Investigation of Effective Modification Treatments for Thin Titanium Membranes

Authors list: Reiko Kobatake, Kazuya Doi, Yoshifumi Oki, Hanako Umehara, Hiromichi Kawano, Takayasu Kubo, Kazuhiro Tsuga Affiliation: Department of Advanced Prosthodontics, Hiroshima University Graduate School of Biomedical and Health Sciences Corresponding author: Kazuya Doi Address: Department of Advanced Prosthodontics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan Tel: +81 82 257 5677 Fax: +81 82 257 5679 E-mail address: kazuya17@hiroshima-u.ac.jp


Introduction
The existence of sufficient bone volume is a factor for successful treatment with dental implants [1].In cases with insufficient bone volume at the implant placement site due to bone absorption or trauma, implant threads can be partially exposed when the implant is placed into the bone tissue, and in these cases, guided bone regeneration (GBR) is applied to augment the bone tissue [2,3].Barrier membranes play a crucial role in GBR, because epithelial tissues recover more quickly than bone, and can invade into the space required for new bone formation and inhibit the process [4].Therefore, positioning the barrier membrane at the interface between the epithelium and periosteum retains the space required for bone healing.
Absorbent membranes, such an atelocollagen or polyglycolic acid, display excellent operability and do not require removal, and are used as barrier membranes in periodontal therapy.These absorbent membranes are suitable for small bone defects; however, their application to the grafting of large bone defects is problematic because of their insufficient mechanical strength [5,6].
Non-absorbent membranes, such as those made from titanium, are superior in mechanical strength to absorbent membranes.Titanium membranes are used as barrier membrane for GBR because of their superior biocompatibility, mechanical strength, and operability.Therefore various studies have demonstrated that they make and retain space well in grafts of large bone defects [7,8,9].However, titanium does not have the ability to accelerate bone formation because it is bioinert [10,11].
It is well known that titanium surface topography can be improved by various modification methods [12,13].Improved titanium surfaces have bioactive ability, and can promote cell adhesion and osteoinduction [14,15,16].In particular, chemical methods, such as acid etching or alkali treatment, are often used because of their simplicity [17,18,19].A titanium surface modified by a strong acid or alkali solution can form an apatite layer when soaked in body fluid [18,21,22].For the reason, these modified treatments are already being applied as dental implants and titanium plates used in bone reconstruction.Therefore, the creation of bioactive thin titanium membranes would be beneficial for GBR.
Chemical treatments corrode the titanium surface, making it rough, extremely hydrophilic, and suitable for bone formation [23].The corrosion depth is not a serious problem for solid titanium materials such as implant

2-2 Corrosion depth
Table 1 shows the thickness of each sample.The acid group displayed a significantly large corrosion depth compared to the other groups, while the degree of corrosion in the alkali group was slight and comparable to the control.image in Fig 2c .Table 3 shows the contact angle of each sample.The angle in the alkali group was almost 0°, significantly lower than other groups, suggesting that the alkali membranes had much higher hydrophilicity.
Figure 2 The shapes of the water drops applied to each sample.To regenerate large sections of bone, the GBR membrane needs to be malleable enough to easily conform to bone morphology, and have adequate mechanical strength to maintain its form until the new bone has formed.
There is a correlation between mechanical strength and thickness; thicker Thickness and mechanical tensile strength of acid treatment was significantly decreased compare with non-treated membrane and alkali membrane.The SEM image was significant, according to the results of corrosion depth and tensile strength measurements.
Several studies have reported that acid treatment modifies the wettability of titanium surfaces [13,23].In wettability test, the contact angle of acid treatment was significantly higher than non-treated and alkali treatment.
Currently, we have no clear explanation for this discrepancy.However, these past studies were performed using finely polished, smoothly surfaced titanium disks as control samples.In our study, the thin membrane control had an irregular rough surface caused by the manufacturing process.Thus, acid treatment seems to be altered the thin titanium membrane from a rough surface to a smooth surface, acid membrane showed high contact angle.These results indicated that acid treatment changed slightly rough surface and reduced the mechanical strength for titanium thin membrane.
Conversely, the surface of alkali-treated membranes displayed a regular, rough surface and uniformly dense nanoscale pore structures, consistent with previous reports [22,27].The alkali-treated membranes displayed enhanced hydrophilicity, which may be attributed to the nanoscale pore structure.Increased hydrophilicity promotes cell adhesion and nutrient supply, and is advantageous for bone regeneration [28].Previous studies have compared alkali-and acid-treated implants, and found that implants treated with alkali displayed enhanced mineralization of the implant surface [29].These results indicate that alkali treatment produces a hydrophilic topography with nanoscale pore network.Moreover, the influence of alkali treatment on the strength and thickness of thin titanium

4-3 Corrosion depth
Each membrane thickness was measured by digital micro meter instrument (MDH-25M, Mitutoyo co. Ltd., Kanagawa, Japan).The degree of corrosion depth was compared with before and after treatment.

4-4 Tensile strength
Mechanical strength evaluation used a rectangular membrane from each group.Both the top and bottom sides of each membrane were fixed to the testing machine (AUTO GRAPH AGS-X, Shimadzu), and the samples were pulled at a constant speed (5 mm/min) until their breaking points were reached.The maximum tensile stress value was used to represent the mechanical strength of the membrane.

4-5 Evaluation of wettability
First, each square membrane sample was divided into four 10 µm squares, which were fixed to the stage.Then, a 10-µL drop of pure water was gently applied to each sample.Ten seconds after the water and the membrane touched, an image was taken with an S-image device.Then, the contact angles of the dropped water were measured using ImageJ (National Institutes of Health, USA).These were obtained using a half-angle method, by measuring the angle of the straight line connecting the end point and the vertex of the droplet, and then doubling this value.

4-6 Statistical analyses
All data were analyzed at the 5% significance level using one-way analysis of variance followed by Tukey's test, and are expressed as the mean ± standard deviation (SD).

Table 2
shows the tensile strength of each sample.The tensile strength was significantly decreased by acid treatment.Conversely, there was no significant difference in strength between the alkali and control groups, indicating that alkali treatment did not weaken the membranes.alkaline membrane was extended dramatically, and is not visible in the Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 4 September 2017 doi:10.20944/preprints201709.0010.v1Peer-reviewed version available at Appl.Sci.2017, 7, 1022; doi:10.3390/app7101022

Table 3 .
Contact angle

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 September 2017 doi:10.20944/preprints201709.0010.v1
[24]-reviewed version available at Appl.Sci.2017, 7, 1022; doi:10.3390/app7101022membraneshavehigher strength[24].However, thick membranes are less flexible and formable, creating sharp edges when cutting, trimming, and bending them along the defect site.Thick membranes show less tissue adhesion, which permits penetration of soft tissue from the gap, preventing new bone formation.Thin membranes follow the bone morphology and do not create air pockets, which is advantageous for bone formation.However, [25] decreased mechanical strength, thin membranes can collapse into the defect cavity, decreasing the bone formation space and consequently, the volume of new bone formed.There are reports that membranes of 100 to 200 μm thickness are suitable for healing large-scale bone defects[25].However, the lack of flexibility of membranes of this thickness gives them poor operability.An advantage of titanium membranes is that they maintain their mechanical strength even when thin.A thickness of 20 μm is most suitable for GBR treatment and accordingly, commercially available and clinically applied titanium membranes are 20 μm thick.Our experiments were conducted with membranes of this thickness as well.Thin titanium membranes are manufactured through the extension of a titanium metal mass by applying pressure through the gap between two rollers, until the irregular roughed surface and groves which observed in non-treatment were not detected.The aspect consider that corrosion by strong acid changed slightly smooth topography rather than irregularly roughed non-treatment membrane.Similarly aspect indicate the result of corrosion depth.