**5. Conclusion**

The decomposition of the C 1s and O 1s peak provided a distinct evaluation of oxygen present in inorganic oxide, inorganic hydroxide and organic compounds. This led to a stoichiometry close to (Fe, Cr)OOH for iron and chromium species in the passive layer. The elemental composition of the organic adlayer was converted into concentration of model organic compounds: protein, silane and contaminants. For the latter, extreme poles of hydrocarbons and polysaccharides were taken as models.

Silanization increased markedly the retention of glucose oxidase, whether or not using a linker expected to couple the enzyme with the silane. Direct retention of the enzyme by the silane may be attributed to electrostatic attractions with the protonated amine groups (Jasienak et al., 2009). It is thus demonstrated, with the same biomolecule, that its retention by using an APTES-treated substrate and a linker does not infer covalent grafting through the linker. The occurrence of covalent binding might possibly be evaluated by examining the retention upon aging in electrolytes, keeping in mind that the silane layer itself may alter in these conditions (Dekeyser et al., 2008).

The thickness of the organic adlayer was of the order of 3 nm. In all cases, the concentration of contaminants exceeded the concentration of silane and protein. Angleresolved measurements did not reveal any stratification in the organic adlayer, but this was not conclusive since the effect of a stratification may be masked owing to the roughness created by nanoparticles of inorganic oxyhydroxide present at the stainless steel surface. The presence of organic contaminants is unavoidable when high energy materials are exposed to air for a few hours or to vacuum for a very short time. However the influence of contaminants on the desired surface reactions is not known and is difficult to establish. The best to reduce contamination is to thoroughly clean the substrate by oxidation, to minimize the time of contact of substrates with air at any stage, and to strictly avoid outgassing. Water contact angle measurement is the best way to assess the cleanliness of the native substrate but the information will not be unambiguous after silanization and further treatments.

#### **6. Acknowledgments**

The authors thank Simon Degand for his help in statistics. They acknowledge the support of the "Conseil Régional de Picardie" (France) and the National Fondation for Scientific Research (F.N.R.S. – Belgium).

#### **7. References**

120 Biomaterials – Physics and Chemistry

photoelectron collection angles is due to the approximation of a smooth surface while the analyzed surface is rough at the scale of the inelastic mean free paths. The real organic adlayer thickness should be between the values given in Table 4. Comparison between nat and sil samples suggests that silane just adds up to the contaminants. A 3.0 nm thick adlayer containing 25 wt.% silane corresponds to 4.5 molecules.nm-2. This value is consistent with a monolayer of silane, however the retained silane is mixed with a much larger amount of contaminants. The protein treatment of silanized substrates (compare sil+Gox and sil+BS+Gox to sil) led to a significant decrease of the amount of hydrocarbon-like compounds, while the adlayer thickness did not change appreciably. This suggests that the protein adsorption caused the displacement of part of contamination present on the

The decomposition of the C 1s and O 1s peak provided a distinct evaluation of oxygen present in inorganic oxide, inorganic hydroxide and organic compounds. This led to a stoichiometry close to (Fe, Cr)OOH for iron and chromium species in the passive layer. The elemental composition of the organic adlayer was converted into concentration of model organic compounds: protein, silane and contaminants. For the latter, extreme poles of

Silanization increased markedly the retention of glucose oxidase, whether or not using a linker expected to couple the enzyme with the silane. Direct retention of the enzyme by the silane may be attributed to electrostatic attractions with the protonated amine groups (Jasienak et al., 2009). It is thus demonstrated, with the same biomolecule, that its retention by using an APTES-treated substrate and a linker does not infer covalent grafting through the linker. The occurrence of covalent binding might possibly be evaluated by examining the retention upon aging in electrolytes, keeping in mind that the silane layer itself may alter in

The thickness of the organic adlayer was of the order of 3 nm. In all cases, the concentration of contaminants exceeded the concentration of silane and protein. Angleresolved measurements did not reveal any stratification in the organic adlayer, but this was not conclusive since the effect of a stratification may be masked owing to the roughness created by nanoparticles of inorganic oxyhydroxide present at the stainless steel surface. The presence of organic contaminants is unavoidable when high energy materials are exposed to air for a few hours or to vacuum for a very short time. However the influence of contaminants on the desired surface reactions is not known and is difficult to establish. The best to reduce contamination is to thoroughly clean the substrate by oxidation, to minimize the time of contact of substrates with air at any stage, and to strictly avoid outgassing. Water contact angle measurement is the best way to assess the cleanliness of the native substrate but the information will not be unambiguous after

The authors thank Simon Degand for his help in statistics. They acknowledge the support of the "Conseil Régional de Picardie" (France) and the National Fondation for Scientific

silanized stainless steel surface in the form of hydrocarbon-like compounds.

hydrocarbons and polysaccharides were taken as models.

these conditions (Dekeyser et al., 2008).

silanization and further treatments.

**6. Acknowledgments** 

Research (F.N.R.S. – Belgium).

**5. Conclusion** 


Silanization with APTES for Controlling the Interactions

46, pp. 11962-11968.

pp. 278-289.

508-519.

292.

Between Stainless Steel and Biocomponents: Reality vs Expectation 123

Koh, I., Wang, X., Varughese, B., Isaacs, L., Ehrman, S.H. & English, D.S. (2006) Magnetic

activity, *The Journal of Physical Chemistry B*, Vol. 110, No. 4, pp. 1553-1558. Kohli, P., Taylor, K.K., Harris, J.J. & Blanchard, G.J. (1998) Assembly of covalently-coupled

Landoulsi, J., Dagbert, C., Richard, C., Sabot, R., Jeannin, M., El Kirat, K. & Pulvin, S. (2009)

Landoulsi, J., Genet, M.J., Richard, C., El Kirat, K., Rouxhet, P.G. & Pulvin, S. (2008b)

Landoulsi, J., Kirat, K.E., Richard, C., Féron, D. & Pulvin, S. (2008c) Enzymatic approach in

Le Bozec, N., Compère, C., L'Her, M., Laouenan, A., Costa, D. & Marcus, P. (2001) Influence

Li, G., Yang, P., Qin, W., Maitz, M.F., Zhou, S. & Huang, N. (2011) The effect of

Libertino, S., Giannazzo, F., Aiello, V., Scandurra, A., Sinatra, F., Renis, M. & Fichera, M.

Ma, L., Zhou, J., Gao, C. & Shen, J. (2007) Incorporation of basic fibroblast growth factor by a

Mantel, M., Rabinovich, Y.I., Wightman, J.P. & Yoon, R.H. (1995) A Study of Hydrophobic

Martin, H.J., Schulz, K.H., Bumgardner, J.D. & Walters, K.B. (2007) XPS Study on the Use of

Matinlinna, J., P., Lassila, L., V. J., Özcan, M., Yli-Urpo, A. & Vallittu, P., K. (2004) An

endothelialization, *Biomaterials*, Vol. 32, No. 21, pp. 4691-4703.

on SiO2 surfaces, *Langmuir*, Vol. 24, No. 5, pp. 1965-1972.

*Journal of Prosthodontics*, Vol. 17, No. 211, pp. 155-164.

Waters, *Environmental Science & Technology*, Vol. 42, No. 7, pp. 2233-2242. Lapin, N.A. & Chabal, Y.J. (2009) Infrared characterization of biotinylated silicon oxide

*Physical Chemistry B*, Vol. 113, No. 25, pp. 8776-8783.

*Corrosion Science*, Vol. 43, No. 4, pp. 765-786.

Vol. 23, No. 12, pp. 6645-6651.

corrosion behavior, *Electrochimica Acta*, Vol. 54, No. 28, pp. 7401-7406. Landoulsi, J., Genet, M.J., Richard, C., El Kirat, K., Pulvin, S. & Rouxhet, P.G. (2008a)

iron oxide nanoparticles for biorecognition: Evaluation of surface coverage and

disulfide multilayers on gold, *Journal of the American Chemical Society*, Vol. 120, No.

Enzyme-induced ennoblement of AISI 316L stainless steel: Focus on pitting

Evolution of the passive film and organic constituents at the surface of stainless steel immersed in fresh water, *Journal of Colloid and Interface Science*, Vol. 318, No. 2,

Ennoblement of stainless steel in the presence of glucose oxidase: Nature and role of interfacial processes, *Journal of Colloid and Interface Science*, Vol. 320, No. 2, pp.

microbial-influenced corrosion: A review based on Stainless Steels in Natural

surfaces, surface stability, and specific attachment of streptavidin, *The Journal of* 

of stainless steel surface treatment on the oxygen reduction reaction in seawater,

coimmobilizing heparin and fibronectin on titanium on hemocompatibility and

(2008) XPS and AFM characterization of the enzyme glucose oxidase immobilized

layer-by-layer assembly technique to produce bioactive substrates, *Journal of Biomedical Materials Research Part B: Applied Biomaterials*, Vol. 83B, No. 1, pp. 285-

Interactions between Stainless Steel and Silanated Glass Surface Using Atomic Force Microscopy, *Journal of Colloid and Interface Science*, Vol. 170, No. 1, pp. 203-214.

3-Aminopropyltriethoxysilane to Bond Chitosan to a Titanium Surface, *Langmuir*,

introduction to silanes and their clinical applications in dentistry, *International* 


Dupont, I., Féron, D. & Novel, G. (1998) Effect of glucose oxidase activity on corrosion

El-Ghannam, A.R., Ducheyne, P., Risbud, M., Adams, C.S., Shapiro, I.M., Castner, D.,

Genet, M.J., Dupont-Gillain, C.C. & Rouxhet, P.G. (2008) XPS analysis of biosystems and

Gooding, J.J. & Ciampi, S. (2011) The molecular level modification of surfaces: from self-

Haensch, C., Hoeppener, S. & Schubert, U.S. (2010) Chemical modification of self-assembled

Hanawa T. 2002. Metallic biomaterials. In: Ikada Y, editor. Recent research and

Howarter, J.A. & Youngblood, J.P. (2006) Optimization of silica silanization by 3- Aminopropyltriethoxysilane, *Langmuir*, Vol. 22, No. 26, pp. 11142-11147. Iucci, G., Dettin, M., Battocchio, C., Gambaretto, R., Bello, C.D. & Polzonetti, G. (2007) Novel

Jasienak, M., Suzuki, S., Montero, M., Wentrup-Byrne, E., Griesser, H.J. & Grondahl, L.

Jin, L., Horgan, A. & Levicky, R. (2003) Preparation of end-tethered DNA monolayers on

Katsikogianni, M.G. & Missirlis, Y.F. (2010) Interactions of bacteria with specific biomaterial

Killian, M.S., Wagener, V., Schmuki, P. & Virtanen, S. (2010) Functionalization of metallic

Kim, J., Cho, J., Seidler, P.M., Kurland, N.E. & Yadavalli, V.K. (2010) Investigations of

controlled protein immobilization, *Langmuir*, Vol. 26, No. 4, pp. 2599-2608. Kim, J., Seidler, P., Wan, L.S. & Fill, C. (2009a) Formation, structure, and reactivity of amino-

Kim, W.-J., Kim, S., Lee, B.S., Kim, A., Ah, C.S., Huh, C., Sung, G.Y. & Yun, W.S. (2009b)

hydroxyl functional group, *Langmuir*, Vol. 25, No. 19, pp. 11692-11697.

*Materials Science and Engineering: C*, Vol. 27, No. 5-8, pp. 1201-1206.

*Biodegradation*, Vol. 41, No. 1, pp. 13-18.

177-307.

DOI: 10.1039/c0cs00139b.

6, pp. 2323-2334.

2, pp. 1011-1019.

pp. 6968-6975.

12044-12048.

Vol. 329, No. 1, pp. 114-119.

1118.

*Materials Research Part A*, Vol. 68A, No. 4, pp. 615-627.

developments in biomaterials, Research Signpost, p. 11-31.

potential of stainless steels in seawater, *International Biodeterioration &* 

Golledge, S. & Composto, R.J. (2004) Model surfaces engineered with nanoscale roughness and RGD tripeptides promote osteoblast activity, *Journal of Biomedical* 

biomaterials, in: M. E. (Ed) *Medical Applications of Colloids* (New York, Springer), pp.

assembled monolayers to complex molecular assemblies, *Chemical Society Reviews*,

silane based monolayers by surface reactions, *Chemical Society Reviews*, Vol. 39, No.

immobilizations of an adhesion peptide on the TiO2 surface: An XPS investigation,

(2009) Time-of-flight secondary ion mass spectrometry study of the orientation of a bifunctional diblock copolymer attached to a solid substrate, *Langmuir*, Vol. 25, No.

siliceous surfaces using heterobifunctional cross-linkers, *Langmuir*, Vol. 19, No. 17,

surface chemistries under flow conditions, *Acta Biomaterialia*, Vol. 6, No. 3, pp. 1107-

magnesium with protein layers via linker molecules, *Langmuir*, Vol. 26, No. 14, pp.

chemical modifications of amino-terminated organic films on silicon substrates and

terminated organic films on silicon substrates, *Journal of Colloid and Interface Science*,

Enhanced protein immobilization efficiency on a TiO2 surface modified with a


Silanization with APTES for Controlling the Interactions

*Analysis*. (In press).

pp. 718-724.

pp. 1085-1098.

2, pp. 573-581.

2011-2024.

Vol. 19, No. 23, pp. 9845-9849.

Between Stainless Steel and Biocomponents: Reality vs Expectation 125

Rouxhet, P.G. & Genet, M.J. (2011) XPS analysis of bio-organic systems, *Surface and Interface* 

Rouxhet, P.G., Misselyn-Bauduin, A.M., Ahimou, F., Genet, M.J., Adriaensen, Y., Desille, T.,

Sarath Babu, V.R., Kumar, M.A., Karanth, N.G. & Thakur, M.S. (2004) Stabilization of

Sasou, M., Sugiyama, S., Yoshino, T. & Ohtani, T. (2003) Molecular flat mica surface

Schuessele, A., Mayr, H., Tessmar, J. & Goepferich, A. (2009) Enhanced bone morphogenetic

Scofield, J.H. (1976) Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV, *Journal of Electron Spectroscopy and Related Phenomena*, Vol. 8, No. 2, pp. 129-137.

Siperko, L.M., Jacquet, R. & Landis, W.J. (2006) Modified aminosilane substrates to evaluate

Son, K.J., Ahn, S.H., Kim, J.H. & Koh, W.-G. (2011) Graft copolymer-templated mesoporous

Sordel, T., Kermarec-Marcel, F., Garnier-Raveaud, S., Glade, N., Sauter-Starace, F., Pudda,

Subramanian, A., Kennel, S.J., Oden, P.I., Jacobson, K.B., Woodward, J. & Doktycz, M.J.

Suzuki, N. & Ishida, H. (1996) A review on the structure and characterization techniques of silane/matrix interphases, *Macromolecular Symposia*,Vol. 108, No. 1, pp. 19-53. Suzuki, S., Whittaker, M.R., Grøndahl, L., Monteiro, M.J. & Wentrup-Byrne, E. (2006)

Tanuma, S., Powell, C.J. & Penn, D.R. (1997) Calculations of electron inelastic mean free

IMFP equation, *Surface and Interface Analysis*, Vol. 25, No. 1, pp. 25-35. Tesson, B., Genet, M.J., Fernandez, V., Degand, S., Rouxhet, P.G. & Martin-Jézéquel, V.

*Materials Research Part A*, Vol. 90A, No. 4, pp. 959-971.

*Materials Research Part A*, Vol. 78A, No. 4, pp. 808-822.

adhesion, *Biomaterials*,Vol. 28, No. 8, pp. 1572-1584.

*Biomacromolecules,* Vol. 7, No. 11, pp. 3178-3187.

*Enzyme and Microbial Technology*,Vol. 24, No. 1-2, pp. 26-34.

Bodson, P. & Deroanne, C. (2008) XPS analysis of food products: toward chemical functions and molecular compounds, *Surface and Interface Analysis*,Vol. 40, No. 3-4,

immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications, *Biosensors and Bioelectronics*,Vol. 19, No. 10, pp. 1337-1341. Sargeant, T.D., Rao, M.S., Koh, C.-Y. & Stupp, S.I. (2008) Covalent functionalization of NiTi

surfaces with bioactive peptide amphiphile nanofibers, *Biomaterials*,Vol. 29, No. 8,

silanized with methyltrimethoxysilane for fixing and straightening DNA, *Langmuir*,

protein-2 performance on hydroxyapatite ceramic surfaces, *Journal of Biomedical* 

osteoblast attachment, growth, and gene expression in vitro, *Journal of Biomedical* 

TiO2 films micropatterned with Poly(ethylene glycol) hydrogel: Novel platform for highly sensitive protein microarrays, *ACS Applied Materials & Interfaces*,Vol. 3, No.

C., Borella, M., Plissonnier, M., Chatelain, F., Bruckert, F. & Picollet-D'hahan, N. (2007) Influence of glass and polymer coatings on CHO cell morphology and

(1999) Comparison of techniques for enzyme immobilization on silicon supports,

Synthesis of soluble phosphate polymers by RAFT and their in vitro mineralization,

paths (IMFPs) VI. Analysis of the gries inelastic scattering model and predictive

(2009) Surface chemical composition of diatoms, *ChemBioChem*, Vol. 10, No. 12, pp.


Matinlinna, J.P. & Vallittu, P.K. (2007) Silane based concepts on bonding resin composite to metals, *The Journal of Contemporary Dental Practice*, Vol. 8, No. 2, pp. 1-8. Meng, S., Liu, Z., Shen, L., Guo, Z., Chou, L.L., Zhong, W., Du, Q. & Ge, J. (2009) The effect

Minier, M., Salmain, M.l., Yacoubi, N., Barbes, L., Méthivier, C., Zanna, S. & Pradier, C.-M.

Mosse, W.K.J., Koppens, M.L., Gengenbach, T.R., Scanlon, D.B., Gras, S.L. & Ducker, W.A.

Müller, R., Abke, J., Schnell, E., Macionczyk, F., Gbureck, U., Mehrl, R., Ruszczak, Z., Kujat,

Nanci, A., Wuest, J.D., Peru, L., Brunet, P., Sharma, V., Zalzal, S. & McKee, M.D. (1998)

North, S.H., Lock, E.H., Cooper, C.J., Franek, J.B., Taitt, C.R. & Walton, S.G. (2004) Plasma-

Olefjord, I. & Wegrelius, L. (1996) The influence of nitrogen on the passivation of stainless

Palestino, G., Agarwal, V., Aulombard, R., PeÌ rez, E.a. & Gergely, C. (2008) Biosensing and

Pasternack, R.M., Rivillon Amy, S. & Chabal, Y.J. (2008) Attachment of 3-

Porté-Durrieu, M.C., Guillemot, F., Pallu, S., Labrugère, C., Brouillaud, B., Bareille, R.,

osteoprogenitor cells adhesion, *Biomaterials*, Vol. 25, No. 19, pp. 4837-4846. Puleo, D.A. (1997) Retention of enzymatic activity immobilized on silanized Co-Cr-Mo and Ti-6Al-4V, *Journal of Biomedical Materials Research*, Vol. 37, pp. 222-228. Quan, D., Kim, Y. & Shin, W. (2004) Characterization of an amperometric laccase electrode

Ratner B.D., Hoffman A.S., Schoen F.J. & Lemons J.E. (Eds) (2004) *Biomaterials Science: An Introduction to Materials in Medecine* (Academic Press, San Diego, U.S.A., 2d Ed.).

Version 3.4 (Web Version), http://srdata.nist.gov/xps/index.htm.

steels, *Corrosion Science*, Vol. 38, No. 7, pp. 1203-1220.

temperature, *Langmuir*, Vol. 24, No. 22, pp. 12963-12971. Plueddemann, E.W. (1991) *Silane Coupling Agents* (New York, Plenum).

*Langmuir*, Vol. 24, No. 23, pp. 13765-13771.

2276-2283.

pp. 2884-2891.

Vol. 561, pp. 181-189.

*Langmuir*,Vol. 21, No. 13, pp. 5957-5965.

*Langmuir*, Vol. 25, No. 3, pp. 1488-1494.

*Biomaterials*, Vol. 26, No. 34, pp. 6962-6972.

of a layer-by-layer chitosan-heparin coating on the endothelialization and coagulation properties of a coronary stent system, *Biomaterials*, Vol. 30, No. 12, pp.

(2005) Covalent Immobilization of Lysozyme on Stainless Steel. Interface spectroscopic characterization and measurement of enzymatic activity,

(2009) Peptides grafted from solids for the control of interfacial properties,

R., Englert, C., Nerlich, M. & Angele, P. (2005) Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility,

Chemical modification of titanium surfaces for covalent attachment of biological molecules, *Journal of Biomedical Materials Research*, Vol. 40, No. 2, pp. 324-335. NIST X-ray Photoelectron Spectroscopy Database, NIST Standard Reference Database 20,

based surface modification of polystyrene microtiter plates for covalent immobilization of biomolecules, *ACS Applied Materials & Interfaces*, Vol. 2, No. 10,

protein fluorescence enhancement by functionalized porous silicon devices,

(Aminopropyl)triethoxysilane on silicon oxide surfaces: Dependence on solution

Amédée, J., Barthe, N., Dard, M. & Baquey, C. (2004) Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human

covalently immobilized on platinum surface, *Journal of Electroanalytical Chemistry*,


**6** 

**Human Dentin as Novel Biomaterial** 

Masaru Murata1, Toshiyuki Akazawa2, Masaharu Mitsugi3, In-Woong Um4, Kyung-Wook Kim5 and Young-Kyun Kim6

Human dentin autograft was reported in 2003 as a first clinical case (Murata et al., 2003), while human bone autograft was done in 1820. There was a long-long time lag between the autografts of dentin and bone. In 2009, Korea Tooth Bank was established in Seoul for the processing of the tooth-derived materials in Seoul, and an innovative medical service has begun for bone regeneration. Recently, the tooth-derived materials have been becoming a

The regeneration of lost-parts of the skeleton has been generally carried out with fresh, autogenous bone as a gold standard. To obviate the need for harvesting of grafts and thus, to avoid morbidity resulting from it, the researches for bone substitutes (Kuboki et al., 1995; Asahina et al., 1997; Takaoka et al., 1991; Artzi et al., 2004; Kim et al., 2010) or bone production via bio-engineering have begun (Wozney et al., 1988; Wang et al., 1990; Murata et al., 1999). In the regenerative field, there is a medical need for biomaterials that both allow for bone formation and also gradually absorb as to be replaced by bone. Non-absorbable materials are never replaced by bone and thus, reveal chronic inflammation in tissues as

As bone and dentin consist of fluid (10%), collagen (20%) and hydroxyapatite (70%) in weight volume, our attention for biomaterials is collagenous and ceramic materials (Murata et al., 2000; Murata et al., 2002; Akazawa et al., 2006; Murata et al., 2007). Generally, extracted teeth have been discarded as infective medical dusts in the world. We have thought the non-functional teeth as native resource for self and family (Fig. 1). Therefore, we noticed on bone-inductive, absorbable properties of dentin, and have been studying a medical recycle of human teeth as a novel graft material for bone regeneration in Japan and Korea (Akazawa et al. 2007; Kim et al. 2010). Biomaterial science should support and develop the advanced regenerative therapy using enamel and dentin matrix for patients in

**1. Introduction** 

foreign bodies.

the near future.

realistic alternative to bone grafting.

**for Bone Regeneration** 

*1Health Sciences University of Hokkaido,* 

*3Takamatsu Oral and Maxillofacial Surgery* 

*6Seoul National University Bundang Hospital, 1,2,3Japan* 

*2Hokkaido Organization,* 

*4Tooth Bank Co. Ltd, 5Dankook University,* 

*4,5,6Korea* 

