The study on material surface on the effect of the Low-Intensity Pulsed Ultrasound (LIPUS) for Osseointegration of the Hydroxyapatite coating dental implant

Excellent osseointegration of biomaterial is very important for the stability of dental implants in clinical fi eld. Much has been learned about this concept and signifi cant improvements on the design and surface modifi cation of implants have been done in the implant dentistry. Recently, some clinical studies have also reported that low-intensity pulsed ultrasound Irradiation (LIPUS) might enhance the osseointegration (bone-bonding) by means of X-ray radiographic evaluation. We have already reported that the LIPUS could accelerate the bone-like hydroxyapatite precipitation on the bioactive material surface, which corresponds to osseointgration function. In this review, the mechanism of osseointegration enhanced by LIPUS was presented by the data of our in vitro and in vivo study on hydroxyapatite surface using a Scanning electron microscope and X-ray diffraction analysis. This review suggested that the formation of bone-like apatite on material surface is very important for rapid and fi rm osseointegration success, which might depend on the initial epitaxial nucleation and crystal growth of apatite on the surface, and LIPUS could activate this crystallization process. Mini Review The study on material surface on the effect of the LowIntensity Pulsed Ultrasound (LIPUS) for Osseointegration of the Hydroxyapatite coating dental implant Masanori Kobayashi* Department of Physical Therapy, Takarazuka University of Medical and Health Care, Japan Received: 25 August, 2020 Accepted: 07 September, 2020 Published: 08 September, 2020 *Corresponding author: Masanori Kobayashi, Professor, Department of Physical Therapy, Takarazuka University of Medical and Health Care, Japan, Tel: 81-72-736-8600; Fax: 81-72-736-8659; E-mail: ORCID: https://orcid.org/0000-0003-3874-9263


Introduction
Nowadays, the favorable clinical performance of dental implants has been attributed to their fi rm "osseointegration" as a direct contact between living bone and the surface of a load-carrying implant at the histological level introduced by Brånemark [1,2]. Especially, Hydroxyapatite (HA; Ca 10 (PO 4 ) 6 (OH) 2 ) has an excellent biocompatibility and osseointegration properties, therefore have been widely used as dental implants coating material.
Considering the clinical view, more early bonding between dental implant and maxilla bone will accelerate the onset of activity of patients soon after an operation, with subsequent good long-term results, therefore, some approach has been tried to accelerate the osseointegration or the bone bonding to HA material [3][4][5].
Against these backgrounds, to enhance the osseointegration of dental implant to bone tissue, Low-Intensity Pulsed Ultrasound (LIPUS) stimulation has been focused [6][7][8][9], some clinical studies have reported the LIPUS might enhance the bone-bonding [10,11]. However, as these data of reports have been evaluated by means of indirect analysis such as X-ray radiography measures of bone tissue surround implant. Therefore, it is still unclear whether the LIPUS can promote the direct attachment between bone and implant surface defi ned as an osseointegration though the reduce of dead space of bone around dental implant is certifi cated.
We have also already reported that the osseointegration of some bioactive materials was enhanced by the pulsed ultrasound radiation [12][13][14], the present review demonstrates the fact that LIPUS could accelerate the osseointegration (direct bone-bonding ability) of implant surface side due to the induce of bone-like hydroxyapatite formation on the surface.

Method
To evaluate the enhancement of osseointegration of the HA material by LIPUS irradiation, two experiments, in vitro simulated experiment and in vivo tests using rabbits.

Simulated body fl uid soaking test
As a basic bone-bonding ability test , the Simulated Body Fluid (SBF) soaking method was performed as a simulation experiment according to Kokubo's studies in order to evaluate the apatite-forming ability of bio-active titanium and Bioglass ceramics [15][16][17].
Hank's balanced solution (®Lonza; USA) was used as a SBF solution, and maintained at pH over 7.0 and 37 °C and replaced every two days. μsec signal term, and spatial average intensity of 30 mW/cm 2 .
As for a hydroxyapatite specimen, commercial HA pellet (®CELLYARD, HOYA, Japan; 99.9% HA: Ø 5mm ×2mm) was used. Each samples were soaked in SBF and subjected to ultrasound stimulation for 20 min daily during the operation term for three days, one week, and two weeks, respectively. As a control, the same samples were left in SBF without ultrasound radiation under the same experimental conditions ( Figure 1).
After the above-mentioned processes were completed, the HA surface was subjected to Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD), and these analyses were focused on the top surfaces, on which the ultrasound wave was directly radiated.
Furthermore, in order to compare with the effect of ultrasound waves, the change of the sample's mass before/ after the SBF treatment was measured. The sample mass were measured periodically, the value obtained by dividing the increased mass by the surface area was defi ned as the amount of precipitates per unit material surface area. Statistics analysis of the data was examined through t-test and the evaluation and comparison between two groups and the level of signifi cance was determined to be at p <0.05.

In vivo experiment (Animal experiment)
As an implant sample for animal experiment, the HA pallets (Ø 5mm ×2mm ) were cut into the 3 mm ×3 mm ×2 mm cuboid dice, and implanted in the bilateral iliac region of eight mature female rabbits.
After operation, one side of iliac region of rabbits was radiated with the ultrasound waving by using SAFHS as a LIPUS group, another side was not radiated as a control group. All rabbits were reared in cages with no postoperative immobilization, and sacrifi ced at four weeks after operation, HA samples were resected from rabbits, the bone contact surface was examined by SEM and energy dispersive spectroscopy (EDS).

In vitro simulated body fl uid soaking test
Measurement of the mass of Ca-P crystallization: Figure 2 shows the measured sample mass change as a hydroxyapatitelike precipitation (calcium phosphate crystalline) before/after the SBF soaking. In ultrasound waving group, this bone-like crystalline showed signifi cant increases as compared with that in control group in one week and two weeks soaking in SBF.

XRD ( X-ray diffraction) analysis
These compounds on HA surface have also identifi ed by XRD . The XRD patterns of the layer on the HA surface after one week of control groups is shown in Figure 4. Though the typical XRD pattern of hydroxyapatite was not observed, some peaks of hydroxyapatite were observed.

In vivo experiment (Animal experiment)
Macro appearance of the animal specimens: Figure 5 shows the macro-appearance of HA implant on rabbits iliac crest of LIPUS group postoperative 4 weeks. Neither infl ammation nor infection sign was observed in this area. Figure 6 shows the micro-appearance of the surface of HA sample implanted

Discussion
The simulation method using SBF soaking in this study is well known as a in vitro assay for evaluation of bone-bonding ability of biomaterials, and has been already used widely for the study regarding the osteoconductivity [18][19][20][21] .
According to Kokubo who have developed SBF, the osseointegration of biomaterial mainly due to some step mechanisms, at fi rst the calcium-phosphate precipitate and grows on implant material surface as bone-like apatite, this apatite layer secondary progress the bonding to the bone tissue side grown by the biological mechanism in vivo, and the

Conclusion
This study suggested that the clinical application of Low Intensity Ultrasound (LIPUS) irradiation has a great potential for enhancement of osseointegration of hydroxyapatite dental implant through the promoting mechanism of the nucleation and crystallization of bone-like apatite on implant surfaces.