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\begin{document}

\title{Bio-functionalized titanium surfaces with the modified silk fibroin
carrying titanium binding motif enhanced the ossific differentiation of
MC3T3-E1}



\author[1]{Mai Watanabe}%
\author[2]{Ujjal Bhawala}%
\author[3]{Shinji Takemoto}%
\author[2]{Norihiro Nishiyama}%
\author[4]{Yuichi Nakahara}%
\author[4]{Ken-ichiro Tatematsu}%
\author[4]{Hideki Sezutsu}%
\author[5]{Nobuo Kuwabara}%
\author[1]{Tamiko Minamisawa}%
\author[1]{Kiyotaka Shiba}%
\author[6]{Tetsuo Asakura}%
\affil[1]{Public Interest Incorporated Foundation Japanese Foundation for Cancer Research}%
\affil[2]{Nihon University School of Dentistry Graduate School of Dentistry at Matsudo}%
\affil[3]{Iwate Medical University}%
\affil[4]{National Agriculture and Food Research Organization}%
\affil[5]{Gunma Sericultural Technology Center}%
\affil[6]{Tokyo University of Agriculture and Technology}%


\vspace{-1em}



  
  \date{May 04, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Silk fibroin (SF) from \emph{Bombyx mori} has superior properties as
both a textile and a biomaterial, and has been used to functionalize the
surfaces of various medical inorganic materials including titanium (Ti).
In this paper, we endowed SF with reversible binding ability to Ti by
embedding a titanium binding motif (minTBP-1, RKLPDA). Artificial SF
proteins were first created by conjugating gene cassettes for SF motif
(AGSGAG) and minTBP-1 motif with different ratios, which have been shown
to bind reversibly to Ti surfaces in quartz crystal microbalance
analyses. Based on these results, the functionalized SF (TiBP-SF)
containing the designed peptide
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDAS{]}\textsubscript{8}
was prepared from the cocoon of transgenic \emph{B. mori}, which
accelerates the ossific differentiation of MC3T3-E1 cells when coated on
titanium substrates. Thus, TiBP-SF presents an alternative for endowing
the surfaces of titanium materials with osseointegration functionality,
which would allow the exploration of potential applications in the
medical field.%
\end{abstract}%



\sloppy


Bio-functionalized titanium surfaces with modified silk fibroin carrying
titanium binding motif to enhance the ossific differentiation of
MC3T3-E1.

Mai Watanabe\textsuperscript{+++}, Ujjal K. Bhawala\selectlanguage{ngerman}\textsuperscript{¶},
Shinji Takemoto\textsuperscript{\&}, Norihiro
Nishiyama\textsuperscript{\%}, Yuichi Nakahara\selectlanguage{english}\textsuperscript{[?]},
Ken-ichiro Tatematsu\selectlanguage{english}\textsuperscript{[?]}, Hideki
Sezuts\selectlanguage{english}\textsuperscript{[?]}, Nobuo Kuwabara\selectlanguage{polish}\textsuperscript{ę}, \selectlanguage{english}Tamiko
Minamisawa\textsuperscript{+}, Kiyotaka Shiba\textsuperscript{+*} and
Tetsuo Asakura\textsuperscript{++*}

\textsuperscript{+}Division of Protein Engineering, Cancer Institute,
Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto, Tokyo
125-8550, Japan, \textsuperscript{++}Department of Biotechnology, Tokyo
University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei,
Tokyo 184-8588, Japan, \selectlanguage{ngerman}\textsuperscript{¶}Department of Biochemistry and
Molecular Biology, and Research Institute of Oral Science, Nihon
University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi,
Matsudo, Chiba 271-8587, Japan, \textsuperscript{\&}Department of
Biomedical Engineering, Iwate Medical University, Iwate 028-3694,
Japan,\textsuperscript{\%}Department of Dental Biomaterials, Nihon
University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi,
Matsudo, Chiba 271-8587, Japan, \selectlanguage{english}\textsuperscript{[?]}Transgenic Silkworm
Research Unit, National Agriculture and Food Research Organization,1-2
Owashi, Tsukuba, Ibaraki 305-8634, Japan,\begin{CJK}{UTF8}{gbsn}　\end{CJK}\selectlanguage{english}\selectlanguage{polish}\textsuperscript{ę}\selectlanguage{english}Gunma
Sericultural Technology Center, 2326osteo-2 Soja-machi, Maebashi, Gunma
371-0852, Japan

\textbf{ABSTRACT.} min-TBP-1 modified silk fibroin

Silk fibroin (SF) from \emph{Bombyx mori} has superior properties as
both a textile and a biomaterial, and has been used to functionalize the
surfaces of various medical inorganic materials including titanium (Ti).
In this paper, we endowed SF with reversible binding ability to Ti by
embedding a titanium binding motif (minTBP-1, RKLPDA). Artificial SF
proteins were first created by conjugating gene cassettes for SF motif
(AGSGAG) and minTBP-1 motif with different ratios, which have been shown
to bind reversibly to Ti surfaces in quartz crystal microbalance
analyses. Based on these results, the functionalized SF (TiBP-SF)
containing the designed peptide
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDAS{]}\textsubscript{8}was
prepared from the cocoon of transgenic \emph{B. mori} , which
accelerates the ossific differentiation of MC3T3-E1 cells when coated on
titanium substrates. Thus, TiBP-SF presents an alternative for endowing
the surfaces of titanium materials with osseointegration functionality,
which would allow the exploration of potential applications in the
medical field.

\textbf{KEYWORDS.} Peptide aptamer, transgenic silk fibroin, artificial
protein, biomaterial, 13C solid-state NMR

\textbf{Running title.} minTBP-1 functionalized silk fibroin.

\textbf{INTRODUCTION}

``Motif'' is the versatile functional unit for constructing artificial
biomacromolecules. In some cases, motif can endow non-biomolecules with
its assigned functions, while in other cases different motifs can be
combined to create artificial proteins having novel combinations of
functions (Shiba, 2010). Motif sequences are identified both from
natural sequences of proteins and from \emph{in vitro} evolution
systems. In the latter cases, a phage display system (Scott \& Smith,
1990)) has been employed to create an artificial peptide that binds to
biomolecules and non-biological materials such as inorganic materials.
The hexapeptidic RKLPDA sequence is one such inorganic material binding
peptides. It was originally isolated as a 12-mer peptide binder to a
titanium (Ti) surface by using a peptide display system (Sano \& Shiba,
2003). Subsequent analyses have revealed that the N-terminal RKLPDA
(termed minTBP-1) is both necessary and sufficient for the binding
(Sano, Sasaki, \& Shiba, 2005). This minTBP-1 motif has been widely
employed as an appendix to provide an affinity to Ti in ferritin (Sano,
Ajima, et al., 2005), artificial matrix proteins (Kokubun, Kashiwagi,
Yoshinari, Inoue, \& Shiba, 2008), BMP-2 (Kashiwagi, Tsuji, \& Shiba,
2009), antimicrobial peptide (Geng et al., 2018; Yoshinari, Kato,
Matsuzaka, Hayakawa, \& Shiba, 2010), calcification protein (Tsuji,
Oaki, Yoshinari, Kato, \& Shiba, 2010), DNA-polymerase (Nishida et al.,
2015), carbon nanomaterial-binding peptide (Kokubun, Matsumura,
Yudasaka, Iijima, \& Shiba, 2018), and self-assembled chemical compounds
(Ikemi et al., 2010). Interestingly, it has been revealed that the
interaction between minTBP-1 and the surfaces of Ti is governed by
reversible interactions (Brandt \& Lyubartsev, 2015; Hayashi et al.,
2009; Sano, Ajima, et al., 2005; Schneider \& Ciacchi, 2012; Skelton,
Liang, \& Walsh, 2009). In biological systems, weak, specific,
reversible binding plays a pivotal role in various biological
activities. Indeed, previous studies have shown certain biological
activities has been enhanced by adding minTBP-1 to parental
biomolecules(Kashiwagi et al., 2009; Kokubun et al., 2008).

Silk fibroin (SF) from \emph{Bombyx mori} possesses many superior and
inherent properties as a biomaterial, including advantageous mechanical
properties, environmental stability, biocompatibility, low
immunogenicity, and biodegradability (Aigner, DeSimone, \& Scheibel,
2018; Asakura \& Kaplan, 1994; Asakura, Tanaka, \& Tanaka, 2019; Koeppel
\& Holland, 2017; Koh et al., 2015; Velema \& Kaplan, 2006; Vepari \&
Kaplan, 2007). SF fiber has been used in sutures in the surgical field
for more than 2,000 years (Thurber, Omenetto, \& Kaplan, 2015).
Recently, the representative repeating unit of the crystalline fraction
(56\% of whole SF) (Strydom, Haylett, \& Stead, 1977; Zhou et al., 2001;
Zhou et al., 2000), AGSGAG sequence, has been used as a motif to create
artificial SFs (Asakura et al., 2005; Asakura \& Yao, 2002; Yu Suzuki,
Aoki, Nakazawa, Knight, \& Asakura, 2010; Yao, Ohgo, Sugino, Kishore, \&
Asakura, 2004). Motif-based artificial protein constructions provide us
with opportunities to create artificial biomolecules having novel
biological activities or novel combinations of exiting activities (M.
Yang \& Asakura, 2005; Mingying Yang, Kawamura, Zhu, Yamauchi, \&
Asakura, 2009; M. Yang, Muto, Knight, Collins, \& Asakura, 2008; M.
Yang, Tanaka, et al., 2008; M. Yang, Yamauchi, Kurokawa, \& Asakura,
2007). However, the production of these artificial proteins has
generally been dependent on microorganisms or cell culture systems,
which are obstacles to bulk production.

Recently, a germ line transformation method for the silkworm has been
developed using the transposon piggyback (Kojima et al., 2007; Tamura et
al., 2000). This enabled the production of functionalized transgenic
(TG) SF in a large quantity such as cell-adhesive silks (Asakura et al.,
2014; Yanagisawa et al., 2007), colored fluorescent silk (Iizuka et al.,
2013), calcium binding silk (Nagano et al., 2011), and silk better
suited to artificial blood vessels (Saotome et al., 2015). Such an
improvement of the properties of SF for biomaterials has been attained
by incorporating suitable DNA sequences. SF consists of a heavy chain
(H-chain) and light chain (L-chain) connected by a disulfide bond, as
well as a glycoprotein named P25. It is secreted into the posterior silk
gland and is thought to be assembled into a high molecular mass
elementary unit with a ratio of 6:6:1 (Couble, Chevillard, Moine,
Ravel-Chapuis, \& Prudhomme, 1985; Takei, Kikuchi, Kikuchi, Mizuno, \&
Shimura, 1987; Tanaka et al., 1999; Tanaka, Mori, \& Mizuno, 1993). The
new gene that follows the SF gene is expressed as a new functionalized
SF in the posterior silk gland. Subsequently, the SF stored in the
middle silk gland is spun out through the anterior silk gland and
converted into SF fibers, being coated by silk sericin that emerges in
the middle silk gland. After removal of silk sericin by a degumming
process, we obtained pure SF. There are many advantages in preparing
functionalized SF suitable for biomaterials by the TG technique: (1) the
TG silkworms are easy to handle and the larvae are well adapted for
artificial rearing; (2) the adult moths cannot fly away and therefore
cannot live in the wild; (3) the expression of the recombinant silk
proteins can be confirmed visually by fusion of green fluorescent
proteins as a marker.

We were interested in the possibility that a combination of the AGSGAG
and minTBP-1 motifs could further enhance the functionality of SF when
used as a coating agent for titanium substrates. While titanium has
already been widely used in dental implants, artificial joints, and
vessel stents (Brunette, Tengvall, Textor, \& Thomsen, 2001), further
improvement of its biocompatibility is still required (Pagel et al.,
2016; Schuler et al., 2006; Vidal et al., 2013; Zhang, Zhang, Zhu, Kang,
\& Neoh, 2008). In the present study, we first synthesized artifice
polypeptides containing both AGSGAG and minTBP-1 motifs, and confirmed
their reversible interacting ability to titanium, which characterizes
the minTBP-1 motif. Then, we constructed the transgenic \emph{B.
mori}that spins the cocoon containing the artificial SF. The modified SF
prepared from the cocoons enhanced the ossific differentiation of
MC3T3-E1 cells grown on the titanium plates coated by the artificial SF.
The binding mechanism of silk-TBP on the surface of
TiO\textsubscript{2}nanoparticles was examined briefly using the model
peptides made from the AGSGAG and minTBP-1 motifs, and
\textsuperscript{13}C solid-state NMR.

\textbf{MATERIALS AND METHODS}

\textbf{Production of silk-like proteins,
TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{n}RKLPDAS and
TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{n} by\emph{Escherichia
coli}.}

The recombinant silk-like proteins
TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{n}RKLPDAS (n = 2 and
4) and TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{n} (n = 2 and
4) were prepared according to the method described previously
(Yanagisawa et al., 2007; M. Yang \& Asakura, 2005; Mingying Yang et
al., 2009; M. Yang, Muto, et al., 2008; M. Yang, Tanaka, et al., 2008;
M. Yang et al., 2007). Here, (AGSGAG)\textsubscript{3} (Bc3) and RKLPDA
represent the crystalline region of SF and minimal sequence of titanium
binding peptide, respectively. The construction scheme is shown in
Figure 1a. The pUC-Bc3 is the recombinant vector, in which a single unit
of DNA sequence coding (AGSGAG)\textsubscript{3}AS was inserted into
pUC118 vector plasmid (Yanagisawa et al., 2007; M. Yang \& Asakura,
2005). Because this unit contains \emph{Spe} I and \emph{Nhe} I
restriction enzyme recognition sites, and because these enzymes produce
complemental sticky ends, directional multiplexing of the unit is
possibly though repeated cut-and-paste manipulations (Prince, McGrath,
DiGirolamo, \& Kaplan, 1995; M. Yang \& Asakura, 2005). In this study,
we constructed 2-mer and 4-mer of
{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{n} repeats, which were
named pUC-(Bc3) \textsubscript{2} and pUC-(Bc3) \textsubscript{4},
respectively. To add RKLPDA motif at the end of repeats of
(AGSGAG)\textsubscript{3}AS, oligonucleotide KY-1775,
pCTAGTCGGAAGCTGCCCGACG, and pCTAGCGTCGGGCAGCTTCCGA were annealed and
ligated into the \emph{Nhe} I site of pUC-(Bc3) \textsubscript{2} and
pUC-(Bc3) \textsubscript{4} to obtain pUC-(Bc3) \textsubscript{2}T and
pUC-(Bc3) \textsubscript{4}T. Integrities of constructed artificial
genes were confirmed by sequencing using the Dye Terminator Cycle
Sequencing Quick Start Kit (Beckman Coulter, Brea, CA). For the
expression in \emph{Escherichia coli} , the inserts of these four
plasmids were digested by \emph{Bam} HI and \emph{Hind} III, and were
re-cloned into the same sites of the expression vector pET30a (Nobagen
Inc Darmstadt, Germany) to obtain pET-(Bc3)\textsubscript{2},
pET-(Bc3)\textsubscript{4}, pET-(Bc3)\textsubscript{2}T, and
pET-(Bc3)\textsubscript{4}T. In pET30a vectors, artificial silk fibroin
proteins are expressed as the fusion proteins that carry a vector
derived 6xHis-tag sequence at the N-terminal.

Because the pET30a vector is driven by a T7 promoter, the expression
vectors were introduced in the \emph{E. coli} strain BL21(DE3)pLysS
(Nobagen Inc.), which has T7 polymerase, and the transformed cells were
grown in 200--500 mL of LB (Sambrook, Fritsch, \& Maniatis, 1989)
containing kanamycin and chloramphenicol at 37 \selectlanguage{ngerman}°C. At
OD\textsubscript{660} = 0.5, isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1 thiogalactopyranoside
(IPTG) was added to a final concentration of 1 mM to induce protein
expression for 4 h. After induction, cells were harvested and frozen and
stored at -80 \selectlanguage{ngerman}°C until purification. The protein expression was induced
at 25 °C by adding IPTG to a final concentration of 1 mM. The
recombinant proteins were purified by nickel-chelate chromatography
using an ÄKTA explorer 10S/100 (GE Healthcare, Waukesha, WI, USA) column
according to the manufacturer's protocol. The optimized conditions for
elution were 15 mM imidazole, pH 4.5, and 50 °C. The eluted protein
solutions (6--8 mL) were dialyzed against 10 mM
NaH\textsubscript{2}PO\textsubscript{4}, pH 8.0 and 100 mM NaCl using a
Slide-A-Lyzer Dialysis Cassette (3,500 MWCO, Pierce, Waltham, MA),
followed by concentration into 500 \selectlanguage{greek}µ\selectlanguage{english}L by using Amicon Ultra
Centrifugation Filters-4 (Merck Millipore, Billerica, MA). The purified
proteins from pET-(Bc3)\textsubscript{2}, pET-(Bc3)\textsubscript{4},
pET-(Bc3)\textsubscript{2}T, and pET-(Bc3)\textsubscript{4}T were termed
(Bc3) \textsubscript{2}, (Bc3) \textsubscript{4},
(Bc3)\textsubscript{2}T, and (Bc3) \textsubscript{4}T, respectively
(Figure 1 a).

\begin{CJK}{UTF8}{gbsn}\textbf{QCM analyses.　}

\end{CJK}\selectlanguage{english}Quartz Crystal Microbalance (QCM) measurements were carried out on
Qsense D300 equipment with a Ti sensor (Q-Sense, Vastra Frolunda) that
was intensively cleaned with a UV-ozone ProCleaner system (BioForce
Nanosciences, Ames, IA) prior to use. Before measurements, the Ti-sensor
and the chamber were equilibrated in 50 mM Tris-HCl (pH 8.0) and 150 mM
NaCl (TBS) containing 0.05\% polyoxyethylene sorbitan monolaurate (Tween
20) (TBST) at 25 \selectlanguage{ngerman}°C. The proteins were prepared in a 2 mL volume at a
concentration of 0.5 \selectlanguage{greek}µ\selectlanguage{english}M in TBST. The data were obtained and analyzed by
Q soft 301 and q-tool (Q-Sense).

\textbf{Construction of transgenic silk worm that spins the cocoons
containing minTBP-1 appended silk fibroin (TiBP-SF).}

To introduce the RKLPDA motif (minTBP-1) into the SF heavy chain (HC), a
DNA fragment coding
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDA{]}\textsubscript{8}was
synthesized (GenScript, Piscataway, NJ, USA) and inserted into
the\emph{Bam} HI and \emph{Sal} I sites of plasmid pHC-EGFP (Kojima et
al., 2007). The resultant plasmid was digested with \emph{Asc} I
and\emph{Fse} I and inserted into the \emph{Asc} I and \emph{Fse} I
sites of the plasmid pBac{[}3xP3-EGFPafm{]}(Horn, Schmid, Pogoda, \&
Wimmer, 2002) to produce the vector plasmid pBac{[}HC-TiBP-3xP3EGFP{]}
(Figure 1 b). TG silkworms were produced by injecting the vector plasmid
pBac{[}HC-TiBP-3xP3EGFP{]} into the eggs of silk worms in order to
produce TG silkworms, as reported previously (Tamura et al., 2000). TG
silkworms were established using a non-diapausing strain (w1-pnd) and
were mated with the diapausing strain (w1) to maintain TG silkworms as
diapausing strains. The silkworms were reared on an artificial diet
(Nosan Corporation, Yokohama, Japan) or on fresh mulberry leaves at 25
\selectlanguage{ngerman}°C. Due to the poor yield and robustness of the silk of TG silkworm
strains, they were backcrossed repeatedly with the commercial strains
Gunma and 200, which have been shown to have high silk productivity. The
resulting hybrid strains were then used for mass rearing of silkworms
and collection of silk from the cocoons. The wild-type \emph{B. mori} SF
was used for comparison with the TG SF (TiBP-SF). The SF and TiBP-SF
were dissolved by lithium bromide and desalinated using PD-10 column (GE
Healthcare, Buckinghamshire, UK). Then, the samples were analyzed by
4\%--12\% gradient SDS-PAGE. The gel was stained with Coomassie
Brilliant Blue R250 or transferred to polyvinylidene fluoride (PVDF)
membranes. The membrane was treated with an anti-His tag antibody
(A190-214A, BETHYL, Montgomery, USA). The immunoreactive bands were
visualized using ECL Plus (GE Healthcare, Buckinghamshire, UK) and an
LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).

\textbf{Surface modification of the Ti surface with SF or TiBP-SF.}

Pure wrought Ti disks (ø7 mm × 1 mm) were purchased from Furuuchi
Chemistry (Tokyo, Japan). The surface of each Ti disk was polished with
a sequence of 320-, 600-, and 1200-grit silicon carbide papers (RIKEN,
Saitama, Japan) under a stream of water. The polished Ti surface was
used after being rinsed with ultrapure water
(MilliQ\textsuperscript{®}), ultrasonically cleaned with acetone, and
then dried at room temperature. For coating, the disk was immersed in 5
ml 2 mass\% SF or TiBP-SF aqueous solution at 20 °C for 24 h. After
being air-dried at 20 °C for 1 h, the disk was rinsed with 70\% ethanol
and then air-dried again at 20 °C for 24 h.

\textbf{Cell culture.}

Osteoblast-like cells from rat calvaria (MC3T3-E1) cells were cultured
in sterile cell culture plates containing \selectlanguage{greek}α-\selectlanguage{english}minimal essential medium
(\selectlanguage{greek}α-\selectlanguage{english}MEM) with 10\% heat-inactivated fetal bovine serum (FBS) (Biowest,
Riverside, MO, USA), 2 mM L-glutamine and 10 U/mL
penicillin/streptomycin (Gibco, Grand Island, NY, USA) in a humidified
95\% air/5\% CO\textsubscript{2} incubator at 37 \selectlanguage{ngerman}°C. When the cells
reached sub-confluence, they were harvested and sub-cultured. The cell
suspension (5.0 × 10\textsuperscript{4} cells) was slowly dripped onto
the substrates (non-coated, SF-coated and TiBP-SF coated) in 48-well
plates. Three replicates of each sample were tested during the culture
period. Plates were shaken using gentle agitation for 10 min and were
then incubated for 3 h at 37 °C. Then, 0.5 mL of medium was added along
the side of each well.

\textbf{Scanning electron microscopy observations.}

At the end of the various incubation times, the non-attached cells on
the different substrates were removed from the substrates by rinsing
twice with 0.1 M sodium cacodylate buffered solution, dehydrated in a
graded series of ethanol (50\%, 70\%, and 90\%), and then placed in
100\% ethanol. The dehydrated samples were then immersed in tert-butyl
alcohol under vacuum (FDU-1200, EYELA, Tokyo, Japan). The freeze-dried
samples were mounted on aluminum stubs and were then sputter-coated with
a gold-palladium alloy. Scanning electron microscope (SEM, S-2150,
Hitachi, Tokyo, Japan) observations of each sample were then performed
at numerous magnifications at 10 kV.

\textbf{ALP assay}

Next, 5 × 10\textsuperscript{4} MC3T3-E1 cells/mL in \selectlanguage{greek}α-\selectlanguage{english}MEM were seeded
in 6-well plates overnight. The medium in each well was removed the next
day and replaced with osteoblast differentiation-inducing medium (OIM)
(MK430, Takara Bio Inc., Tokyo, Japan), and then incubated at 37 \selectlanguage{ngerman}°C for
3 weeks in a 5\% CO\textsubscript{2} incubator. Day 0 is the date the
OIM medium was added. ALP activity was determined using an ALP assay kit
(ab83369, Abcam, Cambridge, MA, USA) following the manufacturer's
instructions. In brief, untreated control and NaF-treated MC3T3-E1 cells
were homogenized in assay buffer after washing with cold PBS and
centrifuged at 15,000 rpm for 15 min at 4 °C. For each measurement, the
supernatants were added in triplicate in 96-well plates and each well
was brought to a total volume of 80 \selectlanguage{greek}µ\selectlanguage{english}L with assay buffer, followed by
the addition of 20 \selectlanguage{greek}µ\selectlanguage{english}L stop-solution for a background control. Standard
curves were generated as detailed in the manufacturer's manual. The ALP
activity of each sample was calculated based on a comparison between the
standard and sample curves and is reported as Unit/mL (U/mL).

\textbf{Cell differentiation, Alizarin Red S staining, and
quantification.}

Here, 5 \selectlanguage{ngerman}× 10\textsuperscript{4} MC3T3-E1cells/ml in \selectlanguage{greek}α-\selectlanguage{english}MEM were seeded
overnight with SF or TiBP-SF. The medium was removed the next day,
replaced with OIM, and incubated at 37 \selectlanguage{ngerman}°C for 3 weeks in a 5\%
CO\textsubscript{2} incubator. Day 0 is the date the differentiation
medium was added to cells. The entire medium was replaced every 3 days.
Alizarin red S staining was performed to analyze calcium deposits. Cells
were washed twice with Hanks balanced salt solution (HBSS) (Gibco 14025,
Thermo Fisher Scientific, Waltham, MA, USA) and then fixed with 4\%
paraformaldehyde for 15 min. The cells were stained with 1\% Alizarin
Red S solution for 3 min at 37 °C and rinsed with distilled water to
exclude non-specific staining. For quantitation, cells stained with
Alizarin red were de-stained with 10\% cetylpyridinium chloride
(Sigma-Aldrich, Saint Louis, MO, USA) for 15 min, after which the
extracted stain was transferred to 96-well plates, and the absorbance at
562 nm was measured using a microplate reader. After 2 and 3 weeks of
osteogenic induction, the level of OC in the supernatant was directly
measured using an ELISA kit (USCN Life Science, Wu Han, China) according
to the manufacturer's instructions and a microplate reader at 450 nm.
The assays were performed in triplicate.

\textbf{Quantitative RT-PCR analysis.}

At weeks 2 and 3 after incubation with OIM, total RNAs were isolated
using an miRNeasy Mini kit (Qiagen, Hilden, Germany). First-strand cDNAs
were synthesized from 1\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}g of each total RNA using Super Script VILO
Master Mix (Life Technologies, Carlsbad, CA, USA). One \selectlanguage{greek}µ\selectlanguage{english}L of each cDNA
was subjected to real-time RT-PCR using TaqMan Gene Expression Assays
(Applied Biosystems, Foster City, CA, USA) for Runx2 (Mm00501584\_m1),
Osterix (Mm04209856\_m1), ALP (Mm04209856\_m1), Collagen Type 1 (Col I)
(Mm04209856\_m1), OPN (Mm00436767\_m1) and OC (Mm03413826\_m1), and
Pre-Developed TaqMan Assay Reagents (Applied Biosystems) for \selectlanguage{greek}β-\selectlanguage{english}actin
(Mm02619580\_g1) as an internal control. Two-step PCR cycling was
carried out as follows: 2 min at 50 \selectlanguage{ngerman}°C for 1 cycle, 10 min at 95 °C for
1 cycle, then 15 s at 95 °C and 1 min at 60 °C for 40 cycles. Two
independent measurements were averaged, and relative mRNA expression
levels were calculated as a ratio to \selectlanguage{greek}β-\selectlanguage{english}actin expression of each sample.

\textbf{Statistical analysis.}

All quantitative data are expressed as means \selectlanguage{ngerman}± SD. Statistical analyses
were performed using the statistics program SPSS 20. Results were
analyzed by one-way ANOVA, followed by post hoc tests for pair-wise
comparisons (Tukey's test). Differences were considered to be
significant for p \textless{} 0.05.

\textbf{Synthesis of the Model Peptides of the AGSGAG and minTBP-1
motifs.}

To examine the binding mechanism of silk-TBP on the surface of
TiO\textsubscript{2} nonoparticles, the model peptides of the AGSGAG and
minTBP-1 motifs, i.e., RKLPDA (minTBP-1) , (AGSGAG)\textsubscript{2},
AGSGAGGRKLPDAGGAGSGAG (Silk-TBP), and
AGSG{[}1-\textsuperscript{13}C{]}AGGRKLPD{[}3-\textsuperscript{13}C{]}AGGAGSGAG
(\textsuperscript{13}C Silk-TBP) were synthesized in a stepwise fashion
on Fmoc-amino acid-PEG-PS resin by a Pioneer Peptide Synthesis System
(PE Biosystems, USA) using Fmoc solid phase method in our laboratory
(Asakura et al., 2005; Asakura \& Yao, 2002; Yu Suzuki et al., 2010; Yao
et al., 2004). The purity was more than 95\%, as verified by
high-performance liquid chromatography and \textsuperscript{13}C
solution NMR. The {[}1-\textsuperscript{13}C{]}Ala
(99.9\%\textsuperscript{13}C enrichment) and
{[}3-\textsuperscript{13}C{]}Ala (99.9\% \textsuperscript{13}C
enrichment) were purchased from Cambridge Isotopes Laboratories,
Andover, MA, USA.

\textbf{Preparation of TiO\textsubscript{2} particles
with\textsuperscript{13}C Silk-TBP on the surface.}

The nanoparticles of TiO\textsubscript{2} (Aeroxide
TiO\textsubscript{2}P25), which were kindly provided by NIPPON AEROSIL
co., JP, had an average particle size of 21 nm and a surface area of 50
± 15 m\textsuperscript{2}/g. The TiO\textsubscript{2} nanoparticles were
suspended in water and sonicated twice for 30 min with a 50\% duty
cycle. The suspension was then centrifuged at 10,000 rpm for 15 min, and
the precipitated TiO\textsubscript{2} nanoparticles were washed in
distilled water. This process was repeated, and the particles were dried
overnight. The dried particles were suspended in 20 mM phosphate buffer
at pH = 7.0 and sonicated again for 10 min with a 50\% duty cycle. The
peptides (TBP, (AGSGAG)\textsubscript{2}, and Silk-TBP) were dissolved
in 20 mM phosphate buffer and mixed with the suspended
TiO\textsubscript{2} nanoparticle solutions, respectively. The weight
ratio of the peptide and suspended TiO\textsubscript{2} nanoparticle was
1:1 for each case. Finally, the state of dispersion of the mixture after
standing overnight was observed.

\textbf{Solid-state \textsuperscript{13}C CP/MAS NMR Measurement} .

The \textsuperscript{13}C Silk-TBP (20 mg) was dissolved in 20 mM
phosphate buffer at pH = 7.0 (10 ml) and mixed with the
TiO\textsubscript{2} nanoparticles (50 mg). The solution was stirred for
4 h at 100 rpm to reach equilibrium. Then equilibrated. Then the
suspension was centrifuged at 10,000 rpm for 10 min and excess peptide
dissolved in the supernatant was removed. This process was repeated.
Thus, TiO\textsubscript{2} particles with \textsuperscript{13}C Silk-TBP
adsorbed on the surface were obtained after drying.
The\textsuperscript{13}C CP/MAS NMR spectra of the \textsuperscript{13}C
Silk-TBP and \textsuperscript{13}C Silk-TBP adsorbed on the surface of
TiO\textsubscript{2} nanoparticles were recorded on a Bruker Avance 400
NMR spectrometer with an operating frequency of 100.0 MHz
for\textsuperscript{13}C at a sample spinning rate of 8 kHz in a 4 mm
diameter ZrO\textsubscript{2} rotor. The numbers of scans were 1 K
for\textsuperscript{13}C Silk-TBP and 40 K for \textsuperscript{13}C
Silk-TBP adsorbed on the TiO\textsubscript{2} nanoparticles. The pulse
delay was 5 s and contact time was 2 ms. The \textsuperscript{13}C
chemical shifts were referenced to TMS using adamantane as a secondary
standard (\textsuperscript{13}CH peak at 28.8 ppm).



\textbf{RESULTS AND DISCUSSION}

\textbf{QCM analyses}

Designer proteins (Bc3) \textsubscript{2}, (Bc3) \textsubscript{4},
(Bc3) \textsubscript{2}T, and (Bc3) \textsubscript{4}T (Figure 1 a) were
synthesized and purified as described in MATERIALS AND METHODS. Using
QCM, the mode of interactions between the crated artificial proteins and
titanium surface was assessed. In the QCM assay, binding of molecules on
the surface of the sensor decreases the resonance frequency of the
sensor and detachment of molecules from the surface is observed as the
resonance frequency increases. Steps of QCM measurements are
schematically illustrated in Figure 2 a. First, the Ti sensor was
equilibrated with water for 30--60 min and the measurement was started
(time = 0). After 5 min, TBST (a solution buffer of the proteins) was
injected 3 times (time = 5, 10, and 15 min). Then, protein solution was
injected into the measurement chamber (time = 20 min). After 30 min, the
sensor was washed with TBST 3 times (time = 50, 55, and 60 min),
followed by washing with water 3 times (time = 65, 70, and 75 min).
Results obtained from (Bc3)\textsubscript{2} and (Bc3)\textsubscript{2}T
are shown in Figure 2 b. Injections of the protein solutions rapidly
decreased the resonance frequency of the sensor, indicating that both
(Bc3)\textsubscript{2} and (Bc3)\textsubscript{2}T have bound to the
surfaces of Ti. Because TBST washing only slightly increased the
frequency, the binding of these proteins to Ti is stable under the
conditions. When the measurement sensor was washed with water (time = 65
min), an increase of frequencies was observed, which was due to buffer
change (not detachment of proteins from the sensor). When the rates of
frequency increase in water were compared between
(Bc3)\textsubscript{2}and (Bc3)\textsubscript{2}T (time = 65--80 min),
it can be recognized that (Bc3)\textsubscript{2}T was detaching faster
than B(Bc3)\textsubscript{2}. Similar results were obtained from
(Bc3)\textsubscript{4} and (Bc3)\textsubscript{4}T (Figure 2 c).

\textbf{Construction of transgenic \emph{Bombyx mori} that spins the
cocoons containing the functionalized silk fibroin (TiBP-SF).}

The QCM analyses indicated that the artificial proteins composed of SF
core motifs and minTBP-1 motif have the abilities to bind to (and detach
from) the titanium surface. To evaluate the effect of the appended
minTBP-1 motif on the nourishing ability of SF for cells, we decided to
construct the large-scale production system for SF-minTBP-1 proteins.
For this experiment, we selected the (Bc3)\textsubscript{2}T =
{[}TS{[}(AGSGSG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDA{]} as a
core unit. This was because in the QCM experiments,
(Bc3)\textsubscript{2}T bound in larger quantities and detached faster
(especially in water) than (Bc3)\textsubscript{4}T, suggesting the
properties of minTBP-1 was more manifested in this formula.

We have already established the transgenic \emph{B. mori} system, which
has enabled us to prepare large amounts of engineered SF (Asakura et
al., 2014; Iizuka et al., 2013; Kojima et al., 2007; Nagano et al.,
2011; Saotome et al., 2015; Tamura et al., 2000; Yanagisawa et al.,
2007). In this study, a DNA fragment coding
{[}TS{[}(AGSGSG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDA{]}\textsubscript{8}((Bc3)\textsubscript{2}T)
was inserted into the plasmid pHC-EGFR(Kojima et al., 2007) to construct
pBac{[}HC-TiBP-3xP3EGFP{]} (Figure 1 b), which was injected into the
eggs of the silkworm, and transgenic silkworms were selected as
described previously (Kojima et al., 2007; Tamura et al., 2000;
Yanagisawa et al., 2007). The established transgenic silkworms spin
cocoons that are apparently indistinguishable from wild-type samples
(data not shown). From the cocoons, SF proteins were prepared following
standard procedures described in MATERIALS AND METHODS, and the obtained
fibroin proteins (named TiBP-SF) were analyzed by SDS-PAGE followed by
CBB staining (Figure 3 a). From the TiBP-SF sample, in addition to SF
H-chain and SF L-chain proteins, an additional protein band was observed
that was not detected from the wild-type SF sample. Western blotting
using an anti-His tag antibody confirmed that the protein was derived
from a transgenic gene (Figure 3 b).

The apparent molecular weight of the appendix protein (75 K) was larger
than that of the estimated molecular weight (52 kD). Similar abnormal
mobilities of the modified SFs in SDS-PAGE have also been reported in
other experiments (Asakura et al., 2014; Iizuka et al., 2013; Kojima et
al., 2007; Saotome et al., 2015; Teule et al., 2012; Yanagisawa et al.,
2007), which may be due to either skewed amino acid compositions of the
proteins or particular secondary structures.\begin{CJK}{UTF8}{gbsn}　\end{CJK}\selectlanguage{english}Because the ratio of
minTBP-1 motif to SF motif should be extremely small (approximately 0.1
to 0.9 \% in a similar system (Yanagisawa et al., 2007)) compared to the
model artificial proteins used in the experiments of Figure 2, we were
interested in the effect of minTBP-1 on the binding ability of SF to
titanium. As shown in Figure 4 a, QCM analyses indicated that TiBP-SF
decreased the amounts of protein absorbed on the titanium sensor, which
agreed well with the results obtained from (Bc3)\textsubscript{2}T and
(Bc3)\textsubscript{4}T. This means that with a decreased ratio of
minTBP-1/silk motif, the minTBP-1 motif masks the binding ability of
sill peptide to the material surface. Thus, doping SF with minTBP-1
dramatically changed the binding properties of SF. This notion was
further confirmed by the observation that the Silk model peptide
(AGSGAG)\textsubscript{2} was not able to disperse Ti particles in
aqueous solution, whereas Silk-TBP model peptide AGSGAGGRKLPDAGGAGSGAG
can endow the Ti particle with dispersibility similar to the mimTBP-1
peptide (Figure 4 b).

\textbf{Osteoblast differentiation on the TiBP-SF-coated titanium
surfaces.}

The above data demonstrated that TiBP-SF and SF have distinct behaviors
in terms of binding to the titanium surface. However, it was not clear
whether the altered properties of TiBP-SF can improve the performance of
titanium for medical devices. To address this question, we performed
biological assessments using an osteoblastic cell line (MC3T3-E1), which
has often been used to evaluate osteoblast differentiation. After
coating titanium plates with either SF or TiBP-SF (as described in
MATERIALS and METHODS), MC3T3-E1 was grown on the plates for 14 and 21
days in the differentiation-inducing medium (OIM). The morphologies of
the cells on the plates were then observed by SEM. As shown in Figure 5
a, the enhanced differentiation of MC3T3-E1, which is characterized by
an elongated sheet-like cell structures with multiple filopodia-like
intercellular connections protruding from their leading edges, was
evident after 21 days when the titanium plates were coated with TiBP-SF.
Without coating (or SF-coating), these characteristic morphologies were
not evident.

The formation of filopodia from MC3T3-E1 after 21 days of growth on the
TiBP-SF coated Ti plate suggested the cells were differentiated into a
hydroxyapatite-mineralizing state, which was further confirmed by the
increased alkaline phosphatase (ALP) activity and osteocalcin (OC)
production. As shown in Figure 5 b, both the ALP activity and OC
production from the cells grown on the TiBP-SF coated Ti substrate were
the highest among the 3 groups after 21 days of growth. Moreover, the
ALP activity of the SF group was lower than that of the TiBP-SF group (p
\textless{} 0.05), indicating that TiBP-SF was more beneficial than SF
for inducing cell differentiation and promoting cell mineralization. The
mineralization of MC3T3-E1 cells was also evaluated by alizarin red
staining (Figure 5 c). Cells grown on TiBP-SF displayed higher
mineralization capacities than those grown on SF (p \textless{} 0.01),
as well as the control after culture for 21 days. The calcified deposits
were uniform and clustered in all locations. To evaluate the cell
differentiation state in more detail, the expression levels of
osteoblast-related genes (including Runx2, Osterix, OC, OPN, ALP, and
Col I) were evaluated by real-time PCR analyses after culture for 14 and
21 days (Figure 5 d). Runx2 and Osterix are master transcription factors
involved in regulating the process of MC3T3-E1cell differentiation.
After culture for 14 days, the expression levels of both the Runx2 and
Osterix genes in cells cultured on TiBP-SF were up-regulated more than
those cultured on SF or on the control. Although the expression level of
the Runx2 gene decreased in all groups after culture for 21 days, the
TiBP-SF group remained much higher than the SF group. A similar trend
was also found for Osterix expression. The early markers of osteoblasts
(such as the transcription of ALP and Col I) were also up-regulated
after culture for 21 days on TiBP-SF compared with the other groups.
Furthermore, culture on TiBP-SF up-regulated the mRNA expression levels
of OC and OPN at most time intervals. Thus, TiBP-SF was shown to have an
ability for enhancing the differentiation of an osteoblast model cell
line \emph{in vitro} .

\textbf{Conformations of \textsuperscript{13}C Silk-TBP
and\textsuperscript{13}C Silk-TBP adsorbed on the surface of
TiO\textsubscript{2} nanoparticles in the solid state.}

To obtain further insights into the binding mechanisms of the
SF-minTBP-1 proteins, we synthesized the \textsuperscript{13}C -labeled
model peptide for solid state NMR analyses. Figure 6
shows\textsuperscript{13}C CP/MAS NMR spectra (10-70 ppm) of (a)
\textsuperscript{13}C Silk-TBP
(AGSG{[}1-\textsuperscript{13}C{]}AGGRKLPD{[}3-\textsuperscript{13}C{]}AGGAGSGAG)
and (b) \textsuperscript{13}C Silk-TBP adsorbed on the surface of
TiO\textsubscript{2} nanoparticles together with the assignment (Asakura
et al., 2005; Asakura \& Yao, 2002; Yu Suzuki et al., 2010; Y. Suzuki,
Shindo, \& Asakura, 2016; Wishart, 2011; M. Yang \& Asakura, 2005; Yao
et al., 2004). The chemical shifts and the assignments are summarized in
Table 1. According to the chemical shifts of the peaks, Ala C\selectlanguage{greek}β \selectlanguage{english}and Ala
C\selectlanguage{greek}α \selectlanguage{english}carbons, the conformation of the \textsuperscript{13}C Silk-TBP
sample in a solid state is considered to be random coil. This is due to
the sample preparation by freeze-dry treatment (Asakura et al., 2005;
Yao et al., 2004). After adsorption treatment of the peptides on the
surface of the TiO\textsubscript{2} nanoparticles, a
small\textsuperscript{13}C labeled methyl peak of
{[}3-\textsuperscript{13}C{]}Ala\textsuperscript{13} residue in the
partial sequence (RKLPD{[}3-\textsuperscript{13}C{]}A) in the peptide
could be still observed. Interestingly, the Ala\textsuperscript{13}C\selectlanguage{greek}β
\selectlanguage{english}chemical shift changed from a random coil (16.3 ppm) to an \selectlanguage{greek}α-\selectlanguage{english}helix (14.4
ppm) (Figure 6). Namely, the Ala\textsuperscript{13} residue adsorbed on
the TiO\textsubscript{2} particle took an \selectlanguage{greek}α-\selectlanguage{english}helix form. The \selectlanguage{greek}α-\selectlanguage{english}helix peak
also became remarkably sharper compared to the Ala C\selectlanguage{greek}β \selectlanguage{english}random coil peak
of the peptide before adsorption. This could indicate that the
interaction between the minTBP-1 and the surfaces of
TiO\textsubscript{2} is governed by reversible interactions. In
addition, relatively sharp peaks at 21.0 and 25.6 ppm could be observed,
which are assigned to the side chain carbons of natural abundant Leu and
Lys residues in the sequence RKLPDA. Namely, the peak at 21.0 ppm is
assigned to the Leu C\selectlanguage{greek}δ\selectlanguage{english}\textsubscript{1},C\selectlanguage{greek}δ\selectlanguage{english}\textsubscript{2}, and Lys C\selectlanguage{greek}γ
\selectlanguage{english}carbons, and the peak at 25.6 ppm to the Lys C\selectlanguage{greek}δ \selectlanguage{english}carbon (according to the
chemical shifts). Thus, the Leu and Lys residues are considered to be
attached to the surface of TiO\textsubscript{2} particles, although the
backbone peaks of these residues could not be observed.

\textbf{CONCLUSIONS}

By using a transgenic \emph{B. mori} system, we synthesized the modified
silk fibroin TiBP-SF, which is doped with an artificial protein composed
of silk fibroin motif (AGSGAG) and titanium binding motif (RKLPDAS) in
the
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDAS{]}\textsubscript{8}formula.
The TiBP-SF enhanced the ossific differentiation of osteoblast-like
cells when coated on the surfaces of a titanium plate. Accordingly,
TiBP-SF has potential applications in the medical field.

\textbf{Acknowledgments}

T. A. and K. S acknowledge the supports of a JSPS KAKENHI, Grant-in-Aid
for Scientific Research (C), Grant Number JP19K05609 and Grant-in-Aid
for Scientific Research (A), Grant Number 23240135, respectively.

\textbf{Figure captions}

\textbf{Figure 1.} (a) Construction flow of the recombinant plasmids
from genetic blocks coding for the crystalline region of silk fibroin
((AGSGAG)\textsubscript{3}AS, blue arrow) and for the minimal titanium
binding motif (RKLPDA , orange arrow). B, N, S, and H
represent\emph{Bam} HI, \emph{Nhe} I, \emph{Spe} I, and \emph{Hind} III,
respectively. From the resultants plasmids, the His-tagged proteins,
(Bc3) \textsubscript{2}, (Bc3) \textsubscript{4},
(Bc3)\textsubscript{2}T, and (Bc3) \textsubscript{4}T were produced in
BL21(DE3)pLysS and purified by nickel-chelate chromatography, whose
domain structures are schematically illustrated by the colored ellipses
(indicated in the inner box). (b) The structure of the vector for
constructing the transgenic silkworm. The gene for
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDA{]}\textsubscript{8}is
under the control of Fibroin H promoter. (c) The structure of proteins
translated from pBac{[}HC-TiBP-3xP3EGFP{]}, which has the N-terminal
domain (NTD), the C-terminal domain (CTD), and C-terminal His-tag, which
were derived from the parental vector, pBac{[}3xP3-EGFPafm{]} (Horn et
al., 2002). Arrows indicate right and left arms of the transposon
piggyBac. EGFP was used for screening the transgenic silkworm.

\textbf{Figure 2.} (a) Schematic drawings representing the changes in
frequency ([?]f; left axis) of titanium sensor upon (i) buffer injection,
(ii) sample injection, (iii) buffer wash and (iv) water wash in QCM
analyses. (b) Changes in resonance frequency with 0.5 \selectlanguage{greek}µ\selectlanguage{english}m
(Bc3)\textsubscript{2} (blue) and (Bc3) \textsubscript{2}T (green). (c)
Changes in resonance frequency with 0.5 \selectlanguage{greek}µ\selectlanguage{english}m (Bc3) \textsubscript{4}(blue)
and (Bc3) \textsubscript{4}T (green).

\textbf{Figure 3.} (a) SDS-PAGE and Coomassie Brilliant Blue R250
staining for the functionalized SF, TiBP-SF, and its parental SF. The
arrow indicates the artificial protein containing
{[}TS{[}(AGSGAG)\textsubscript{3}AS{]}\textsubscript{2}RKLPDA{]}\textsubscript{8}(Figure
1 c). The open circle, the closed circle, and the triangle indicate
Fibroin H-chain protein, Fibroin L-chain protein, and Fibro hexamerin
protein (Inoue et al., 2000), respectively. (b) Western blotting using
anti-His antibody, which recognizes the C-terminal His-tag of the
artificial SF.

\textbf{Figure 4.} (a) Changes in resonance frequency of titanium sensor
with 1 mg/mL of SF (blue) and TiBP-SF (green). Details of experimental
procedures are described in Figure 1 caption and MATERIALS AND METHODS.
(b) Dispersion of TiO\textsubscript{2} particles (21 nm) with TBP
(RKLPDA) peptide, Silk-TBP (AGSGAGGRKLPDAGGAGSGAG) peptide, Silk
((AGSGAG)\textsubscript{2} ) peptide and no peptide (control). Details
of experiments are described in MATERIALS AND METHODS.

\textbf{Figure 5.} (a) SEM images (magnification of 2000\selectlanguage{ngerman}×) of non-coated
(cont), SF-coated and TiBP-SF-coated polished Ti surfaces after
cultivating MC3T3-E1 cells for 14 or 21 days. The bars indicate 10 \selectlanguage{greek}µ\selectlanguage{english}m.
(b) Effects of TiBP-SF coating on the alkaline phosphatase (ALP)
activity and the expression level of osteocalcin (OC) of MC3T3-E1 cells
after 14 and 21 days incubation. Values are expressed as means \selectlanguage{ngerman}± SD (n =
3). Asterisks indicate P \textless{} 0.05 in the t-test. (c) Effect of
TiBP-SF coating on the mineralization in MC3T3-E1 cells. Cells were
stained with Alizarin Red (left) and quantified as described in the
MATERIALS AND METHODS after 14 and 21 days incubation. Asterisks
indicate P \textless{} 0.05 in the t-test. (d) Expression levels of mRNA
for Runx2, Osterix, ALP) Collagen Type I (Col I), Osteopontin (OPN) and
OC, which were quantified by quantitative RT-PCR, in MC3T3-E1 cells
after 14 and 21 days incubation on non-coated (control), SF-coated, and
TiBP-SF-coated polished Ti surfaces. The relative mRNA expression levels
were calculated as a ratio to \selectlanguage{greek}β-\selectlanguage{english}actin expression of each sample. Values
represent means \selectlanguage{ngerman}± SD from three independent experiments. Asterisks
indicate P \textless{} 0.05 in the t-test.

\textbf{Figure 6.} \textsuperscript{13}C CP/MAS NMR spectra (10--70 ppm)
of (a) \textsuperscript{13}C Silk-TBP
(AGSG{[}1-\textsuperscript{13}C{]}AGGRKLPD{[}3-\textsuperscript{13}C{]}AGGAGSGAG)
and (b) \textsuperscript{13}C Silk-TBP adsorbed on the surface of
TiO\textsubscript{2} nanoparticles together with the assignments.

Table 1. \textsuperscript{13}C chemical shifts and assignments
of\textsuperscript{13}C CP/MAS NMR spectra of (a) \textsuperscript{13}C

Silk-TBP and (b) \textsuperscript{13}C Silk-TBP adsorbed on the surface
of TiO\textsubscript{2} nanoparticles.

Chemical Shift~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~Assignment

(a)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~(b)

\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}..

(b) 14.5
ppm~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ala~\textsuperscript{13}Cb~~~~~a-helix

(a) 16.2 ppm~~~~~~~~~~~~~~~~~~~~~~Ala~\textsuperscript{13}Cb~~~random
coil

(b) 21
ppm~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Leu
Cd\textsubscript{1},d\textsubscript{2}~~Lys Cg

(b)~25.5
ppm~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Lys
Cd

(a) 42 ppm (broad)~~~~~~~~~~~~Gly Ca

(b) 42--43 ppm
(broad)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Gly Ca

(a) 49 ppm (broad)~~~~~~~~~~~~~Ala Ca~~~random coil~~~~~~~~~~~~~~-

\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}\ldots{}..

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