WO2011056013A2

WO2011056013A2 - Dental bone graft comprising 4-hexylresorcinol and implant coating with the same

Info

Publication number: WO2011056013A2
Application number: PCT/KR2010/007787
Authority: WO
Inventors: Seong-Gon Kim; Chang-Hoon Chae; Je-Yong Choi; Dong-Heon Baek; Dong-Su Park; Byung-Dong Hahn; Mulyukin Andrey
Original assignee: Lee, Yong Chan
Priority date: 2009-11-06
Filing date: 2010-11-05
Publication date: 2011-05-12
Also published as: WO2011056013A3; KR101178939B1; KR20110050308A

Abstract

The present invention relates to a dental bone graft containing 4-hexylresorcinol and an implant having a surface modification of the dental bone graft. Using 4-hexylresorcinol as an additive, an anti-microbial activity and alkaline phosphatase activity in osterblasts were highly enhanced in a dental bone graft of the present invention. In addition, the surface modification of the graft and implant significantly resulted in enhancement of attachment strength and rate of osteoblasts and adhesion to the bone.

Description

DENTAL BONE GRAFT COMPRISING 4-HEXYLRESORCINOL AND IMPLANT

COATING WITH THE SAME

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a dental bone graft containing 4- hexylresorcinol and a dental implant having a surface modification with the dental bone graft. DESCRIPTION OF THE RELATED ART

Dental implant has been widely used for the restoration of missing teeth. As other bone implants, causative factors on success of dental implant include patient's body state, properties of used implants or dentist's skill. The patient's state includes the patient's age, pathogenic state, habit or height or bone qualities. The properties of used implants include diameter, length, shape and surface modifications. Thus, there have been attempted various techniques to enhance success rate of dental implants. Although the patient's body state may not provide perfect conditions for implant, the success rate of dental implants may be strikingly improved by the techniques focusing on materials for implantation.

The upper portion of the fixture may be exposed into the oral mucosa according to the implant design. As every implant has a structure corresponded to the tooth crown, some portion of dental implant must be uncovered with biological barrier such as mucosa. Because many kinds of bacteria live in the oral cavity, the exposed surface of the dental implant may provide the bacterial attachment and induce the inflammation. Though some implant failure is caused by mechanical properties of dental implant, most failures are due to the peri-implant inflammation

(1).

As the surface of fixture mainly faces to the alveolar bone, rapid bone regeneration around the fixture is very important in successful osseointegration. However, there are wide interpersonal differences in healing capacity. Some systemic diseases such as diabetes meilitus and osteoporosis influence on the success rate of dental implant (2). Therefore, the surface modification for dental implant has been aimed to the rapid bone regeneration around dental implant. If the surface roughness is increased until some point, the cellular attachment can be accelerated (3). Accordingly, the alkaline phosphatase activity is also increased (4). However, rough surface may provide the bacterial attachment more easily than smooth surfaced materials (5). The rough surface is also hardly cleansed. Therefore, some dental implants have highly polished surface around the neck of the fixture (6).

Some bioactive molecules can be coated on dental implant to accelerate new bone formation instead of making rough surface. The hydroxyapatite (HA) is main component of bone and it has been used as a graft material (7). HA coated dental implant also has been developed and used. In case of ophthalmology application, HA-coated implant has often failed due to the infection (8, 9). HA is highly hydrophilic and provides bacterial attachment (10, 11).

In case of HA coated dental implant, its success rate is somewhat controversial. There have been many reports of high success rate in the HA coated dental implant (12, 13). However, there have been negative reports in the long term follow-up study concerning HA coated dental implant (14). Recently, direct comparison study shows that HA coated dental implant appears higher failure rate when its surface is to be exposed to oral cavity (15). Considering that HA is highly biocompatible and rapid osseointeg ration in poor quality bone, the development of the infection resistant HA coating may be valuable for the clinical applications. Therefore, HA coating with bio-inert antiseptics can be considered the next generation of the HA coating.

Alkylresorcinols, natural non-isoprenoid lipids found in various plant and bacterial species, attract attention due to a variety of biological functions as nonspecific antioxidants, antimutagens, and regulatory molecules (16). In microorganisms, chemical analogs of 4-hexylresorcinol (4-HR) occur in dormant cysts (17, 18) and resting cyst-like cells of some bacteria (19, 20). Recognizing that the formation of dormant cells by bacteria in the experimental conditions can be favored by exogenous administration of these autoregulatory factors, as well as their analogs - alkylresorcinols (21) and the fact that they are similarly active with respect to not only bacteria but also anti-cancer effects on eukaryotic cells (ras-transformed fibroblasts) (22; Submitted paper). 4-HR is well known antiseptics (23) and it does not indicate any toxic effect up to a concentration of 5(Vg/ml in Caco-2 cells (24). As 4-HR is organic material, it is weak to heat treatment. Thus, it will be difficult to develop combination coating with HA on the metal surface. If the coating can be done under room temperature, HA with 4-HR can be coated on the metallic surface without losing its biological properties.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains. DETAILED DESCRIPTION OF THIS INVETNION

The present inventors have done intensive studies to develop functional additives capable of being mixed with a conventional dental bone graft. As results, we have discovered that a bone graft modified with 4-hexylresorcinol or a dental implant having surface modification with the bone graft may significantly result in an increase of alkaline phosphatase activity, enhancement of attachment strength and rate of osteoblasts on the surface of implant, and adhesion to the bone compared with a conventional dental bone graft.

Accordingly, it is an object of this invention to provide a dental bone graft.

It is another object of this invention to provide a dental implant having a surface modification with the dental bone graft.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a dental bone graft comprising 4-hexylresorcinol as an active ingredient. The present inventors have done intensive studies to develop functional additives capable of being mixed with a conventional dental bone graft. As results, we have discovered that a bone graft modified with 4-hexylresorcinol or a dental implant having surface modification with the bone graft may significantly result in an increase of alkaline phosphatase activity, enhancement of attachment strength and rate of osteoblasts on the surface of implant, and adhesion to the bone compared with a conventional dental bone graft.

The term "dental bone graft" used herein refers to a graft which is used to fill a cavity within a bone tissue and to facilitate a new bone formation for substitution when a loss portion of bone tissue is generated by various dental disorders.

In addition, the term "implant" refers to a substitute in itself for natural teeth loss, or a dental procedure for restoring an original function of teeth, which a screw implant fixture is linked to alveolar bone and fused with a bone for a certain period of time, followed by fixing a dental prosthesis such as an abutment and an artificial tooth crown on the surface of the implant fixture. However, the implant used in the specification refers to a substitute in itself for natural teeth loss. 4-hexylresorcinol contained as an additive in the dental bone graft of the present invention is called as IUPAC nomenclature of 4-hexylbenzene-l,3-diol, and is a compound represented by the following Formula 1:

Conventionally, 4-hexylresorcinol was dissolved in olive oil and taken for treatment of enteritis, or was added to prevent discoloration in shrimp. However, 4- hexylresorcinol has not been utilized as a dental bone graft in body or an additive of coating material for a dental implant until now.

According to a preferable embodiment, the 4-hexylresorcinol of the present invention is contained in an amount of 0.01-10 wt%, more preferably 0.1-5 wt%, and most preferably, 0.1-2 wt% based on the total weight of the dental bone graft

It is difficult to expect an anti-microbial activity of 4-hexylresorcinol with a weight range of not more than 0.01 wt%. In addition, the anti-microbial activity of 4-hexylresorcinol with a weight range of not less than 0.01 wt% is very slightly enhanced depending on increasing dose whereas the implant surface is not able to be coated due to low stability of formations.

According to another preferable embodiment, the dental bone graft of the present invention is selected from the group consisting of hydroxyapatite, monocalcium phosphate, tricalcium phosphate and silica, and includes more preferably hydroxyapatite and tricalcium phosphate, and most preferably, hydroxyapatite.

In particular, hydroxyapatite is a component equivalent to human bone and tooth, and has an excellent effect on the restoration of tooth anamel loss. Recently, hydroxyapatite also is in the spotlight of tooth whitening as hydroxyapatite permits to restore an original color of teeth.

According to a preferable embodiment, the dental bone graft of the present invention has a higher osteoblast attachment potency compared to a dental bone graft without 4-hexylresorcinol.

The dental bone graft of the present invention may be utilized by being modified to a putty, paste, moldable strip, block or chip shape via a molding method such as pressure, contraction, pressure contact, packing, compression, stiffening, and so on. Using a chemical additive, the dental bone graft of the present invention may be formulated as a form such as a gel, a powder, a paste, a purification and a pellet. In addition, it is possible to utilize the dental bone graft of the present invention as a powder.

In the formulation of bone graft as described above, it is preferable to add a biologically active material. Preferably, the biologically active material may utilize a growth factor promoting a bone growth, a peptide and protein capable of inducing enhancement of osteogenesis, a fibrin, a bone morphogenetic factor, a bone growth factor, a chemotherapeutic agent, an antibiotics, an analgesic drug, a biphosphonate, a strontium salt, a fluoride salt, a magnesium salt and a sodium salt, and so forth.

The above-mentioned growth factor may use BMP (bone morphogenic protein), PDGF (Platelet-derived growth factor), TGF-beta (Transgenic growth factor), IGF-I (Insulin-like growth factor), IGF-II, FGF (Fibroblast growth factor) and BGDF-II (beta-2-microglobulin). The above-described peptide and protein capable of inducing enhancement of osteogenesis may use various peptides containing a RGD sequence and a variety of proteins such as collagen and fibrinogen.

The bone morphogenetic factor described above may utilize osteocalcin, bonesialo protein, osteogenin, BMP, and so on.

According to a preferable embodiment, the dental bone graft of the present invention has an excellent anti-microbial activity compared to a dental bone graft without 4-hexylresorcinol.

According to a preferable embodiment, the dental bone graft of the present invention induces higher expression of osteocalcin compared to a dental bone graft without 4-hexylresorcinol.

According to a preferable embodiment, the dental bone graft of the present invention remarkably increases an alkaline phosphatase activity.

In another aspect of this invention, there is provided an implant having a surface modification of the dental bone graft described above.

According to a preferable embodiment, the implant of the present invention has a significantly higher bonding power compared to a dental bone graft without 4- hexylresorcinol.

By modifying a surface of the dental bone graft, the dental bone graft may be utilized as various formulations such as a gel, a powder, a paste, a purification and a pellet, and preferably, a powder.

The term "surface modification" used herein refers to various coating modification well-known to those ordinarily skilled in the art for improving a surface of an implant.

According to the present invention, the surface of the dental bone graft of this invention is coated to have a shape of a thin film via a physiochemical thin film deposition known in the art, preferably including a pulsed laser deposition (PLD), a sputtering deposition, a chemical vapor deposition (CVD), a dip-coating deposition, a plating deposition and three-dimensional plasma gun deposition. Using the pulsed laser deposition (PLD), a sputtering deposition, a chemical vapor deposition (CVD), a plating deposition and three-dimensional plasma gun deposition as described above, an outer circumference has to be uniformly coated by rotating an implant or revolving a laser, a sputtering gun, a crucible or a plasma gun. In the dip-coating deposition, a coating may be carried out by dipping an implant into a certain solution.

According to a preferable embodiment, the implant of the present invention includes an implant consisting of numerous materials known to those ordinarily skilled in the art, more preferably titanium (Ti) or titanium alloy. The titanium alloy includes Ti-6AI-4V, Ti-6AI-7Nb and Ti-13Nb-13Zr.

It is another object of this invention to provide a method for improving characteristics of a dental bone graft to be coated on an dental implant, which comprises: combining a main material for the dental bone graft with 4- hexylresorcinol; wherein the characteristics of a dental bone graft is osteoblast attachment potency, anti-microbial activity, osteocalcin expression or alkaline phosphatase activity.

It is still another object of this invention to provide a method for allowing a dental implant to possess improved characteristics, which comprises: coating the dental implant with the dental bone graft of the present invention, wherein the characteristics of the dental implant is osteoblast attachment potency, antimicrobial activity, osteocalcin expression or alkaline phosphatase activity.

Since the present method comprises the dental bone graft of this invention as active ingredients described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification. According to a preferable embodiment, the combination of the main material and 4-hexylresorcinol allows the dental bone graft to have higher osteoblast attachment potency, to have higher anti-microbial activity, to induce higher expression of osteocalcin and to increase an alkaline phosphatase activity.

According to a preferable embodiment, the main material is at least one selected from the group consisting of hydroxyapatite, monocalcium phosphate, tricalcium phosphate and silica.

According to a preferable embodiment, the dental bone graft permits the dental implant to have higher osteoblast attachment potency, to have higher antimicrobial activity, to induce higher expression of osteocalcin and to increase an alkaline phosphatase activity.

According to a preferable embodiment, the coating is performed by aerosol deposition.

According to a preferable embodiment, the implant comprises titanium or titanium alloy.

The features and advantages of this invention are summarized as follows:

(a) The present invention relates to a dental bone graft containing 4- hexylresorcinol and an implant having a surface modification of the dental bone graft.

(b) Using 4-hexylresorcinol as an additive, an anti-microbial activity and alkaline phosphatase activity in osterblasts were highly enhanced in a dental bone graft of the present invention.

(c) In addtion, the surface modification of the graft and implant significantly improved enhancement of attachment strength and rate of osteoblasts and adhesion to the bone.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents an analysis of HA coating and HA with 4-HR combination coating. A. XRD analysis. HA coated titanium is well matched with the characteristic hexagonal phase of the stiochiometric hydroxyapatite (ICDD, 086-0740). B. FT-IR analysis. The measured infrared absorption spectrum of the HA shows characteristic vibrational modes; Two vibrational absorptions at 1090 and 1024 cm^"1. 4-HR coated HA shows intense absorption peaks at 2920 and 2850 cm^"1 and a weak absorption at 1469 cm^"1. C. Cell attachment analysis. The cellular attachment was examined at 1, 4, and 24 hours after seeding G63 cells on coated disc. HA with 4-HR combination coating showed more rapid cellular attachment and spread than HA coating.

Fig. 2 represents an antibacterial effect of 4-HR. A. The anti-microbial test.

Clear inhibitory zone was observed around 4-HR disc in the range of concentration between 10 pg and 10 mg in all tested species. However, 0.1 and 1 g disc did not show the inhibitory zone. A) Aggregatibacter actinomycetemcomitans (ATCC 33384). B) Prevotella intermedia (ATCC 25611). C) Porphyromonas gingivalis (ATCC 49417). D) Staphylococcus epidermidis, (ATCC 14990). B. The antimicrobial test of the coated disc. In case of P. gingivalis, 0.3 mm of inhibitory zone was observed around HA with 4-HR coated disc. However, there was no inhibitory zone around HA coated disc. The inhibitory zone around HA coated disc was 1.5mm in S. aureus and 0.3 mm in P. intermedia. The inhibitory zone around HA with 4-HR coated disc was 1 mm in S. aureus and 0.3 mm in P. intermedia. C. The SEM image of HA with 4-HR coated dental implant after contamination by bacteria. The bacterial attachment (*) on the implant surface was confirmed by SEM image (A:x30 original magnification, B:xl0,000 original magnification).

Fig. 3 represents a cellular response to HA with 4-HR combination coatings. A. MTT assays. The growth of MG63 cells were not inhibited with increasing of 4-HR concentrations, from 1 pg/ml to 10 pg/ml. B. 4-HR increases ALP activity in MG63 cells. When 4-HR was given to MG63 cells, the alkaline phosphatase activity was increased dose-dependently to 10 pg/ml. C. Increased ALP activity by 4-HR application was also observed in the HA with 4-HR combination coating. When the ALP activity of MG63 on bare titanium surface was set as 1, the ALP activity of HA coating was 2.33 ± 0.46 (p=0.036) and 3.03 ± 0.50 for HA with 4-HR coating (p=0.019). D. The osteocalcin expression was increased with increasing 4-HR concentrations, from 1 g/ml to 10 pg/ml. E. When the osteocalcin expression of MG63 on bare titanium surface was set as 1, the osteocalcin expression of HA coating was 2.01 ± 0.07 (p<0.001) and 3.07 ± 0.07 for HA with 4-HR coating (p<0.001).

Fig. 4 shows a torgue and histological analysis of 4-HR coated HA implant after in vivo implantation. A. The removal torque of the experimental group was 65.31 ± 6.46 N.cm and 47.88 ± 12.65 N.cm in the control (p=0.021). B. Histological findings. The peri-implant bone formation of HA with 4-HR coating was higher than that of HA coating in both A) 4 weeks and B) 8 weeks after the implant installation. The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples. EXAMPLES Experimental Materials and Methods

Aerosol Deposition of Hydroxyapatite and 4-Hexylresorcinol Composite Coatings on Titanium Alloy

The coating method was in accord to previous publication (25). Briefly, Ti specimens (Grade IV, Dynamet, WA) that were machined into disks, a dimension of 5 mm (diameter) x 0.4 mm (thickness) was used as substrate. These disks were cleaned ultrasonically in acetone, ethanol, and deionized water. The raw HA powder (Alfar Aesar, Ward Hill, MA) used in this study was subjected to the predeposition treatment consisting of heating to 1050°C for 2 h, ball milling for 24 h. Average particle sizes (d₅o) before deposition was 2.0 pm. HA or HA with 4-HR combination deposition was conducted by using the same aerosol deposition system as in the previous report (26). The powder was placed in a vibrating aerosol chamber that contained fine floating particles. The fine particles were carried by oxygen gas and sprayed onto the Ti disk or implant in the deposition chamber. The chamber was continuously evacuated using a mechanical booster and rotary pumps. Although the particles were sprayed from the nozzle with a slit-type opening (0.5 mm x 25.4 mm), the motored stage with a Ti disk or implant was moved perpendicular to the length of the slit-type opening.

X-ray diffraction and FT-IR

X-ray diffraction (XRD) patterns were performed on a diffractometer (PANalytical X'Pert Pro MPD) equipped with a Pixel (256 channels) detector and using Cu-Κα (λ = 1.5418 A) radiation source generated at a voltage of 45 kV and a current of 40 mA. The samples were placed on a glass holder and examined in the range of 25 - 80° (2Θ) at a step size of 0.026° with 500s counting per step, therefore, a total measuring time of 72 min for each diffraction pattern. Soller and divergent slits were set to 0.04 radian and 0.5°, respectively. The chemical identity of the samples was determined by comparing the experimental XRD patterns to standard reference data compiled by the International Centre for Diffraction Data (ICDD, formerly the Joint Committee on Powder Diffraction Standards, JCPDS).

Fourier transform infrared (FT-IR) absorbance spectra were obtained using a microscope (Hyperion 3000, Bruker Optics) equipped with a germanium (Ge) attenuated total reflectance objective lens (ATR 20x) and a mercury cadmium telluride (MCT) detector and attached to a Fourier transform spectrometer (Vertex 80, Bruker Optics) with a Ge-on-KBr beamsplitter. Spectra were recorded in the spectral range of 600 - 4000 cm-1 at a resolution of 2 cm^"1 and 32 repeated scans were averaged for each spectrum.

Anti-microbial properties test

Two periodontal pathogens (Porphyromonas gingivalis, ATCC 49417, and Prevotella intermedia ATCC 25611) were purchased from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea). And Staphylococcus aureus, ATCC 25923 and Staphylococcus epidermidis, ATCC 14990 were purchased from the American Type Culture Collection (ATCC). The P. gingivalis and P. intermedia were cultured with anaerobic Brain Heart Infusion broth containing 1.0 ug/ml Vit Kl and 5 ug/ml Hemin at anaerobic condition. And S. aureus and S. epidermidis were cultured on the nutrient agar at aerobic condition.

Each of bacteria was cultured and diluted with sterile media at 0.5 absorbance at 450 nm. On the blood agar plate supplemented with 5% sheep blood, 1.0 ug/ml Vit Kl and 5 ug/ml Hemin for P. gingivalis and P. intermedia, 200 ul of bacterial solution was spread. S. aureus and S. epidermidis were spread on the nutrient agar with same manner. HA-coated disc and HA with 0.3% of 4-hexyl- resocinol coated disc were placed on the surface of each plate. The plates were incubated at 37 °C anaerobic condition for 2 days. In case of S. aureus and 5. epidermidis, the plates were incubated at 37 °C aerobic condition for 1 day. The maximum inhibition zone diameter was measured.

Scanning microscopy

MG-63 cells were grown on the HA disc and HA+4-HR disc. The cell culture was stopped at 30 min, 1 hr, 4 hr, and 24 hr after seeding. The samples were fixed. All materials were prepared for the scanning electron microscopic examination. After immobilization of the samples on plate, each sample was coated by gold and examined by the scanning electron microscopy (H-800, Hitachi, Japan). MTT assay, alkaline phosphatase assay, and osteocalcin assay

MTTs assays were performed as follows: MG63 cells were grown on the titanium disc, HA disc, and HA+4-HR disc in 6-well multiplates with yellow tetrazolium salt 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Cell proliferation kit I; Roche Molecular Biochemicals) for 4 h at ambient temperature. Formazan crystals were solubilized overnight, and the product was quantified spectrophotometrically by measuring absorbance at 590 nm using a Victor Multilabel counter (Perkin-Elmer-Wallac, Freiburg, Germany).

ALP was evaluated from the transformation of p-nitriphenylphosphate into p- nitrophenol at 37°C and pH 10.2 using appropriate reactives (all from Sigma, St. Louis, MO, U.S.A.) while the specific activity of ALP was calculated with regard to the protein concentration of lysates determined by means of a commercially available colorimetric assay (#71230, AnaSpec, Freemont, CA, U.S.A.). Animal experiment, torque test, and histomorphometric analysis

Twelve 4-month-old New Zealand white rabbits with average weight of 2.7kg (range 2.5-3.0kg) were used in this experiment. This experiment was approved by Institutional Animal Care and Use Committee of Bioventure Incubation Center, Hanbat National University, Daejeon, Korea (No. 2009-NCT-005).

General anesthesia was induced by intramuscular injection of a combination of 0.4ml of ketamine (lOOmg/ml) (Ketara; Yuhan, Seoul, Korea) and 0.3ml of xylazine (lOmg/kg body weight; Rompun; Bayer Korea, Seoul, Korea). The tibia area was shaved and disinfected with povidine-iodine. Then, an incision was created in the periosteum. Sharp subperiosteal dissection reflected the periosteum, exposing the tibia bones. Two implants (diameter: 3.0mm, length: 10.0mm: MSP30103R, Osstem, Seoul, Korea) were installed into each hole. The implants were contaminated by the bacterial culture medium for 5 min before installation. The medium contained 10⁴ cfu of Aggregatibacter actinomycetemcomitans (ATCC 33384). A half of HA implants were installed into the proximal site of the tibia and HA+4-HR implants into the distal site accordingly. The other HA implants were installed into the distal site and HA+4-HR implants into the mesial site accordingly. These assignments were done to rule out the anatomic factor for osseointegration. Then, periosteum and skin closed in layer with 3-0 silk. Postoperatively rabbit received gentamicin lmg/kg (Kookje Inc., Seoul, Korea) intramuscular 3 times daily for 3 days. Each rabbit was individually caged and received food and water. Three animals were humanely sacrificed at 4 weeks. Six implants from three animals were fixed in 10% formalin and undergone to the histomorphometric evaluation. Twelve animals were humanely sacrificed at 8 weeks. Six implants from three animals were fixed in 10% formalin and undergone to the histomorphometric evaluation. Twelve implants from six animals were used for the measurement of the removal torque. The peak removal torque was measured by a digital torquimeter.

The procedure for the specimen preparation was in accord to previous publication (27). The specimen was stained in a Villanueva bone stain solution for 7 days. After the dehydration procedure, the specimen was embedded in methylmethacylate resin. Then sections were sliced along the long axis of the implant by low speed diamond wheel saw. Finally, the section was grinded to the thickness of 30 μητι. The sections were taken digital image using a digital camera (DP-20; Olympus, Tokyo, Japan). The image was analyzed by Sigma Scan Pro (SPSS Inc., Chicago, IL). First three threads from the abutment portion were used for the image analysis. Total new bone was calculated by a percentage of the total region between threads. The bone to the implant contact was calculated by a percentage of the bony contact to the implant thread.

Results

Surface analysis of 4-HR coated HA implant by X-ray diffraction and FT-IR and better cell attachment XRD pattern for the HA coated titanium is well matched with the characteristic hexagonal phase of the stiochiometric hydroxyapatite (ICDD, 086-0740) (Fig. 1A). Relatively strong peak intensity from the titanium compared to that of the HA is also observed. When the film is further coated with 4-HR, the diffraction pattern of the HA are still conserved.

The measured infrared absorption spectrum of the HA shows characteristic vibrational modes; Two vibrational absorptions at 1090 and 1024 cm^"1 can be assigned to P0₄ ^3" v3 asymmetrical stretching mode of HA (Fig. IB). Absorptions at 964 and 631 cm ¹ can be assigned to P0₄ ^3" vl and labile P0₄ modes, respectively (29). The weak absorption peak observed at 3571 cm^"1 indicates the presence of - OH group of HA. 4-HR coated HA shows intense absorption peaks at 2920 and 2850 cm^"1 and a weak absorption at 1469 cm^"1, which are corresponding to the well known asymmetrical and symmetrical stretching and bending modes of -CH₂- in an aliphatic hydrocarbon, respectively, in addition to the characteristic stretching modes from P0₄ and -OH groups of HA. These additional vibrational absorptions from aliphatic hydrocarbon and hydroxyl group clearly verify 4-HR coating on the HA.

The cellular attachment was examined at 1, 4, and 24 hours after seeding MG63 cells on coated disc (Fig. 1C). HA with 4-HR combination coating showed more rapid cellular attachment and spread than HA coating.

Anti-microbial properties test

In the anti-microbial test, clear inhibitory zone was observed around 4-HR disc in the range of concentration between 10 pg and 10 mg in all tested species (Fig. 2A). However, 0.1 and 1 pg disc did not show the inhibitory zone. The antimicrobial test was also done in the coated disc (Fig. 2B). In case of P. gingivalis, 0.3 mm of inhibitory zone was observed around HA with 4-HR coated disc. However, there was no inhibitory zone around HA coated disc. The inhibitory zone around HA coated disc was 1.5mm in S. aureus and 0.3 mm in P. intermedia. The inhibitory zone around HA with 4-HR coated disc was 1 mm in 5. aureus and 0.3 mm in P. intermedia. Bacterial attachment was observed on top of implant by SEM (Fig. 2C). These results indicate that 4-HR from coated HA implant can inhibit bacterial infection in peri-implant sites.

MTT, alkaline phosphatase, and osteocalcin assays

According to results obtained from MTT assays, the growth of MG63 cells were not inhibited with increasing of 4-HR concentrations, from 1 pg/ml to 10 pg/ml (Fig. 3A). The relative cell viability was not significantly different until 72 hours treatment with 10 pg/ml of 4-HR.

When 4-HR was given to MG63 cells, the alkaline phosphatase activity was increased dose-dependently to 10 g/ml (Fig. 3B). The control was MG-63 cells grown without 4-HR and set their ALP activity as 1. The relative ALP activity after 5, 10, and 20 pg/ml of 4-HR application was 1.60 ± 0.09, 3.16 ± 0.24, and 3.00 ± 0.19 (p=0.006, <0.001, and <0.001, respectively). Increased ALP activity by 4-HR application was also observed in the HA with 4-HR combination coating (Fig. 3C). When the ALP activity of MG63 on bare titanium surface was set as 1, the ALP activity of HA coating was 2.33 ± 0.46 (p=0.036) and 3.03 ± 0.50 for HA with 4-HR coating (p=0.019). When compared ALP activity of HA coating to that of HA with 4- HR, there was not statistically significant difference (p>0.05).

The osteocalcin expression was increased with increasing 4-HR concentrations, from 1 pg/ml to 10 pg/rnl (Fig. 3D). The control was MG63 cells grown without 4-HR and set their osteocalcin expression level as 1. The relative osteocalcin expression after 1, 5, and 10 pg/ml of 4-HR application was 1.20 ± 0.08, 1.18 ± 0.06, and 1.32 ± 0.09 (p=0.019, 0.009, and 0.004, respectively). When the osteocalcin expression of MG63 on bare titanium surface was set as 1, the osteocalcin expression of HA coating was 2.01 ± 0.07 (p<0.001) and 3.07 ± 0.07 for HA with 4-HR coating (p<0.001; Fig. 3E). When compared osteocalcin expression of HA coating to that of HA with 4-HR, there was statistically significant difference (p<0.001).

Torque test and histomorphometric analysis

Next, we tested the removal torque and peri-implant bone formation. The result of torque test was shown in Figure 4A. The average value of all measured variables was higher in the experimental group than in the control at 8 weeks after the operation. The removal torque was significantly different as 65.31 ± 6.46 N.cm and 47.88 ± 12.65 N.cm in the experimental group and control group, respectively.

The peri-implant bone formation of HA with 4-HR coating was higher than that of HA coating in both 4 weeks and 8 weeks after the implant installation (Fig. 4B). The results of histomorphometry were shown in Table 1. Mean new bone formation was significantly different as 19.28 ± 3.12 % and 17.75 ± 1.88% in the experimental group and control group at 4 weeks after operation, respectively. We also observed the significant difference of it as 52.85 ± 9.72 % in the experimental group and 23.47 ± 9.08% in the control at 8 weeks (p=0.037). Besides, mean bone to implant contact seemed higher as 25.27 ± 4.38% in the experimental group than 20.77 ± 5.27% in the control at 4 weeks. Although it was not statistically significant at 4 weeks, it showed significantly difference as 50.97 ± 7.25% in the experimental group and 29.33 ± 2.08% in the control at 8 weeks (p=0.048).

Discussions

Dental implant has been a primary consideration for replacing missing teeth. Compared to the conventional prosthetics, dental implant needs a time for osseointegration between the fixture and the alveolar bone. To reduce the time for osseointegration, there have been numerous modifications on the implant design and the surface technology. HA coating is one of them and has an advantage for early bone regeneration. However, microorganism can attach to the HA coated surface easily as biofilm and it can induce an inflammation around the implant fixture (28). 4-HR is well known ingredient for oral anti-septics (23). HA particles were mixed with 4-HR and coated on the titanium surface. In this study, 4-HR-HA combination coating had clear advantages compared to HA coating. First, 4-HR-HA combination coating showed better antimicrobial properties compared to HA coating. Second, 4-HR-HA combination coating showed better cellular attachment than HA coating. Thirds, 4-HR-HA combination coating showed higher osteocalcin expression and alkaline phosphatase activity compared to HA coating. Fourth, 4-HR-HA combination coating showed higher bonding power to the bone in case of infection than HA coating.

XRD pattern for HA coated titanium shows a combination of diffraction peaks from both the coated film as well as titanium substrate (Fig. 1). The observed diffraction pattern of the coated film is well matched with the characteristic hexagonal phase of the stiochiometric hydroxyapatite (ICDD, 086-0740). Relatively strong peak intensity from the titanium compared to that of the HA indicates that the coated HA has a thin film thickness. When the film is further coated with 4-HR, the diffraction pattern of the HA are still conserved. Any diffraction peaks from calcite, β-tricalcium phosphate, calcium oxide, and calcium nitrate do not detected as by-products, indicating high quality crystalline phase of the deposited HA film. The measured infrared absorption spectrum of the HA shows characteristic vibrational modes (Fig. 1). 4-HR coated HA shows intense absorption peaks, which are corresponding to the well known asymmetrical and symmetrical stretching and bending modes of -CH₂- in an aliphatic hydrocarbon. XRD pattern and FT-IR pattern demonstrated that HA with 4-HR combination was well coated on titanium surface.

4-HR has been an ingredient of oral gargling solution and well known antiseptics. Historically, 4-HR was used for the therapeutic drug for infectious disease and its solution in olive oil had been taken by human without any complication (30, 31). Now, it is a component of topical antiseptics on oral mucosa (C0691518: information was derived from the NIH UMLS). In current study, clear inhibitory zone was observed around 4-HR disc in the range of concentration between 10 pg and 10 mg in all tested species (Fig. 2). In case of coated disc experiment, the antimicrobial properties seemed to be reduced compared to the paper disc (Fig. 2). As 4-HR was bound to HA, release from the coated surface was impaired compared to the paper disc. Interestingly, HA disc was also showed the antimicrobial properties to S. aureus and P. intermedia. This might be due to the release of free calcium ion from the coated surface. Free calcium ion is toxic to some bacterial species (32, 33). Considering that the P. gingivalis inhibitory zone was not observed in HA coating, but in 4-HR combination coating, 4-HR combination coating had beneficial effect in the antibacterial spectrum when compared to HA coating.

The cellular attachment was examined at 1, 4, and 24 hours after seeding MG63 cells on coated disc (Fig. 4). HA with 4-HR combination coating showed more rapid cellular attachment and spread than HA coating. Accordingly, ALP activity and OC expression were also up-regulated (Fig. 3). The early cellular attachment on 4- HR combination coating might let MG63 cells be activated. However, rapid cellular attachment on 4-HR combination coating is not fully explained. 4-HR can induce the intracellular calcium influx in many types of cells. It has been known that alkylresorcinols are able to modify the structural state of cell membranes, resulting in changes of permeability for ions and thereby ionic homeostasis (34), and the intracellular content of calcium is bacterial cells, including those generated under an influence of 4-HR (35). Other bacterial lipids also have similar properties. Transglutaminase-1 (TG-1) can be induced by the intracellular calcium influx (36). When 4-HR is given to MG63 cells, TG-1 gene transcription is increased dose dependency (data not shown). TG-1 can increase the cellular attachment (37). Therefore, the rapid cellular attachment of 4-HR combination coating might be due to TG-1 mediated mechanism.

In the animal experiment, 4-HR combination coating showed superior peri- implant bone regeneration in A. actinomycetemcomitans infection state compared to HA coated implant. The bacterial attachment on the implant surface was confirmed by SEM image (Fig. 2C). The removal torque and mean bone to implant contact were better in the 4-HR combination coated implant than HA-coated implant. The mechanism of the superior bone regeneration around 4-HR combination coated dental implant could be explained as follows. First, 4-HR is antiseptic agent and it has more advantages in infection state compared to HA only coating. Second, 4-HR may increase the cellular attachment on the implant surface like in vitro experiment. Third, 4-HR can affect the regulation of NF-kB functions in bone cells (data not shown). Similarly, N-(3-oxo-dodecanoyl) homoserine lactone, produced by Pseudomonas aeruginosa, selectively impairs the regulation of NF-kB functions in activated mammalian cells (38). Therefore, 4-HR may impair osteoclastogenesis by impairing RANK mediated NF-kB signaling pathway. Unlike the bisphosphonate, 4-HR can increase angiogenesis by the up-regulation of VEGF (data not shown). When 4- HR is used chronically, it may cause osteosclerosis (39). This also might be due to the impaired osteoclastogenesis.

Collectively, 4-HR combination coated dental implant had clear advantages compared to HA only coated dental implant. First, it showed antimicrobial properties. Therefore, it can be used for the immediate dental implant installation after the extraction of the infected teeth. Second, it showed superior bone formation ability compared to HA coated dental implant. Therefore, it can be considered for the patients who had poor quality bone.

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Claims

What is claimed is:

1. A dental bone graft comprising 4-hexylresorcinol as an active ingredient.

2. The dental bone graft according to claim 1, wherein 4-hexylresorcinol is contained in an amount of 0.01-10 wt% based on the total weight of the dental bone graft.

3. The dental bone graft according to claim 1, wherein the dental bone graft comprises a main material selected from the group consisting of hydroxyapatite, monocalcium phosphate, tricalcium phosphate and silica.

4. The dental bone graft according to claim 1, wherein the dental bone graft has a higher osteoblast attachment potency compared to a dental bone graft without 4-hexylresorcinol.

5. The dental bone graft according to claim 1, wherein the dental bone graft has an anti-microbial activity compared to a dental bone graft without 4- hexylresorcinol.

6. The dental bone graft according to claim 1, wherein the dental bone graft induces higher expression of osteocalcin compared to a dental bone graft without 4-hexylresorcinol.

7. The dental bone graft according to claim 1, wherein the dental bone graft increases an alkaline phosphatase activity.

8. A dental implant having a surface modification with the dental bone graft according to any one of claims 1-7.

9. The dental implant according to claim 8, wherein the implant comprises titanium or titanium alloy.

10. A method for improving characteristics of a dental bone graft to be coated on an dental implant, which comprises: combining a main material for the dental bone graft with 4-hexylresorcinol; wherein the characteristics of a dental bone graft is osteoblast attachment potency, anti-microbial activity, osteocalcin expression or alkaline phosphatase activity.

11. The method according to claim 10, wherein the combination of the main material and 4-hexylresorcinol allows the dental bone graft to have higher osteoblast attachment potency, to have higher anti-microbial activity, to induce higher expression of osteocalcin and to increase an alkaline phosphatase activity.

12. The method according to claim 10, wherein the main material is at least one selected from the group consisting of hydroxyapatite, monocalcium phosphate, tricalcium phosphate and silica.

13. A method for allowing a dental implant to possess improved characteristics, which comprises: coating the dental implant with the dental bone graft according to any one of claims 1-7, wherein the characteristics of the dental implant is osteoblast attachment potency, anti-microbial activity, osteocalcin expression or alkaline phosphatase activity.

14. The method according to claim 13, wherein the dental bone graft permits the dental implant to have higher osteoblast attachment potency, to have higher antimicrobial activity, to induce higher expression of osteocalcin and to increase an alkaline phosphatase activity.

15. The method according to claim 13, wherein the coating is performed by aerosol deposition.

16. The method according to claim 13, wherein the implant comprises titanium or titanium alloy.