time-independent. The amount of bone formation predicted by the model of Carlier et al. corresponded to the amount measured experimentally under similar conditions. Moreover, experimentally impaired bone formation due to conditions such as insufficient cell seeding and scaffold decalcification, was also retrieved in silico. Subsequently, this model was used to optimise CaP scaffold selection to make their use in combination with cells more clinically relevant.

Although computational models can contribute to the general knowledge on bone formation inside CaP structures and useful to customise the CaP carriers to the patient-specific needs as well as the particular bone application, experimental research is necessary to establish and validate the mathematical models. The most important parameters and their respective parameter values should be experimentally quantified. Consequently, the multidisciplinary problem of optimizing scaffold architecture and seeding protocols for bone tissue engineering strategies requires an integrative approach which was nicely summarized by Bohner et al. [165]: a combination of mathematical modelling to explain a mechanism of biomaterial-cell interactions with experimental research to provide data for the determination of model parameters as well as the validation of the mathematical model. This integrative approach requires a careful design and an extensive characterisation of the scaffold. Moreover, this process is intrinsically iterative: new experimental results can be fed to the model and new research hypotheses can result from thorough model analysis.

(7) Limitations, Future Perspectives and Conclusion

As discussed above, it is clear that the bone induction by CaP biomaterials is influenced by the physicochemical properties of the material and thus the subsequent cellular events of osteogenesis. However, the exact key determinant(s) of CaP osteoinduction, meaning the molecular mechanisms involved and the stem cell-material-host interactions upon implantation is/are still underdetermined. Therefore, further study to decipher the molecular signalling at the cellular level (such as receptor binding of the released Ca2+ and PO43- and the activation of critical intracellular osteogenesis pathway), and to understand the critical biological parameters that are essential for implanted cells to communicate and integrate effectively with the host system are required [166]. Unfortunately, the translation of the complex in vivo osteogenesis environment into an in vitro system is far from trivial, and the predictiveness of in vitro observations often does not correlate well with the in vivo bone formation capability [89]. This is due to some critical issues such as the use of correct cell types for a specific type of CaP-based biomaterial and the selection of the correct culture conditions. Therefore, there is a need for customisation of the CaP-based biomaterial to overcome these limitations. Moreover, other parameters present in vivo, including the compositions and dynamics of body fluids and blood vessel in-growth, are technically challenging to be translated into a simplified in vitro setting. Recently, the reactivity of CaP-based biomaterials in culture medium was revealed to have significant influences on Ca2+ and PO43- dissolution kinetics and the subsequent cellular behaviour, which was not correlated to the ion dissolution behaviour evaluated in simulated body fluid (SBF) or phosphate-buffered saline. This must be carefully considered when evaluating the osteoinductive potential of the material [167]. We propose the standardisation of in vitro dissolution tests to evaluate the ions release kinetics of a CaP-based biomaterial, where the in vitro dissolution experiment should resemble and be customised to the in vivo environment as close as possible [156]. Firstly, the CaP scaffolds should be thoroughly characterised (porosity, pore size distribution, grain size, surface topography and surface area, composition and etc.) before and after the dissolution test. Here, we highlight on the use of non-invasive methods including Raman spectroscopy [158] and micro- or nano-focus X-ray computed tomography to obtain quantitative characterisation of the material properties [168]. Secondly, we propose a dual solution system for the in vitro dissolution testing using simulated body fluid [169] and culture medium (with and without the presence of cells) [170]. This will allow for the characterisation of the intrinsic ionic dissolution behaviour of the biomaterial in a solution having similar ionic composition to the bodily fluid, followed by the understanding on the interactions or the influences of the cells and proteins on the ionic dissolution kinetics. In fact, more and more studies have shown the reduction of Ca2+ in the culture medium, indicating the formation of new CaP crystals onto the existing CaP substrate [128]. Thirdly, the experimental setting should also account for the local in vivo hydrodynamic of the bodily fluid, for instance, at the implantation site. This can be achieved by performing the dissolution test under dynamic condition (e.g. using perfusion system) at physiologically relevant flow rate [118, 127].

Moreover, the in vitro and in vivo experiments should be designed in such a way that they minimise variability and enable quantification, thus providing the essential data for the determination of model parameters as well as for the model validation. It is of note that as mathematical models predict the dynamics at different scales (e.g. molecular, cellular and tissue) as a function of time and space, temporal and spatial quantitative data are crucial to the success of mathematical models. Clearly, computational modelling plays an essential role to further unravel the complex mechanism of CaP osteoinduction in vivo. Most of the current models look either at mechanoregulatory or bioregulatory stimuli, depending on the specific research question that is being answered. In the future, however, these models could be combined to further improve the predictive capabilities of the model.

The limitations mentioned above underline the importance of an interdisciplinary strategy for the optimisation of CaP scaffolds for bone tissue engineering applications [165]. An essential characteristic of these integrative research efforts is their iterativity: model analysis can lead to new research hypotheses and new experimental findings can be used to improve the predictive capacities of the model.

In conclusion, this review discussed the importance of CaP for physiological bone homeostasis as well as its potential for bone tissue engineering. Although numerous studies have been devoted to unravel the mechanisms of osteoinductivity of CaP scaffolds, many questions still remain unresolved. To overcome this bottleneck, it is therefore essential that experimental and computational research are combined so that the complex in vivo biological process of bone regeneration inside CaP constructs can be deciphered in an effective and systematic manners. This includes computational modelling and correlation analysis between the physicochemical properties of CaP constructs and the influences on in vitro and in vivo biological outcomes, thus identifying the critical parameters and thorough understanding of the underlying biological that govern osteoinductivity of CaP constructs. Only in this way, it will be possible to make CaP a clinically relevant and predictive tissue engineering construct for effective bone defect repair.


This work is funded by the KU Leuven IDO project 05/009 – QuEST, Stem Cell Institute of Leuven – KU Leuven, ENDEAVOUR project G.0982.11N. Aurélie Carlier and Johanna Bolander are PhD fellows of the Research Foundation Flanders (FWO Vlaanderen). The work is part of Prometheus, the Leuven Research and Development Division of Skeletal Tissue Engineering of KU Leuven: The authors declare that they have no potential conflict of interest and they had and will have no financial relationships with companies whose products are relevant to the subject of this study.


[1] Murugan R, Ramakrishna S. Development of nanocomposites for bone grafting. Compos Sci Technol 2005;65:2385-406.

[2] Fratzl P. Bone fracture: When the cracks begin to show. Nat Mater 2008;7:610-2.

[3] Rho JY, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys 1998;20:92-102.

[4] Athanasiou KA, Zhu C, Lanctot DR, Agrawal CM, Wang X. Fundamentals of biomechanics in tissue engineering of bone. Tissue Eng 2000;6:361-81.

[5] Goldstein SA. The mechanical properties of trabecular bone: dependence on anatomic location and function. J Biomech 1987;20:1055-61.

[6] Keaveny TM, Morgan EF, Niebur GL, Yeh OC. Biomechanics of trabecular bone. Annu Rev Biomed Eng 2001;3:307-33.

[7] Minns RJ, Atkinson A, Steven FS. The role of calcium in the mechanical behaviour of bone. Phys Med Biol 1983;28:1057-66.

[8] Currey JD. Bones: Structure and Mechanics. New Jersey: Princeton University Press; 2006.

[9] Fantner GE, Hassenkam T, Kindt JH, Weaver JC, Birkedal H, Pechenik L, et al. Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture. Nat Mater 2005;4:612-6.

[10] Fantner GE, Adams J, Turner P, Thurner PJ, Fisher LW, Hansma PK. Nanoscale ion mediated networks in bone: osteopontin can repeatedly dissipate large amounts of energy. Nano Lett 2007;7:2491-8.

[11] Nakahara H, Dennis JE, Bruder SP, Haynesworth SE, Lennon DP, Caplan AI. In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells. Exp Cell Res 1991;195:492-503.

[12] Colfen H. Biomineralization: A crystal-clear view. Nat Mater 2010;9:960-1.

[13] Stewart AJ, Roberts SJ, Seawright E, Davey MG, Fleming RH, Farquharson C. The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization. Bone 2006;39:1000-7.

[14] Anderson HC, Garimella R, Tague SE. The role of matrix vesicles in growth plate development and biomineralization. Front Biosci 2005;10:822-37.

[15] Golub EE. Role of matrix vesicles in biomineralization. Biochim Biophys Acta 2009;1790:1592-8.

[16] Siffert RS. The role of alkaline phosphatase in osteogenesis. J Exp Med 1951;93:415-26.

[17] Golub EE, Harrison G, Taylor AG, Camper S, Shapiro IM. The role of alkaline phosphatase in cartilage mineralization. Bone Miner 1992;17:273-8.

[18] Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nihon Med Sch 2010;77:4-12.

[19] Houston B, Stewart AJ, Farquharson C. PHOSPHO1-A novel phosphatase specifically expressed at sites of mineralisation in bone and cartilage. Bone 2004;34:629-37.

[20] Roberts S, Narisawa S, Harmey D, Millan JL, Farquharson C. Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization. J Bone Miner Res 2007;22:617-27.

[21] Meyer U, Meyer T, Vosshans J, Joos U. Decreased expression of osteocalcin and osteonectin in relation to high strains and decreased mineralization in mandibular distraction osteogenesis. J Craniomaxillofac Surg 1999;27:222-7.

[22] Monfoulet L, Malaval L, Aubin JE, Rittling SR, Gadeau AP, Fricain JC, et al. Bone sialoprotein, but not osteopontin, deficiency impairs the mineralization of regenerating bone during cortical defect healing. Bone 2010;46:447-52.

[23] Roach HI. Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biol Int 1994;18:617-28.

[24] Rosenthal AK, Gohr CM, Uzuki M, Masuda I. Osteopontin promotes pathologic mineralization in articular cartilage. Matrix Biol 2007;26:96-105.

[25] Mochida Y, Parisuthiman D, Pornprasertsuk-Damrongsri S, Atsawasuwan P, Sricholpech M, Boskey AL, et al. Decorin modulates collagen matrix assembly and mineralization. Matrix Biol 2009;28:44-52.

[26] Gopalakrishnan R, Suttamanatwong S, Carlson AE, Franceschi RT. Role of matrix Gla protein in parathyroid hormone inhibition of osteoblast mineralization. Cells Tissues Organs 2005;181:166-75.

[27] Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res 2003;18:1842-53.

[28] Suzuki A, Ghayor C, Guicheux J, Magne D, Quillard S, Kakita A, et al. Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells. J Bone Miner Res 2006;21:674-83.

[29] Hughes-Fulford M, Li CF. The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization. J Orthop Surg Res 2011;6:8.

[30] Clase CM, Norman GL, Beecroft ML, Churchill DN. Albumin-corrected calcium and ionized calcium in stable haemodialysis patients. Nephrol Dial Transplant 2000;15:1841-6.

[31] Payne RB, Carver ME, Morgan DB. Interpretation of serum total calcium: effects of adjustment for albumin concentration on frequency of abnormal values and on detection of change in the individual. J Clin Pathol 1979;32:56-60.

[32] Orwar O, Lobovkina T, Gozen I, Erkan Y, Olofsson J, Weber SG. Protrusive growth and periodic contractile motion in surface-adhered vesicles induced by Ca(2+)-gradients. Soft Matter 2010;6:268-72.

[33] Breitwieser GE. Extracellular calcium as an integrator of tissue function. Int J Biochem Cell Biol 2008;40:1467-80.

[34] Dvorak MM, Riccardi D. Ca2+ as an extracellular signal in bone. Cell Calcium 2004;35:249-55.

[35] Olszak IT, Poznansky MC, Evans RH, Olson D, Kos C, Pollak MR, et al. Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo. J Clin Invest 2000;105:1299-305.

[36] Godwin SL, Soltoff SP. Extracellular calcium and platelet-derived growth factor promote receptor-mediated chemotaxis in osteoblasts through different signaling pathways. J Biol Chem 1997;272:11307-12.

[37] Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 2006;439:599-603.

[38] Aguirre A, Gonzalez A, Planell JA, Engel E. Extracellular calcium modulates in vitro bone marrow-derived Flk-1+ CD34+ progenitor cell chemotaxis and differentiation through a calcium-sensing receptor. Biochem Biophys Res Commun 2010;393:156-61.

[39] Duncan RL, Akanbi KA, Farach-Carson MC. Calcium signals and calcium channels in osteoblastic cells. Semin Nephrol 1998;18:178-90.

[40] Huiskes R, Ruimerman R, van Lenthe GH, Janssen JD. Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 2000;405:704-6.

[41] Zayzafoon M. Calcium/calmodulin signaling controls osteoblast growth and differentiation. J Cell Biochem 2006;97:56-70.

[42] Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, et al. Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc Natl Acad Sci U S A 2004;101:5140-5.

[43] Ozkucur N, Perike S, Sharma P, Funk RH. Persistent directional cell migration requires ion transport proteins as direction sensors and membrane potential differences in order to maintain directedness. BMC Cell Biol 2011;12:4.

[44] Ozkucur N, Monsees TK, Perike S, Do HQ, Funk RH. Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells. PLoS One 2009;4:e6131.

[45] Yamauchi M, Yamaguchi T, Kaji H, Sugimoto T, Chihara K. Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells. Am J Physiol Endocrinol Metab 2005;288:E608-16.

[46] Barradas AM, Fernandes HA, Groen N, Chai YC, Schrooten J, van de Peppel J, et al. A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials 2012;33:3205-15.

[47] Valerio P, Pereira MM, Goes AM, Leite MF. BG60S dissolution interferes with osteoblast calcium signals. J Mater Sci Mater Med 2007;18:265-71.

[48] Dvorak MM, Chen TH, Orwoll B, Garvey C, Chang W, Bikle DD, et al. Constitutive activity of the osteoblast Ca2+-sensing receptor promotes loss of cancellous bone. Endocrinology 2007;148:3156-63.

[49] Tsai JA, Bucht E, Torring O, Kindmark H. Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts. Horm Metab Res 2004;36:22-6.

[50] Titorencu I, Jinga VV, Constantinescu E, Gafencu AV, Ciohodaru C, Manolescu I, et al. Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells. Cytotherapy 2007;9:682-96.

[51] Maeno S, Niki Y, Matsumoto H, Morioka H, Yatabe T, Funayama A, et al. The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. Biomaterials 2005;26:4847-55.

[52] Nakade O, Takahashi K, Takuma T, Aoki T, Kaku T. Effect of extracellular calcium on the gene expression of bone morphogenetic protein-2 and -4 of normal human bone cells. J Bone Miner Metab 2001;19:13-9.

[53] Pekkinen M, Ahlstrom M, Riehle U, Lamberg-Allardt C. Effects of extracellular calcium and phosphate on expression and realese of parathyroid hormone-related protein in osteoblast progenitor cells. Bone 2007;40:S270-S.

[54] Lee YK, Song J, Lee SB, Kim KM, Choi SH, Kim CK, et al. Proliferation, differentiation, and calcification of preosteoblast-like MC3T3-E1 cells cultured onto noncrystalline calcium phosphate glass. J Biomed Mater Res A 2004;69:188-95.

[55] Nayab SN, Jones FH, Olsen I. Modulation of the human bone cell cycle by calcium ion-implantation of titanium. Biomaterials 2007;28:38-44.

[56] Jalota S, Bhaduri SB, Tas AC. Osteoblast proliferation on neat and apatite-like calcium phosphate-coated titanium foam scaffolds. Mat Sci Eng C-Bio S 2007;27:432-40.

[57] Liu Y, Cooper PR, Barralet JE, Shelton RM. Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells. Biomaterials 2007;28:1393-403.

[58] Park JW, Park KB, Suh JY. Effects of calcium ion incorporation on bone healing of Ti6Al4V alloy implants in rabbit tibiae. Biomaterials 2007;28:3306-13.

[59] Piters E, Boudin E, Van Hul W. Wnt signaling: a win for bone. Arch Biochem Biophys 2008;473:112-6.

[60] Conrads KA, Yi M, Simpson KA, Lucas DA, Camalier CE, Yu LR, et al. A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol Cell Proteomics 2005;4:1284-96.

[61] Beck GR, Jr. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J Cell Biochem 2003;90:234-43.

[62] Beck GR, Jr., Knecht N. Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent. J Biol Chem 2003;278:41921-9.

[63] Julien M, Khoshniat S, Lacreusette A, Gatius M, Bozec A, Wagner EF, et al. Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. J Bone Miner Res 2009;24:1856-68.

[64] Liu YK, Lu QZ, Pei R, Ji HJ, Zhou GS, Zhao XL, et al. The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering. Biomed Mater 2009;4:025004.

[65] Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 2005;19:1093-104.

[66] Habibovic P, Bassett DC, Doillon CJ, Gerard C, McKee MD, Barralet JE. Collagen biomineralization in vivo by sustained release of inorganic phosphate ions. Adv Mater 2010;22:1858-62.

[67] Barrere F, van Blitterswijk CA, de Groot K. Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics. Int J Nanomedicine 2006;1:317-32.

[68] Chai YC, Roberts SJ, Schrooten J, Luyten FP. Probing the osteoinductive effect of calcium phosphate by using an in vitro biomimetic model. Tissue Eng Part A 2011;17:1083-97.

[69] Chai YC, Roberts SJ, Van Bael S, Chen Y, Luyten FP, Schrooten J. Multi-level factorial analysis of Ca2+/Pi supplementation as bio-instructive media for in vitro biomimetic engineering of three-dimensional osteogenic hybrids. Tissue Eng Part C Methods 2012;18:90-103.

[70] Bhandari M, Jain AK. Bone stimulators: Beyond the black box. Indian J Orthop 2009;43:109-10.

[71] Eyckmans J, Roberts SJ, Schrooten J, Luyten FP. A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling. J Cell Mol Med 2010;14:1845-56.

[72] Habibovic P, de Groot K. Osteoinductive biomaterials--properties and relevance in bone repair. J Tissue Eng Regen Med 2007;1:25-32.

[73] Pazzaglia UE, Zatti G, Ragni P, Ceciliani L. The role of mineralization in experimental models of osteogenetic induction with decalcified bone matrix. Ital J Orthop Traumatol 1988;14:369-75.

[74] Jukes JM, Both SK, Leusink A, Sterk LM, van Blitterswijk CA, de Boer J. Endochondral bone tissue engineering using embryonic stem cells. Proc Natl Acad Sci U S A 2008;105:6840-5.

[75] Yuan H, Fernandes H, Habibovic P, de Boer J, Barradas AM, de Ruiter A, et al. Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. Proc Natl Acad Sci U S A 2010;107:13614-9.

[76] Yuan H, van Blitterswijk CA, de Groot K, de Bruijn JD. Cross-species comparison of ectopic bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) scaffolds. Tissue Eng 2006;12:1607-15.

[77] Urist MR. Bone: formation by autoinduction. Science 1965;150:893-9.

[78] Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res 1971;50:1392-406.

[79] Jones W, Roberts RE. Pathological Calcification and Ossification in Relation to Leriche and Policard's Theory. Proc R Soc Med 1933;26:853-9.

[80] Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective. Am J Physiol Endocrinol Metab 2004;286:E686-96.

[81] Zebboudj AF, Shin V, Bostrom K. Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells. J Cell Biochem 2003;90:756-65.

[82] Yuan H, Yang Z, Li Y, Zhang X, De Bruijn JD, De Groot K. Osteoinduction by calcium phosphate biomaterials. J Mater Sci Mater Med 1998;9:723-6.

[83] Alvis M, Lalor P, Brown MK, Thorn MR, Block JE, Hornby S, et al. Osteoinduction by a collagen mineral composite combined with isologous bone marrow in a subcutaneous rat model. Orthopedics 2003;26:77-80.

[84] Habibovic P, van der Valk CM, van Blitterswijk CA, De Groot K, Meijer G. Influence of octacalcium phosphate coating on osteoinductive properties of biomaterials. J Mater Sci Mater Med 2004;15:373-80.

[85] Ripamonti U, Crooks J, Khoali L, Roden L. The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs. Biomaterials 2009;30:1428-39.

[86] Yuan HP, de Bruijn JD, Zhang XD, van Blitterswijk CA, de Groot K. Bone induction by porous glass ceramic made from Bioglass (R) (45S5). Journal of Biomedical Materials Research 2001;58:270-6.

[87] Winter GD, Simpson BJ. Heterotopic bone formed in a synthetic sponge in the skin of young pigs. Nature 1969;223:88-90.

[88] Fujibayashi S, Neo M, Kim HM, Kokubo T, Nakamura T. Osteoinduction of porous bioactive titanium metal. Biomaterials 2004;25:443-50.

[89] Barradas AM, Yuan H, van Blitterswijk CA, Habibovic P. Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms. Eur Cell Mater 2011;21:407-29; discussion 29.

[90] Fan H, Ikoma T, Tanaka J, Zhang X. Surface structural biomimetics and the osteoinduction of calcium phosphate biomaterials. J Nanosci Nanotechnol 2007;7:808-13.

[91] Habibovic P, Yuan HP, van der Valk CM, Meijer G, van Blitterswijk CA, de Groot K. 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials 2005;26:3565-75.

[92] Akiyama N, Takemoto M, Fujibayashi S, Neo M, Hirano M, Nakamura T. Difference between dogs and rats with regard to osteoclast-like cells in calcium-deficient hydroxyapatite-induced osteoinduction. Journal of Biomedical Materials Research Part A 2011;96A:402-12.

[93] Habibovic P, Sees TM, van den Doel MA, van Blitterswijk CA, de Groot K. Osteoinduction by biomaterials - Physicochemical and structural influences. Journal of Biomedical Materials Research Part A 2006;77A:747-62.

[94] Li X, Liu H, Niu X, Fan Y, Feng Q, Cui FZ, et al. Osteogenic differentiation of human adipose-derived stem cells induced by osteoinductive calcium phosphate ceramics. J Biomed Mater Res B Appl Biomater 2011;97:10-9.

[95] Lu Z, Zreiqat H. The osteoconductivity of biomaterials is regulated by bone morphogenetic protein 2 autocrine loop involving alpha2beta1 integrin and mitogen-activated protein kinase/extracellular related kinase signaling pathways. Tissue Eng Part A 2010;16:3075-84.

[96] Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J, Lietze A, et al. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci U S A 1984;81:371-5.

[97] Cheng L, Ye F, Yang R, Lu X, Shi Y, Li L, et al. Osteoinduction of hydroxyapatite/beta-tricalcium phosphate bioceramics in mice with a fractured fibula. Acta Biomater 2010;6:1569-74.

[98] Ripamonti U, Roden LC. Induction of bone formation by transforming growth factor-beta2 in the non-human primate Papio ursinus and its modulation by skeletal muscle responding stem cells. Cell Prolif 2010;43:207-18.

[99] Rueger JM, Siebert HR, Dohr-Fritz M, Schmidt H, Pannike A. Time sequence of osteoinduction and osteostimulation elicited by biologic bone replacement materials. Life Support Syst 1985;3 Suppl 1:471-5.

[100] Mulliken JB, Kaban LB, Glowacki J. Induced osteogenesis--the biological principle and clinical applications. J Surg Res 1984;37:487-96.

[101] Low KL, Tan SH, Zein SH, Roether JA, Mourino V, Boccaccini AR. Calcium phosphate-based composites as injectable bone substitute materials. J Biomed Mater Res B Appl Biomater 2010;94:273-86.

[102] Coffin JD, Florkiewicz RZ, Neumann J, Mort-Hopkins T, Dorn GW, 2nd, Lightfoot P, et al. Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol Biol Cell 1995;6:1861-73.

[103] LeGeros RZ. Calcium phosphate biomaterials: An update. Int J Oral-Med Sci 2006;4:117-23.

[104] Bose S, Tarafder S. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review. Acta Biomater 2012;8:1401-21.

[105] Ginebra MP, Traykova T, Planell JA. Calcium phosphate cements as bone drug delivery systems: a review. J Control Release 2006;113:102-10.

[106] LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev 2008;108:4742-53.

[107] Chow LC. Next generation calcium phosphate-based biomaterials. Dent Mater J 2009;28:1-10.

[108] Yoshikawa M, Tsuji N, Shimomura Y, Hayashi H, Ohgushi H. Osteogenesis depending on geometry of porous hydroxyapatite scaffolds. Calcif Tissue Int 2008;83:139-45.

[109] Kuboki Y, Jin Q, Takita H. Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis. J Bone Joint Surg Am 2001;83-A Suppl 1:S105-15.

[110] Mankani MH, Afghani S, Franco J, Launey M, Marshall S, Marshall GW, et al. Lamellar spacing in cuboid hydroxyapatite scaffolds regulates bone formation by human bone marrow stromal cells. Tissue Eng Part A 2011;17:1615-23.

[111] Wilson CE, de Bruijn JD, van Blitterswijk CA, Verbout AJ, Dhert WJ. Design and fabrication of standardized hydroxyapatite scaffolds with a defined macro-architecture by rapid prototyping for bone-tissue-engineering research. J Biomed Mater Res A 2004;68:123-32.

[112] Duan Y, Yao Z, Wang C, Chen J, Zhang X. [A study of bone-like apatite formation on porous calcium phosphate ceramics in dynamic SBF]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2002;19:365-9.

[113] Li X, van Blitterswijk CA, Feng Q, Cui F, Watari F. The effect of calcium phosphate microstructure on bone-related cells in vitro. Biomaterials 2008;29:3306-16.

[114] Fischer EM, Layrolle P, Van Blitterswijk CA, De Bruijn JD. Bone formation by mesenchymal progenitor cells cultured on dense and microporous hydroxyapatite particles. Tissue Eng 2003;9:1179-88.

[115] Balaguer T, Boukhechba F, Clave A, Bouvet-Gerbettaz S, Trojani C, Michiels JF, et al. Biphasic calcium phosphate microparticles for bone formation: benefits of combination with blood clot. Tissue Eng Part A 2010;16:3495-505.

[116] Yuan H, van Blitterswijk CA, de Groot K, de Bruijn JD. A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods. J Biomed Mater Res A 2006;78:139-47.

[117] Daculsi G, LeGeros RZ. Biphasic calcium phosphate (BCP) bioceramics: Chemical, physical and biological properties. Enc Biomat Biomed Eng 2006:1-9.

[118] Duan YR, Zhang ZR, Wang CY, Chen JY, Zhang XD. Dynamic study of calcium phosphate formation on porous HA/TCP ceramics. J Mater Sci Mater Med 2005;16:795-801.

[119] Daculsi G, Laboux O, Malard O, Weiss P. Current state of the art of biphasic calcium phosphate bioceramics. J Mater Sci Mater Med 2003;14:195-200.

[120] Arinzeh TL, Tran T, McAlary J, Daculsi G. A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials 2005;26:3631-8.

[121] Ye F, Lu X, Lu B, Wang J, Shi Y, Zhang L, et al. A long-term evaluation of osteoinductive HA/beta-TCP ceramics in vivo: 4.5 years study in pigs. J Mater Sci Mater Med 2007;18:2173-8.

[122] Lobo SE, Arinzeh TL. Biphasic calcium phosphate ceramics for bone regeneration and tissue engineering applications. Materials 2010;3:815-26.

[123] Garrido CA, Lobo SE, Turibio FM, Legeros RZ. Biphasic calcium phosphate bioceramics for orthopaedic reconstructions: clinical outcomes. Int J Biomater 2011;2011:129727.

[124] El-Ghannam A. Bone reconstruction: from bioceramics to tissue engineering. Expert Rev Med Devices 2005;2:87-101.

[125] Kim KH, Ramaswamy N. Electrochemical surface modification of titanium in dentistry. Dent Mater J 2009;28:20-36.

[126] Bosetti M, Lloyd AW, Santin M, Denyer SP, Cannas M. Effects of phosphatidylserine coatings on titanium on inflammatory cells and cell-induced mineralisation in vitro. Biomaterials 2005;26:7572-8.

[127] Chai YC, Truscello S, Bael SV, Luyten FP, Vleugels J, Schrooten J. Perfusion electrodeposition of calcium phosphate on additive manufactured titanium scaffolds for bone engineering. Acta Biomater 2011;7:2310-9.

[128] Chai YC, Kerckhofs G, Roberts SJ, Van Bael S, Schepers E, Vleugels J, et al. Ectopic bone formation by 3D porous calcium phosphate-Ti6Al4V hybrids produced by perfusion electrodeposition. Biomaterials 2012;33:4044-58.

[129] Mourino V, Cattalini JP, Boccaccini AR. Metallic ions as therapeutic agents in tissue engineering scaffolds: an overview of their biological applications and strategies for new developments. J R Soc Interface 2012;9:401-19.

[130] Guldberg RE. Spatiotemporal delivery strategies for promoting musculoskeletal tissue regeneration. J Bone Miner Res 2009;24:1507-11.

[131] Enomoto-Iwamoto M, Iwamoto M, Mukudai Y, Kawakami Y, Nohno T, Higuchi Y, et al. Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes. J Cell Biol 1998;140:409-18.

[132] Vukicevic S, Grgurevic L. BMP-6 and mesenchymal stem cell differentiation. Cytokine Growth Factor Rev 2009;20:441-8.

[133] Tan TW, Huang YL, Chang JT, Lin JJ, Fong YC, Kuo CC, et al. CCN3 increases BMP-4 expression and bone mineralization in osteoblasts. J Cell Physiol 2012;227:2531-41.

[134] Franceschi RT. The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. Crit Rev Oral Biol Med 1999;10:40-57.

[135] Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci U S A 1981;78:7599-603.

[136] Seeherman H, Wozney J, Li R. Bone morphogenetic protein delivery systems. Spine (Phila Pa 1976) 2002;27:S16-23.

[137] Sampath TK, Reddi AH. Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix. Proc Natl Acad Sci U S A 1983;80:6591-5.

[138] Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery). J Tissue Eng Regen Med 2008;2:81-96.

[139] Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528-34.

[140] Carragee EJ, Ghanayem AJ, Weiner BK, Rothman DJ, Bono CM. A challenge to integrity in spine publications: years of living dangerously with the promotion of bone growth factors. Spine J 2011;11:463-8.

[141] Langer R, Folkman J. Polymers for the sustained release of proteins and other macromolecules. Nature 1976;263:797-800.

[142] Fujita N, Matsushita T, Ishida K, Sasaki K, Kubo S, Matsumoto T, et al. An analysis of bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein 2 from a biodegradable sponge composed of gelatin and beta-tricalcium phosphate. J Tissue Eng Regen Med 2012;6:291-8.

[143] Kang Y, Kim S, Khademhosseini A, Yang Y. Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2. Biomaterials 2011;32:6119-30.

[144] Gilbert M, Shaw WJ, Long JR, Nelson K, Drobny GP, Giachelli CM, et al. Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion. J Biol Chem 2000;275:16213-8.

[145] Raghunathan V, Gibson JM, Goobes G, Popham JM, Louie EA, Stayton PS, et al. Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals. J Phys Chem B 2006;110:9324-32.

[146] Zimmerman LB, De Jesus-Escobar JM, Harland RM. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 1996;86:599-606.

[147] Brunet LJ, McMahon JA, McMahon AP, Harland RM. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 1998;280:1455-7.

[148] Vehof JW, Takita H, Kuboki Y, Spauwen PH, Jansen JA. Histological characterization of the early stages of bone morphogenetic protein-induced osteogenesis. J Biomed Mater Res 2002;61:440-9.

[149] Kuboki Y, Takita H, Kobayashi D, Tsuruga E, Inoue M, Murata M, et al. BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res 1998;39:190-9.

[150] Kuboki Y, Saito T, Murata M, Takita H, Mizuno M, Inoue M, et al. Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell-differentiation. Connect Tissue Res 1995;32:219-26.

[151] van der Meulen MC, Huiskes R. Why mechanobiology? A survey article. J Biomech 2002;35:401-14.

[152] Geris L, Reed AA, Vander Sloten J, Simpson AH, Van Oosterwyck H. Occurrence and treatment of bone atrophic non-unions investigated by an integrative approach. PLoS Comput Biol 2010;6:e1000915.

[153] Faratian D, Goltsov A, Lebedeva G, Sorokin A, Moodie S, Mullen P, et al. Systems biology reveals new strategies for personalizing cancer medicine and confirms the role of PTEN in resistance to trastuzumab. Cancer Res 2009;69:6713-20.

[154] Geris L, Vander Sloten J, Van Oosterwyck H. In silico biology of bone modelling and remodelling: regeneration. Philos Transact A Math Phys Eng Sci 2009;367:2031-53.

[155] Bohner M, Baumgart F. Theoretical model to determine the effects of geometrical factors on the resorption of calcium phosphate bone substitutes. Biomaterials 2004;25:3569-82.

[156] Impens S, Chen Y, Mullens S, Luyten F, Schrooten J. Controlled cell-seeding methodologies: a first step toward clinically relevant bone tissue engineering strategies. Tissue Eng Part C Methods 2010;16:1575-83.

[157] Byrne DP, Lacroix D, Planell JA, Kelly DJ, Prendergast PJ. Simulation of tissue differentiation in a scaffold as a function of porosity, Young's modulus and dissolution rate: application of mechanobiological models in tissue engineering. Biomaterials 2007;28:5544-54.

[158] Gentleman E, Swain RJ, Evans ND, Boonrungsiman S, Jell G, Ball MD, et al. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation. Nat Mater 2009;8:763-70.

[159] Checa S, Prendergast PJ. Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: a mechano-biological model using a lattice approach to simulate cell activity. J Biomech 2010;43:961-8.

[160] Stops AJ, Heraty KB, Browne M, O'Brien FJ, McHugh PE. A prediction of cell differentiation and proliferation within a collagen-glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow. J Biomech 2010;43:618-26.

[161] Sanz-Herrera JA, Garcia-Aznar JM, Doblare M. A mathematical model for bone tissue regeneration inside a specific type of scaffold. Biomech Model Mechanobiol 2008;7:355-66.

[162] Lacroix D, Planell JA, Prendergast PJ. Computer-aided design and finite-element modelling of biomaterial scaffolds for bone tissue engineering. Philos Transact A Math Phys Eng Sci 2009;367:1993-2009.

[163] Carlier A, Chai YC, Moesen M, Theys T, Schrooten J, Van Oosterwyck H, et al. Designing optimal calcium phosphate scaffold-cell combinations using an integrative model-based approach. Acta Biomater 2011;7:3573-85.

[164] Zhang Q, Chen J, Feng J, Cao Y, Deng C, Zhang X. Dissolution and mineralization behaviors of HA coatings. Biomaterials 2003;24:4741-8.

[165] Bohner M, Loosli Y, Baroud G, Lacroix D. Commentary: Deciphering the link between architecture and biological response of a bone graft substitute. Acta Biomater 2011;7:478-84.

[166] Chai YC, Roberts SJ, Desmet E, Kerckhofs G, van Gastel N, Geris L, et al. Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers. Biomaterials 2012;33:3127-42.

[167] Gustavsson J, Ginebra MP, Engel E, Planell J. Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media. Acta Biomater 2011;7:4242-52.

[168] Kerckhofs G, Schrooten J, Van Cleynenbreugel T, Lomov SV, Wevers M. Validation of x-ray microfocus computed tomography as an imaging tool for porous structures. Rev Sci Instrum 2008;79:013711.

[169] Bohner M, Lemaitre J. Can bioactivity be tested in vitro with SBF solution? Biomaterials 2009;30:2175-9.

[170] Lee JT, Leng Y, Chow KL, Ren F, Ge X, Wang K, et al. Cell culture medium as an alternative to conventional simulated body fluid. Acta Biomater 2011;7:2615-22.

Figure Legends:

Figure 1: (a) Composition and (b) mechanical properties of human bone [Athanasiou KA et al. 2000; Keaveny TM et al. 2001].

Figure 2: Theories of biomineralisation of collagen fibril. (a) Direct nucleation of CaP crystal. (i) CaP clusters form complexes with the functional biopolymer (e.g. polyaspartic acid), and (ii) form stable mineral droplets. (iii) The mineral droplets bind to a distinct region on the collagen fibres and enter the fibril, and (iv) diffuse through the interior of the fibril before solidifying into a disordered (amorphous) phase. (v) This amorphous phase is transformed into oriented apatite crystals directed by the collagen orientation [modified from Colfen H. Nat Mater. 2010]. (b) Matrix vesicle (MV) mediated matrix mineralisation. Schematic showing the three possible mechanisms for the initiation of MV-mediated matrix mineralisation [adapted from Golub E.E. Biochimical et Biophysica Acta. 2009]. PPi, pyrophosphate.

Figure 3: The influence of specific surface area of CaP-based biomaterials on in vivo ectopic bone formation. Each data point represents one type of CaP-implant with specific surface area and its in vivo bone forming capacity. Based on these studies, a threshold level of specific surface area required to induce bone formation, was chosen at around 1.0 m2/g (the cut-off point). [Data are adopted from Habibovic et al. 2005; Li et al. 2008; Yuan et al. 2010].

Figure 4: The concept of integrating manufacturing process, stem cell technology and computational modelling as a novel strategy to design and optimise CaP-based bone reparative unit for effective bone regeneration that can also assist the translation to personalised bone regeneration.

1   2   3


Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconThe application of calcium phosphate precipitation chemistry to phosphorus recovery: the influence of organic ligands

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconThe application of calcium phosphate precipitation chemistry to phosphorus recovery: the influence of organic ligands

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconName Current New Explanation Effective

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconRealism is transformative—it can synthesize critical theories to provide effective strategies of change

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconHistory of soul the siberian’s orok, yukagir views the eskimo’s yakut, chuvach views

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconWorking with bone and antler. Cleaning bones. Whitening bones. Sources of bone and antler

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconThis research paper has been commissioned by the International Commission on Nuclear Non-proliferation and Disarmament, but reflects the views of the author and should not be construed as necessarily reflecting the views of the Commission

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconThis research paper has been commissioned by the International Commission on Nuclear Non-proliferation and Disarmament, but reflects the views of the author and should not be construed as necessarily reflecting the views of the Commission

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconGeometrical construction, use of instruments, scales, engineering curves. Orthographic projections, conversion of pictorial views to orthographic views and vice versa. Dimensioning. Unit – II

Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective Bone Regeneration Strategies iconThe copyright of this publication is owned by King’s College London. The views expressed in this report are those of the authors alone and do not in any way represent the views of the Greater London Authority or King’s College London

Разместите кнопку на своём сайте:

База данных защищена авторским правом © 2012
обратиться к администрации
Главная страница