Nanolayer formation on titanium by phosphonated gelatin for cell adhesion and growth enhancement

International Journal of Nanomedicine Dovepress open access to scientific and medical research O r i g in a l R e s e a r c h International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 37.59.46.207 on 13-Jul-2018 For personal use only. Open Access Full Text Article Nanolayer formation on titanium by phosphonated gelatin for cell adhesion and growth enhancement This article was published in the following Dove Press journal: International Journal of Nanomedicine 2 September 2015 Number of times this article has been viewed Xiaoyue Zhou 1,2,* Shin-Hye Park 1,* Hongli Mao 3 Takashi Isoshima 1 Yi Wang 2 Yoshihiro Ito 1,3 Nano Medical Engineering Laboratory, RIKEN, Wako, Saitama, Japan; 2Department of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin, People’s Republic of China; 3 Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, Wako, Saitama, Japan 1 *These authors contributed equally to this work Correspondence: Yoshihiro Ito Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Tel +81 48 467 4979 Fax +81 48 467 9300 Email Introduction Titanium and titanium alloys are widely used in medical applications such as the replacement of hard tissues including bone, joints, and dental implants, because of their nontoxicity, good mechanical properties, and excellent resistance to corrosion.1 However, there is still a need to further investigate their biocompatibility including the interface between titanium and the biological tissue. Because of a lack of bonding of implants to juxtaposed tissues, current orthopedic implants have a variety of problems including infection, extensive inflammation, and overall poor osseointegration. Therefore, many attempts have been made to modify the surface of titanium with functional or biological components to induce tissue responses to biomaterials and provide a set of powerful signals for cell growth and differentiation.2–6 However, there are limited procedures for surface modification with biological molecules. To biologically modify metal surfaces, silane-based coupling methods have been conventionally employed to prepare an initial organic layer on the metal surface.7–10 However, in addition to physicochemical modification,11–13 recent biomimetic approaches inspired by underwater organisms for surface modification have been proposed by many studies.14–23 3,4-Dihydroxyphenylalanine was identified in underwater adhesion proteins, and its simplified compound dopamine has been employed for biological modification 5597 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2015:10 5597–5607 Dovepress © 2015 Zhou et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php http://dx.doi.org/10.2147/IJN.S82166 Powered by TCPDF (www.tcpdf.org) Abstract: Phosphonated gelatin was prepared for surface modification of titanium to stimulate cell functions. The modified gelatin was synthesized by coupling with 3-aminopropylphosphonic acid using water-soluble carbodiimide and characterized by 31P nuclear magnetic resonance and gel permeation chromatography. Circular dichroism revealed no differences in the conformations of unmodified and phosphonated gelatin. However, the gelation temperature was changed by the modification. Even a high concentration of modified gelatin did not form a gel at room temperature. Time-of-flight secondary ion mass spectrometry showed direct bonding between the phosphonated gelatin and the titanium surface after binding. The binding behavior of phosphonated gelatin on the titanium surface was quantitatively analyzed by a quartz crystal microbalance. Ellipsometry showed the formation of a several nanometer layer of gelatin on the surface. Contact angle measurement indicated that the modified titanium surface was hydrophobic. Enhancement of the attachment and spreading of MC-3T3L1 osteoblastic cells was observed on the phosphonated gelatin-modified titanium. These effects on cell adhesion also led to growth enhancement. Phosphonation of gelatin was effective for preparation of a cell-stimulating titanium surface. Keywords: phosphonated gelatin, surface modification, titanium, cell adhesion Dovepress International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 37.59.46.207 on 13-Jul-2018 For personal use only. Zhou et al of metal surfaces.20–22 In addition, as another non-canonical amino acid, phosphonated serine has been applied to underwater adhesion.23–25 Such phosphate groups have been found in the underwater adhesive proteins of the sandcastle worm and caddy silks,16,17 which interact specifically with a titanium surface.26–33 In previous studies, we have anchored various extracellular matrices and growth factors onto metal to provide a source of signals to continuously, stably, and efficiently stimulate cells to reconstitute damaged tissues during long-term regeneration.34–36 Therefore, it may be useful to prepare metal-anchored proteins using biomimetic methods for convenient surface modification. The cell-adhesive protein gelatin has been employed for the chemical modification of titanium.8,37–40 Here, titaniumand cell-adhesive gelatin was prepared by chemical modification with phosphate groups as a biological approach to enhance cell functions on titanium surfaces. We found that the gelation temperature was reduced by the modification and time-of-flight secondary ion mass spectrometry (ToFSIMS) showed direct bonding between the phosphonated gelatin and the titanium surface. In addition, the modified surface promoted cell adhesion and spreading, as well as cell growth. Materials and methods Materials Porcine gelatin (gelatin from porcine skin, Type A, G1890, IEP: 7–9) and 3-aminopropylphosphonic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride was purchased from Wako Pure Chemical Industries, Ltd (Tokyo, Japan). The osteoblast cell line MC-3T3L1 was provided by the RIKEN Cell Bank (Tsukuba, Japan) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Moregate Inc., Brisbane, QLD, Australia). Trypsin (0.25%)-EDTA (1 mmol) solution was purchased from Wako Pure Chemical Industries, Ltd (Tokyo, Japan). 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