Osteoporosis Changes Collagen/Apatite Orientation and Young's Modulus in Vertebral Cortical Bone of Rat.

This study revealed the distinguished changes of preferential orientation of collagen and apatite and Young's modulus in two different types of osteoporotic bones compared with the normal bone. Little is known about the bone material properties of osteoporotic bones; therefore, we aimed to assess material properties in osteoporotic bones. 66 female Sprague-Dawley rats were used. We analyzed the volumetric bone mineral density, collagen/apatite orientation, and Young's modulus of fifth lumbar vertebral cortex for osteoporotic rats caused by ovariectomy (OVX), administration of low calcium and phosphate content (LCaP) diet, and their combination (OVX + LCaP), as well as sham-operated control. Osteocyte conditions were assessed by hematoxylin and eosin and immunohistochemical (matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)) staining. All osteoporotic animals showed bone loss compared with the sham-operated control. OVX improved craniocaudal Young's modulus by enhancing collagen/apatite orientation along the craniocaudal axis, likely in response to the elevated stress due to osteoporotic bone loss. Conversely, LCaP-fed animals showed either significant bone loss or degraded collagen/apatite orientation and Young's modulus. Osteocytes in LCaP and OVX + LCaP groups showed atypical appearance and MEPE- and DMP1-negative phenotype, whereas those in the OVX group showed similarity with osteocytes in the control group. This suggests that osteocytes are possibly involved in the osteoporotic changes in collagen/apatite orientation and Young's modulus. This study is the first to demonstrate that osteoporosis changes collagen/apatite orientation and Young's modulus in an opposite manner depending on the cause of osteoporosis in spite of common bone loss.

Novel evaluation method of dentin repair by direct pulp capping using high-resolution micro-computed tomography.

OBJECTIVES: We evaluated a novel micro-computed tomography (micro-CT) assessment for quality and quantity of dentin repair, which is difficult to visualize by histological analysis, after direct pulp capping under standardized cavity preparation. MATERIALS AND METHODS: Standardized cavities were prepared on Wistar rats and direct pulp capping was performed using two commercial bioceramics, ProRoot MTA, and iRoot BP Plus. After 2 or 4 weeks, quality and quantity of tertiary dentin formation were evaluated using high-resolution micro-CT analyses including dentin mineral density, dentin mineral contents, compactness and integrity of tertiary dentin, and dentin volume with/without void space. Reproducibility of micro-CT analyses was confirmed by histological evaluation of the same specimen. RESULTS: The exposed pulp area sizes were similar between iRoot BP Plus and ProRoot MTA. Micro-CT analysis of 2-week samples showing compactness of tertiary dentin was significantly higher in iRoot BP Plus than ProRoot MTA (p < 0.05). Tertiary dentin volume without void space, dentin mineral contents, and density were not significantly different between the groups. In 4-week samples, a significant increase was observed in dentin mineral density, compactness, and dentin volume with/without void space induced by iRoot BP Plus (p < 0.05). Micro-CT analysis of tertiary dentin integrity demonstrated that some ProRoot MTA specimens had small defects and lacked continuity (6/512 images). No defects were observed with iRoot BP Plus. CONCLUSIONS: Micro-CT analysis was confirmed as an accurate, objective, and inclusive approach for evaluating quality and quantity of dentin repair. CLINICAL RELEVANCE: These multifaceted approaches to evaluate pulp capping materials may accelerate review processes, ultimately improving vital pulp therapy.

Alignment of Biological Apatite c-Axis Under Functional Loading: A Preliminary Report.

PURPOSE: To understand the bone modeling/remodeling process of functionally loaded implants by observing the alignment of the biological apatite (BAp) c-axis. MATERIALS AND METHODS: Commercially available titanium implants with TiO2 grit-blasted surfaces were placed in the fourth premolar and the first molar of the beagle dog and were functionally loaded with casted crowns and were compared to the non-loaded controls. After being killed, the samples were subjected to histological observation and to a microbeam x-ray diffraction (µXRD) analysis with a transmission optical system to evaluate the preferential BAp c-axis alignment. RESULTS: The non-loaded group presented mostly nonoriented BAp c-axis around the implant, whereas for the loaded group, preferential alignment of BAp c-axis was observed along the loading direction of the implant suggesting that the Bap aligned itself based on the direction of the functional mechanical loading. CONCLUSION: The µXRD revealed that the c-axis orientation was perpendicular to the implant thread working against the functional loaded direction, which may contribute in designing an optimal implant macrogeometry.

Promotion of endodontic lesions in rats by a novel extraradicular biofilm model using obturation materials.

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P < 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.

Preparation and characterizations of antibacterial iodine-containing coatings on pure Ti.

Iodine-containing coatings were prepared on pure Ti surfaces via electrochemical deposition to enhance their antibacterial properties. The factors influencing iodine content were analyzed using an orthogonal experiment. The electrochemically deposited samples were characterized using scanning electron microscopy with energy dispersive spectroscopy and X-ray photoelectron spectroscopy, and their antibacterial properties and cytotoxicity were evaluated. The results showed that changing the deposition time is an effective way to control the iodine content. The iodine content, coating thickness, and adhesion of the samples increased with deposition time. Iodine in the coatings mainly exists in three forms, which are I2, I3-, and pentavalent iodine. For samples with iodine-containing coatings, the antibacterial ratios against E. coli and S. aureus were greater than 90% and increased with increasing iodine content. Although the samples with iodine-containing coatings showed some inhibition of the proliferation of MC3T3-E1 cells, the cell viabilities were all higher than 80%, suggesting that iodine-containing coatings are biosafe.

The influence of immediate occlusal loading on micro/nano-structure of peri-implant jaw bone in rats.

PURPOSE: The objective of the present study was to ascertain the effect of immediate occlusal loading after implant placement on osseointegration and the micro/nanostructure of the surrounding bone. METHODS: After extraction of a rat maxillary right second molar, an implant was placed immediately with initial fixation (2 N< ). The implants were placed to avoid occlusal loading due to mastication, and in the loaded group, a superstructure was fabricated and subjected to occlusal loading. Bone morphometry, collagen fiber anisotropy, and biological apatite (BAp) crystallite alignment were quantitatively evaluated in both groups after extraction and fixation of the jaw bone at Days 7 and 21 after surgery. RESULTS: Osseointegration was observed in both groups. Bone morphometry showed significant differences in bone volume, trabecular number, trabecular thickness and bone mineral density (BMD) at Days 21 postoperatively (P < 0.05). A significant difference was also found in the trabecular separation at Days 7 postoperatively (P < 0.05). In the evaluation of collagen fiber anisotropy, collagen fiber bundles running differently from the existing bone were observed in both groups. In terms of BAp crystallite alignment, a specific structure was observed in the reconstructed new bone after implantation, and preferential orientation of BAp crystallite alignment was observed in the longitudinal direction of the implants in the Day 21 postoperative loaded group. CONCLUSION: When sufficient initial fixation is achieved at the time of dental implant placement, then the applied masticatory load may contribute to rapidly achieving not only bone volume, but also adequate bone quality after implant placement.

Water-Mediated On-Demand Detachable Solid-State Adhesive of Porous Hydroxyapatite for Internal Organ Retractions.

Novel adhesives for biological tissues offer an advanced surgical approach. Here, the authors report the development and application of solid-state adhesives consisting of porous hydroxyapatite (HAp) biocompatible ceramics as novel internal organ retractors. The operational principles of the porous solid-state adhesives are experimentally established in terms of water migration from biological soft tissues into the pores of the adhesives, and their performance is evaluated on several soft tissues with different hydration states. As an example of practical medical utility, HAp adhesive devices demonstrate the holding ability of porcine livers and on-demand detachability in vivo, showing great potential as internal organ retractors in laparoscopic surgery.

Alveolar Bone Microstructure Surrounding Orthodontic Anchor Screws with Plasma Surface Treatment in Rats.

A lateral load was applied to anchor screws that had undergone surface treatment, and the structure, cellular dynamics, and quality of the bone surrounding anchor screws were analyzed to investigate the effect of this surface treatment on the peri-implant jawbone. In addition, bone microstructural characteristics were quantitatively evaluated for each site of loading on the bone around the anchor screw. Rats were euthanized after observation on days 3, 5, or 7, and bone quality analyses were performed. Bone-implant contact rate increased more rapidly at an early stage in the treated surface group than in the untreated surface group. Bone lacuna morphometry showed that the measured values adjacent to the screw at the screw neck on the compressed side (A) and at the screw tip on the uncompressed side (D) were significantly lower than those at the screw tip on the compressed side (B) and at the screw neck on the uncompressed side (C). Collagen fiber bundle diameter showed that the measured values adjacent to regions A and D were significantly higher than those at regions B and C. Anchor screw surface activation facilitates initial bone contact of the screw, suggesting that early loading may be possible in clinical practice.

Novel nano-hydroxyapatite coating of additively manufactured three-dimensional porous implants improves bone ingrowth and initial fixation.

Electron beam melting (EBM) has been used to fabricate three-dimensional (3D) porous Ti-6Al-4V surfaces for acetabular cups in total hip arthroplasty. However, there are radiographic concerns regarding poor implant fixation and bone ingrowth around electron beam melted (EBMed) 3D porous cups. We hypothesize that nano-hydroxyapatite (nHA) coating can promote bone ingrowth and thus decrease the occurrence of radiolucent lines around EBMed 3D porous cups. This study aimed to investigate the effect of a novel nHA coating on the biological performance of EBMed 3D porous implants in a beagle transcortical model. Low-porosity (control) and high-porosity 3D porous Ti-6Al-4V implants were manufactured using EBM. Half of the high-porosity implants were coated with nHA without clogging the 3D pores. Implants were inserted into the femoral diaphysis of the beagles. The beagles were euthanized at 4, 8, and 12 weeks postoperatively, and push-out testing was performed. Bone ingrowth was evaluated by histological analysis. Although the increase in porosity alone had no effect on biological behavior, the addition of nHA to high-porosity 3D implants significantly improved early bone fixation and bone ingrowth into the deep region of porous structures compared to low-porosity implants. This is the first report of a novel nHA coating that improved bone ingrowth into the deeper regions of 3D porous implants, which can prevent the occurrence of radiolucent lines around EBMed 3D porous cups.

Effect of the Correction of Bilateral Differences in Masseter Muscle Functional Pressure on the Mandible of Growing Rats.

The objective of this study is to clarify the effect of restoring the lowered masticatory muscle functional pressure and correcting bilateral differences in masticatory muscle functional pressure on jawbone growth during growth and development with a quantitative evaluation of the changes in the micro/nanostructural characteristics of entheses. Male Wistar rats aged 4 weeks were divided into an experimental group injected with a botulinum toxin serotype A (BoNT/A) formulation to reduce muscle function (BTX group) and a control group (CTRL group). They were euthanised after 6, 8, 10, 12, and 16 weeks after measuring the difference between the midline of the upper and lower incisors. The mandibles were harvested for histological examination, second harmonic generation imaging, and the quantitative evaluation of biological apatite (BAp) crystal alignment. The midline difference decreased with age in weeks. In rats from 6 weeks after BoNT/A administration to 12 weeks after administration, the collagen fibre bundle diameter was significantly smaller in the BTX group; the difference between the two groups decreased with increasing age. BAp crystal alignment was significantly different on the x-axis and the y-axis on the BTX group from 6 weeks after BoNT/A administration to 10 weeks after administration. Asymmetry of mandibular bone formation caused by load imbalance during growth could be corrected by the adjustment of the function of the masseter muscle on either side.

The effects of orthodontic anchor screw inserted into the femur of growth-phase or mature rats -Osteoid formation, bone mineral density, collagen fiber bundles, biological apatite crystal orientation.

The purpose of this study is to investigate the effect of orthodontic anchor screws (OASs) inserted into the femur of growth-phase or mature rats using histological observation and bone structure analysis. The experimental animals are growth-phase (6-week-old) or mature (25-week-old) male Wistar rats. OAS was placed into the point one-third of the femoral length from the proximal end of the femur, and the response of the surrounding bone was observed and measured. The results showed at the OAS bone interface, in growth-phase rats, bone mineral density (BMD) was reduced and the running angle of collagen fiber bundles varied significantly. In mature rats, more osteoid was observed and biological apatite (BAp) crystals showed a different orientation. It was suggested that after the insertion of OASs, bone volume and quality are decreased, but after a sufficient healing period, a new bone micro/nano structure, different from the original structure, are reconstructed.

Chronological histological changes during bone regeneration on a non-crosslinked atelocollagen matrix.

Cleavage of the antigenic telopeptide region from type I collagen yields atelocollagen, and this is widely used as a scaffold for bone regeneration combined with cells, growth factors, etc. However, neither the biological effect of atelocollagen alone or its contribution to bone regeneration has been well studied. We evaluated the chronological histological changes during bone regeneration following implantation of non-crosslinked atelocollagen (Koken Co., Ltd.) in rat calvarial defects. One week after implantation, osteogenic cells positive for runt-related transcription factor 2 (Runx2) and osteoclasts positive for tartrate-resistant acid phosphatase (TRAP) were present in the atelocollagen implant in the absence of bone formation. The number of Runx2-positive osteogenic cells and Osterix-positive osteoblasts increased 2 weeks after implantation, and bone matrix proteins (osteopontin, OPN; osteocalcin, OC; dentin matrix protein 1, DMP1) were distributed in newly formed bone in a way comparable to normal bone. Some resorption cavities containing osteoclasts were also present. By 3 weeks after implantation, most of the implanted atelocollagen was replaced by new bone containing many resorption cavities, and OPN, OC, and DMP1 were deposited in the residual collagenous matrix. After 4 weeks, nearly all of the atelocollagen implant was replaced with new bone including hematopoietic marrow. Immunohistochemistry for the telopeptide region of type I collagen (TeloCOL1) during these processes demonstrated that the TeloCOL1-negative atelocollagen implant was replaced by TeloCOL1-positive collagenous matrix and new bone, indicating that new bone was mostly composed of endogenous type I collagen. These findings suggest that the atelocollagen itself can support bone regeneration by promoting osteoblast differentiation and type I collagen production.

Alignment of biological apatite crystallites at first molar in human mandible cortical bone.

The aim of this study was to quantitatively clarify the c-axis alignment of biological apatite (BAp) crystallites (hereafter referred to as BAp alignment) in the cortical bone of the human mandible first molar. Six mandible specimens were collected from the cadavers of six dentulous Japanese adults (mean age, 63.0 +/- 12.1 years) held at the Department of Anatomy, Tokyo Dental College. A microbeam x-ray diffraction system was used to determine BAp alignment in the mesiodistal direction. Bone mineral density (BMD) was also measured using 3-dimensional trabecular structure measurement software. The results showed that the degree of BAp alignment in the mesiodistal direction was low in the alveolar area and high at the base of the mandible, suggesting that BAp alignment in the alveolar area is affected by occlusal force. Moreover, it was observed that the correlation between BAp alignment and BMD was small, indicating that BAp alignment and BMD could be independent factors. Therefore, determining BAp alignment was important in the evaluation of bone quality, including bone strength.

Development of orthophosphosilicate glass/poly(lactic acid) composite anisotropic scaffolds for simultaneous reconstruction of bone quality and quantity.

Reconstruction of organ-specific architecture is necessary to recover the original organ function. The anisotropic structure of bone tissue is strongly related to the collagen fibril alignment and bone apatite crystal direction. Bone regeneration indicates following two main process; first, restoration of bone mineral density (BMD; bone quantity), and second, restoring bone apatite c-axis orientation (bone quality). In addition to BMD, bone quality is the most important factor among bone mechanical properties. Recovery of the original bone function requires development of novel scaffolds with simultaneous reconstruction of bone quality and quantity. Herein, novel orthophosphosilicate glass (PSG)/poly(lactic acid) composite anisotropic scaffolds were developed to control cell alignment and enhance bone formation, which are important for the simultaneous reconstruction of bone quality and quantity. The strategy to control cell alignment and bone formation involved designing anisotropic scaffolds in combination with the release of therapeutic ions by PSGs. The morphology of fibrous scaffolds containing PSGs was quantitatively designed using electrospinning. This successfully modulated cell alignment and subsequent bone apatite c-axis orientation along the fiber-oriented direction. The released silicate and Mg2+ ions from PSGs in scaffolds improved cell adhesion, proliferation, and calcification. To best of our knowledge, this is the first report demonstrating that the anisotropic scaffolds containing bioactive glasses regenerate bone tissues with simultaneous reconstruction of bone quality and quantity via stimulating osteoblasts by inorganic ions and designing morphology of scaffolds.

Structural characteristics of the bone surrounding dental implants placed into the tail-suspended mice.

BACKGROUND: There are many unclear points regarding local structural characteristics of the bone surrounding the implant reflecting the mechanical environment. PURPOSE: The purpose of this study is to quantitatively evaluate bone quality surrounding implants placed into the femurs of mice in an unloading model, and to determine the influence of the mechanical environment on bone quality. METHODS: Twenty 12-week-old male C57BL6/NcL mice (n = 5/group) were used as experimental animals. The mice were divided into two groups: the experimental group (n = 10) which were reared by tail suspension, and the control group (n = 10) which were reared normally. An implant was placed into the femur of a tail-suspended mouse, and after the healing period, they were sacrificed and the femur was removed. After micro-CT imaging, Villanueva osteochrome bone stain was performed. It was embedded in unsaturated polyester resin. The polymerized block was sliced passing through the center of the implant body. Next, 100-µm-thick polished specimens were prepared with water-resistant abrasive paper. In addition to histological observation, morphometric evaluation of cancellous bone was performed, and the anisotropy of collagen fibers and biological apatite (BAp) crystals was analyzed. RESULTS: As a result, the femoral cortical bone thickness and new peri-implant bone mass showed low values in the tail suspension group. The uniaxial preferential orientation of BAp c-axis in the femoral long axis direction in the non-implant groups, but biaxial preferential orientation of BAp c-axis along the long axis of implant and femoral long axis direction were confirmed in new bone reconstructed by implant placement. Collagen fiber running anisotropy and orientation of BAp c-axis in the bone surrounding the implant were not significantly different due to tail suspension. CONCLUSIONS: From the above results, it was clarified that bone formation occurs surrounding the implant even under extremely low load conditions, and bone microstructure and bone quality adapted to the new mechanical environment are acquired.

In-air micro-proton-induced X-ray/gamma-ray emission analysis of the acid resistance of root dentin after applying fluoride-containing materials incorporating calcium.

This study employed an in-air micro-proton-induced X-ray/gamma-ray emission system to assess the effectiveness of fluoride-containing materials (FCMs) incorporating calcium in preventing root caries. Dentin surfaces of human third molars were coated with one of three FCMs: fluoride-releasing glass-ionomer cement (F7) and experimental materials in which half (P1) or all (P2) of the strontium in F7 was replaced with calcium. Dentin without FCM coating served as the control. Specimens were immersed in saline at 37°C for 1 month, sectioned, and then demineralized. Calcium loss after demineralization was lower in the Ca-substituted groups than in the Ca-unsubstituted groups (p<0.05). Calcium loss was negatively correlated with fluoride uptake (p<0.01). In the F7, P1, and P2 groups, the retraction of the dentin surface was significantly suppressed as compared with the control group. FCMs incorporating calcium improved the acid resistance of root dentin and could help prevent root caries.

Proliferation and differentiation potential of pluripotent mesenchymal precursor C2C12 cells on resin-based restorative materials.

This study investigated the proliferation and differentiation potential of pluripotent mesenchymal cells on three resin-based restoratives using a typical pluripotent mesenchymal precursor cell line, C2C12. C2C12 cells were cultured for 3-21 days on cured specimens of a Bis-GMA/TEGDMA-based composite resin (APX; Clearfil AP-X), a 4-META/MMA-based resin cement (SB; Superbond C&B) or a HEMA-containing resin modified glass-ionomer (LC; Fuji Ionomer Type II LC). To examine the influences on differentiation potential, alkaline phosphatase (ALP) activity of the cells cultured on each material was determined. On APX and SB, cells adhered and proliferated well, and no significant influences on ALP activity were observed. In contrast, poor cell proliferation and significant suppression of ALP activity were observed for cells cultured on LC, similar to those cultured on a zinc oxide EBA cement used as a control material. Bis-GMA/TEGDMA-based composite resin and 4-META/MMA-based resin exhibited better biocompatibility for C2C12 cells than HEMA-containing resin modified glass-ionomer, suggesting a potential advantage of the former two resins to show smaller influences on regeneration of periapical or periodontal tissue.

Design and fabrication of Ti-Zr-Hf-Cr-Mo and Ti-Zr-Hf-Co-Cr-Mo high-entropy alloys as metallic biomaterials.

Novel TiZrHfCr0.2Mo and TiZrHfCo0.07Cr0.07Mo high-entropy alloys for metallic biomaterials (bio-HEAs) were developed based on the combination of Ti-Nb-Ta-Zr-Mo alloy system and Co-Cr-Mo alloy system as commercially-used metallic biomaterials. Ti-Zr-Hf-Cr-Mo and Ti-Zr-Hf-Co-Cr-Mo bio-HEAs were designed using (a) a tree-like diagram for alloy development, (b) empirical alloy parameters for solid-solution-phase formation, and (c) thermodynamic calculations focused on solidification. The newly-developed bio-HEAs overcomes the limitation of classical metallic biomaterials by the improvement of (i) mechanical hardness and (ii) biocompatibility all together. The TiZrHfCr0.2Mo and TiZrHfCo0.07Cr0.07Mo bio-HEAs showed superior biocompatibility comparable to that of commercial-purity Ti. The superior biocompatibility, high mechanical hardness and low liquidus temperature for the material processing in TiZrHfCr0.2Mo and TiZrHfCo0.07Cr0.07Mo bio-HEAs compared with the Ti-Nb-Ta-Zr-Mo bio-HEAs gave the authenticity of the application of bio-HEAs for orthopedic implants with multiple functions.

Impaired bone quality characterized by apatite orientation under stress shielding following fixing of a fracture of the radius with a 3D printed Ti-6Al-4V custom-made bone plate in dogs.

Custom-made implants have recently gained attention in veterinary medicine because of their ability to properly fit animal bones having a wide variety of shapes and sizes. The effect of custom-made implants on bone soundness and the regeneration process is not yet clear. We fabricated a 3D printed Ti-6Al-4V custom-made bone plate that fits the shape of the dog radius, and placed it into the radius where an osteotomy had been made. The preferential orientation of the apatite c-axis contributes to the mechanical integrity of the bone and is a reliable measure of bone quality. We determined this parameter as well as the bone shape and bone mineral density (BMD). The bone portion which lies parallel to the bone plate exhibited bone resorption, decreased BMD, and significant degradation of apatite orientation, relative to the portion outside the plate, at 7 months after the operation. This demonstrates the presence of stress shielding in which applied stress is not transmitted to bone due to the insertion of a stiff bone plate. This reduced stress condition clearly influences the bone regeneration process. The apatite orientation in the regenerated site remained different even after 7 months of regeneration, indicating insufficient mechanical function in the regenerated portion. This is the first study in which the apatite orientation and BMD of the radius were evaluated under conditions of stress shielding in dogs. Our results suggest that assessment of bone repair by radiography can indicate the degree of restoration of BMD, but not the apatite orientation.

Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats.

The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-ß1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.