tached to the surrounding alveolar bone via soft periodontal ligament tissues. With the loss of natu­

INTRODUCTION

Osseointegration is currently considered the optimal implant­to­bone interface.

Natural teeth are at­

tached to the surrounding alveolar bone via soft periodontal ligament tissues. With the loss of natu­

ral teeth, the periodontal ligament cells are lost as well. Therefore, those cells cannot participate in wound healing around endosseous implants that are inserted for the replacement of lost teeth. At present, optimal healing around implants is consid­

ered achievable via a bone anchorage with intimate bone­to­implant contacts, called osseointegration.

Since osseointegrated implants are “ankylosed” and do not have the same mobility as natural teeth with a periodontal ligament, efforts have been made during the past few decades to compensate for this obvious difference by integrating “shock absorbing

systems” into the implant or its superstructure.

Ideally, it would be preferable to have a system for anchoring dental implants with the same functional mobility as natural teeth.

There has been a concerted effort among materi­

als scientists and clinicians worldwide to improve the performance of dental implants with the aim of accelerating and maintaining their integration into hard and soft tissues and/or extending their range of applications.

The surface characteristics of the implant material affect the rate and extent of os­

seointegration.

Vandrovcová et al.

recently re­

viewed the growing evidence demonstrating that surface­modified materials are highly effective for promoting the adhesion, growth, and osteogenic differentiation of cells.

The structures that were used in this study are nanostructures similar to the TiO

nanotubes cre­

Effect of nano modified titanium surface on adsorption of rat periodontal ligament cells

Yukari Hara, Satoshi Komasa, Shigeki Yoshimine, Hiroshi Nishizaki and Joji Okazaki

Department of Removable Prosthodontics and Occlusion, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573-1121, Japan

When natural teeth are lost, endosseous implants anchored via intimate bone­to­implant contacts are considered an appropriate replacement. The interactions between implants and host tissues depend on several factors. In particular, a growing body of evidence has demonstrated that the surface texture of an implant influences the response of the surrounding cells. Recent studies have shown that treating titanium with aqueous NaOH produces a nanostructured surface texture termed a titanium nanosheet (TNS). Further­

more, it was recently reported that a TNS surface promoted the osteogenic differentiation of rat bone marrow cells. We investigated whether a TNS surface may improve wound healing around endosseous implants. To test this, we assessed the effects of a TNS sur­

face on the adhesion and differentiation of rat periodontal ligament (RPL) cells in vitro.

The results demonstrated that RPL cells cultured on a TNS surface showed more adhe­

sion and higher levels of osteogenic differentiation markers (Runx2 expression, alkaline phosphatase activity, calcium deposition, and osteocalcin secretion) than cells cultured on unmodified titanium. These findings indicate that RPL cells have the potential for in­

creased bone formation on a TNS surface. Thus, TNS surface modification may warrant consideration for use in endosseous dental implants. (J Osaka Dent Univ 2018 ; 52 : 37­

44)

Key words : Periodontal ligament cells ; Titanium nanostructures ; Implant

ated by titanium deposition using the process of TiO

sputtering,

and are called titanium nanosheets (TNS). Recently, it has been demonstrated that nanotube and TNS structures could be obtained on a titanium metal surface by treating the surface with a 10 M NaOH aqueous solution at 30° C.

We em­

ployed this method here to create TNS structures on modified titanium disks. Recent research has shown that treatment with an NaOH aqueous solu­

tion produces a rough, nanoscale surface,

and scanning electron microscopy images of our modi­

fied disks demonstrated that the TNS­modified sur­

face had a good surface roughness without any cracks. A previous study

reported that TNS pro­

duced via chemical processing promoted the osteo­

genic differentiation of rat bone marrow cells. The surface properties and structures of materials influ­

ence the adsorption of proteins. Thus, the in­

creased adsorption of proteins on a TNS­modified surface might influence cell behavior.

Schroeder et al. demonstrated the successful placement of titanium implants using a nonsub­

merged technique and showed for the first time un­

dermineralized histological sections of titanium with soft and hard tissues.

The reaction of soft tissues to the implant surface has been further examined by Schroeder et al.,

Buser et al.,

and Listgarten et al.

These studies indicated that the surface tex­

ture of an implant significantly affects the type of connective tissue that attaches to it. The purpose of this study was to analyze the reaction of soft tissue cells to an alkali­modified titanium surface.

MATERIALS AND METHODS Specimen production

In the test group, titanium disks that were treated to produce nanostructures on their surfaces were used as the experimental material. Titanium disks (15 mm diameter) were punched from 1­mm thick grade 2 unalloyed titanium sheets (Daido Steel, Osaka, Japan). These disks were immersed in 10 M NaOH (aq) and placed in an oil bath maintained at 30° C for 24 h. Unprocessed titanium disks were used as controls. The NaOH solution in each flask was replaced with distilled water (200 mL), and the

NaOH treatment and wash procedure was repeated until the wash solution reached a conductivity of 5 μS/cm. Specimens were then dried at room tem­

perature. The surface topology and roughness of those groups were evaluated using a scanning electron microscope (S­4800 ; Shimadzu, Kyoto, Japan) and scanning probe microscopy (SPM­

9600 ; Shimadzu) over a surface area of 2×2 μm.

The composition of the coating was analyzed by X­

ray photoelectron spectroscopy (XPS ; ESCA 5600, Ulvac­Phi, Kanagawa, Japan), using surface etch­

ing with ionized argon. XPS analysis was also used in the surface analyses, with an Al K line (15 kV, 300 W) used as an X­ray source. Argon ion sput­

tering was performed during XPS to estimate the thickness and structure of the surface layers.

Cell culture

Rat periodontal ligament (RPL) cells were pur­

chased from Lonza (Walkersville, MD, USA). The medium (BulletKit

; Lonza) was prepared at an appropriate concentration (1 mL/cm

) and the cells were carefully thawed from cryovials. According to the recommended seeding density (3500 cells/cm

), the cells were placed into prepared tissue culture flasks and incubated in a 5% CO

humidified incu­

bator at 37° C. The growth medium was changed the day after seeding and every other day thereaf­

ter. When the cells reached 70­80% confluency throughout the flask, they were subcultured using 4­

(2­hydroxyethyl)­1­piperazineethanesulfonic acid­bu­

ffered saline solution, trypsin/ethylenediaminete­

traacetic acid, and trypsin neutralizing solution.

Then, the harvested cells were pelleted by centrifu­

gation at 220×g for 5 min. This study protocol was performed following the Guidelines for Animal Ex­

perimentation at Osaka Dental University (Approval No.16­08001).

Cell adhesion

Cell adhesion was measured using the CellTiter­

Blue

cell viability assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

RPL cells were seeded on the samples at a density

of 4×10

cells/cm

and allowed to attach for 1, 3, 6

or 24 h. At each prescribed time point, nonadherent cells were removed by rinsing with phosphate­

buffered saline (PBS). CellTiter­Blue

reagent (50 μL) and PBS (250 μL) were then added to each well. After incubation at 37° C for 1 h, the solution was removed from the 24­well tissue culture plates (Falcon

, Corning, Corning, NY, USA) and 100 μL was added to a new 96­well tissue culture plate (Falcon

; Corning). The optical densities of the re­

maining solution at 560 and 590 nm were meas­

ured. The difference between the two optical densi­

ties was defined as the proliferation value.

Runx2 mRNA expression

Total RNA was extracted from cells and 1 μg was used to synthesize cDNA using a high­capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA) after 3 days. Runx2 mRNA expression was investigated by quantitative reverse transcrip­

tion (RT)­polymerase chain reaction (PCR) on a StepOnePlus

real­time RT­PCR system (Applied Biosystems). TaqMan

fast universal PCR master mix (10 μL), 1 μL of the primer probe set (20×

TaqMan

gene expression assays), sample cDNA (2 μL), and diethylpyrocarbonate­treated water (7 μL ; Nippongene, Toyama, Japan) were added to each well of a fast 96­well reaction plate (0.1­mL well volume ; Applied Biosystems). The plate was subjected to 40 reaction cycles of 95° C for 1 s and 60° C for 20 s. The expression level of each target gene was calculated with the

Ct method and nor­

malized to that of the negative control group.

Alkaline phosphatase (ALP) activity

RPL cells were washed with PBS and lysed with 200 μL of 0.2% Triton

X­100 (Sigma­Aldrich, St.

Louis, MO, USA). The lysate was transferred to a microcentrifuge tube containing a 5­mm hardened steel ball after 7 and 14 days of culture. Tubes were agitated on a shaker (Mixer Mill Type MM 301 ; Retsch GmbH, Haan, Germany) at 29 Hz for 20 sec to homogenize the sample. ALP activity was measured using an ALP luminometric enzyme­

linked immunosorbent assay (ELISA) kit (Sigma­

Aldrich) according to the manufacturer’s protocol.

The reaction was terminated by adding 3 N NaOH to achieve a final concentration of 0.5 N NaOH and p ­nitrophenol production was determined by meas­

uring the absorbance at 405 nm on a 96­well mi­

croplate reader (SpectraMax

M5 ; Molecular De­

vices, Sunnyvale, CA, USA). DNA content was measured with the PicoGreen

dsDNA assay kit (Invitrogen/Life Technologies) according to the manufacturer’s protocol. The amount of ALP was normalized to the amount of DNA in the cell lysate.

Osteocalcin production

After 21 or 28 days of culture, a commercial sand­

wich ELISA kit (Rat Osteocalcin ELISA kit ; DS Pharma Biomedical, Osaka, Japan) was used to determine osteocalcin levels directly in the cell cul­

ture supernatant according to the manufacturer’s in­

structions.

Calcium deposition

Calcium deposition was assessed using a calcium E­test kit (Wako Pure Chemical Industries, Osaka, Japan). At each tested time point (21 or 28 days of culture), 1 mL calcium E­Test reagent and 2 mL buffer were added to 50 μL of the collected me­

dium. The absorbance of the reaction products was measured at 610 nm using a 96­well microplate reader (Falcon

; Corning). The concentration of calcium ions was calculated according to the manu­

facturer’s instructions.

Statistical analysis

All experiments were performed in triplicate. Data are presented as mean and standard deviation. In all analyses, statistical significance was determined by Student’s t­test at p＜0.05.

RESULTS

Characterization of materials

Scanning electron microscopy images are shown in Fig. 1. After modification in NaOH at 30° C, the tita­

nium surface of the test disks showed a nanometer

­scale network structure. Scanning probe micros­

copy was used to estimate the vertical height of the

surface roughness of the samples. The surface

Control Test

Control Test

Control Test

morphologies of the test and control titanium sur- faces are shown in Fig. 2. The scanning probe mi- croscopy images showed that there were many nanonodules on the surfaces of the test group. The surface roughness (Ra) values were 4.6 and 13.7 nm for the titanium surfaces of the test and control groups, respectively. Figure 3 shows the results of wide-scan XPS surface chemical analyses of the test and control titanium surfaces. The presence of Ti and O was confirmed on both titanium surfaces.

In addition, the presence of Na was confirmed on the surface of the test group. The concentrations of Ti and O on the surface of the test group were greater than those on the surface of the controls.

Cell adhesion

Cell adhesion on the disks after 1, 3, 6 and 24 h of incubation was assessed (Fig. 4). There were sig- nificant differences in cell adhesion and proliferation

Fig. 1 Scanning electron microscopy images.

Fig. 2 Scanning probe microscopy images.

Fig. 3 X-ray photoelectron spectroscopy analysis.

Fig. 4 Rat periodntal ligament cell adhesion.

between the alkali-treated and control samples for all time periods.

Gene expression

The mRNA expression levels of osteogenesis- related genes including runt-related transcription factor (Runx2) in RPL cells grown on the different surfaces for 3 days were assessed by qRT-PCR.

The expression of Runx2 was upregulated in the cells grown on the test disks as compared to those grown on the control disks (Fig. 5).

ALP activity

ALP activity was observed in RPL cells grown on the different substrates for 7 days, and was found to increase over time. There were significant differ- ences in ALP activity between the alkali-treated and untreated titanium surfaces at 7 and 14 days (Fig.

6), with greater activity levels in the former group.

Osteocalcin secretion

We measured osteocalcin levels in the culture su- pernatant of RPL cells grown on the different sub- strates for 21 and 28 days. The supernatants of cells grown on the test disks showed higher levels of secreted osteocalcin at both time points as com- pared with the control cultures (Fig. 7).

Calcium deposition

Calcium deposition in cells cultured on the different surfaces for 21 and 28 days was assessed as a measure of osteogenic differentiation. Mineraliza- tion was greater in the cells grown on the test disks than in those grown on the control disks (Fig. 8).

DISCUSSION

Dental implants, which are artificial tooth roots that support dental prostheses, are a popular approach for dental restoration.

The immediate placement of dental implants into fresh extraction sockets is an

Fig. 5 Expression of Runx2 mRNA (p＜0.05).

Fig. 6 Alkaline phosphatase activity.

Fig. 7 Osteocalcin production.

Fig. 8 Calcium deposition.

attractive alternative to delayed implant placement after alveolar healing because of advantages that include a shorter rehabilitation time, fewer surgical episodes, an improved preservation of bone tis- sues, and improvement in the peri-implant gingival tissue aesthetics.

Immediate implantation, which involves the placement of the implant in the peri- odontium before the healing of the tooth extraction site, is followed by the formation of connective tis- sue by periodontal ligament cells in the fresh ex- traction socket.

Periodontal ligament cells are involved in the complex process of osseointegration, i.e., the es- tablishment of a direct bone-to-implant anchorage without intervening connective tissue, which is es- sential for the success of immediate implantation.

This study investigated whether RPL cells would re- spond differently to titanium implants that had un- dergone a chemical surface modification compared with those that had not. We found that the initial adhesion of cells, the expression of the Runx2 tran- scription factor, and the concentrations of RPL cell differentiation markers such as ALP and osteocal- cin were elevated in cells grown on a TNS-modified titanium disk as compared with those grown on an unmodified, polished titanium disk. We also found that calcium deposition in the extracellular matrix of the RPL cells was increased in the presence of a TNS-modified disk as compared with the cultures grown on unmodified control disks. Our results sug- gest that a TNS-structured titanium surface pro- motes RPL cell adhesion, differentiation, and acti- vation, which augments calcium deposition.

The surface properties of dental implants have long been recognized as a critical factor for achiev- ing clinical success.

The topographical proper- ties of nanostructures on titanium alloy surfaces play important roles in modulating cell responses at the implant-tissue interface, which can have a large effect on tissue integration with the implant.

Re- cently, we showed that TiO

nanotubes and TNSs could be formed on titanium metal surfaces by treatment with 10 M NaOH aqueous solution at 30° C.

We used this method here to prepare TNS- modified disks. Komasa et al.

suggested that TNS

-structured titanium surfaces modulate the osteo- genic differentiation of bone marrow cells and en- hance mineralization.

Ra is a height parameter that is commonly used to describe the surface roughness of implants. The Ra of the TNS-modified titanium surface was 17 nm, which was smaller than that of the untreated ti- tanium surface. Xing, Fujino, and colleagues showed that a surface roughness between 13-16 nm was optimal for RBM cell culture.

The nanonetwork structure formed on the titanium disks here is similar to the hierarchical structure reported by Lingzhou and coworkers.

In their work, hierar- chical nanotextured titanium surface topographies with titania nanostructures that mimicked the hierar- chical structure of bone tissues were produced by etching followed by anodization.

Natural tissues are hierarchical structures of nanoscale building blocks assembled in an organ- ized manner. Hierarchical structures composed of nanocomponents may provide a more suitable sur- face topography for bone marrow cell functions than simpler structures because they can better mimic the structure of natural tissues. In this study, our research revealed that NaOH treatment led to the formation of a Ti-O-Na titanate layer on the tita- nium surface.

Thus, we expect that NaOH treat- ment caused a thick oxide film to form on the TiO

layer on the titanium surface. The deconvolution procedure suggested that this may have been due to surface contamination resulting from the binding of O to C.

Our findings demonstrated that an alkali-modified

titanium surface induced more adhesion of RPL

cells than an untreated titanium surface. Cell adhe-

sion influences several cellular functions including

proliferation, migration, and extracellular matrix pro-

duction. The enhanced adhesion may be related to

the increased adsorption from the culture medium

of serum proteins such as fibronectin and vi-

tronectin, which play important roles in cell adhe-

sion. In a recent study, a TNS surface promoted

the adsorption of albumin and fibronectin.

This ob-

servation suggests that the TNS structure promotes

cell adhesion and spreading on the titanium sur-

face. The periodontal ligament cells that support the teeth within the alveolar socket are composed primarily of osteoblasts, osteoclasts, cementoblasts, and fibroblasts. These results show that TNS struc- tures have the potential to improve periodontal re- generation in dental clinics.

Runx2 mRNA expression, ALP activity, osteocal- cin production, and calcium deposition were all ele- vated by the presence of TNSs on the implant sur- face. Runx2 mediates several early osteogenic gene responses for cellular adhesion.

Several lines of evidence have shown that certain surface modifications lead to a high expression of Runx2 mRNA.

The data from this study suggests that the elevated expression of Runx2 mRNA in cells grown on the nanostructured titanium surfaces compared with that on untreated surfaces is a causative factor in the differentiation of RBM cells into osteogenic cells. Importantly, the functional phenotypes expressed in the middle and late stages of culture, such as ALP activity and miner- alization, were considerably increased.

A substantial body of research has confirmed the ALP-activating effect of surface-modified materi-

als.

The surface modification of implants has

been found to affect ALP activation,

and our re- sults support this conclusion. The increase of os- teocalcin production in the presence of a nanos- tructured surface that we observed is also in agree- ment with previous findings.

Periodontal ligament cells are useful cells for the reconstruction of peri- odontal tissues because they contain osteogenic and fibrogenic progenitor cells, which differentiate into fibroblasts, cementoblasts, or osteoblasts de- pending on the surrounding conditions.

Another study showed that a rough titanium sur- face improved periodontal ligament cell and osteo- genic differentiation. To avoid the interference of connective tissues, the implant surface should be engineered to promote osteogenic differentiation and a faster healing process. In our previous study, the investigation of several different implant surface nanostructures demonstrated that modifying the im- plant surface at the nanometer scale can enhance the adhesion of endothelial cells and the expres-

sion of genes encoding angiogenic factors and ad- hesion molecules.

Therefore, applying the TNS structural modification to a titanium surface could be useful for dental implants because it can pro- mote osteogenic differentiation and a faster healing process.

Titanium and titanium alloy implants have be- come an essential treatment modality in reconstruc- tive surgery in orthopedics and dentistry. However, patient morbidity and treatment complications need to be minimized, and outcome predictability and treatment indications should be maximized. There- fore, considerable effort has been expended on de- veloping new technologies to modify the surface of titanium to assist the biointegration of titanium im- plants with bone. The surface modification method used here is effective and simple, involving incuba- tion in NaOH at room temperature without a tem- plate. The resulting TNS-modified titanium surface induces the osteogenic differentiation of RBM cells.

CONCLUSIONS

Our study demonstrated that periodontal ligament cells cultured on a nano-modified titanium surface showed higher adhesion and were osteogenic dif- ferentiation than those cultured on a non-modified titanium surface. These findings indicate that RPL cells have the potential for increased bone forma- tion on a TNS surface. The TNS surface modifica- tion may warrant consideration when manufacturing the materials for use in dental implants intended for immediate implantation.

We would like to thank Tohru Sekino of Osaka University for preparing the nanosheets and for helpful suggestions.

We are also grateful to the members of the Department of Removable Prosthodontics and Occlusion. We also thank Toshio Tamaki and Hirokazu Hojyo. This work was sup- ported by a grant from the Japan Society for the Promotion of Science (16K20524) and an Oral Implant Research Grant (17-01) from Osaka Dental University.

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