Critical-sized marginal defects around implants in the rabbit mandible.

BACKGROUND: The mandible of the rabbit is considered a reliable model to be used to study bone regeneration in defects. The aim of the present study was to evaluate the formation of new bone around implants installed in defects of either 5 or 10 mm in the mandible of rabbits. MATERIALS AND METHODS: In 12 rabbits, 3 mm deep circumferential defect, either 5 or 10 mm in diameter, were prepared bilaterally and an implant was placed in the center. A collagen membrane was placed to close the entrance. After 10 weeks, biopsies were taken, histological slides were prepared, and different regions of the defects were analyzed. RESULTS: Similar amounts of new bone were found in both defects. However, most of the 5 mm defects were filled with new bone. New bone was observed closing the entrance of the defect and laid onto the implant surface. Only in a few cases the healing was incomplete. Despite a similar percentage of new bone found within the 10 mm defects, the healing was incomplete in most of the cases, presenting a low rate of bone formation onto the implant surface within the defect. Only one case presented the closure of the entrance. CONCLUSIONS: The dimensions of the defect strongly influenced the healing so that a circumferential marginal defect of 10 mm around an implant in the mandible body should be considered a critical-sized defect. The presence of the implant and of residues of teeth might have strongly influenced the healing.

Biocompatibility of dental implants coated with hydroxyapatite using pulsed Er:YAG laser deposition.

We aimed to improve the biocompatibility and osteoinductive potential of Ti implants using a simulated intraoral hydroxyapatite (HAp) coating. We devised a novel surface treatment for aggressive induction of osteoblast adhesion and bone regeneration on the implant surface. A thin α-tricalcium phosphate (α-TCP) film was deposited on the implant surface using a pulsed Er:YAG laser. The coating was converted to HAp through artificial saliva immersion, which was confirmed using scanning electron microscopy (SEM) and X-ray diffraction (XRD). SEM showed needle-like HAp crystals on the Ti disks and sandblasted implant surfaces after immersion in artificial saliva for 96 h. Microcomputed tomography and histological evaluation 4 and 8 weeks after implantation into beagle dog mandibles showed that the HAp-coated implant was biocompatible and exhibited superior osteoinduction compared to that of sandblasted implants. Coating the implant surface with HAp using an Er:YAG laser has potential as a new method of the implant-surface debridement.

Critical-sized marginal defects around implants treated with xenografts in rabbits.

BACKGROUND: Healing of critical-size defects is a well-known problem that has been challenged in several studies. The aim of the experiment was to evaluate bone formation and osseointegration of implants installed in critical defects of the mandibular body simultaneously grafted with Bio-Oss® or Cerabone®. MATERIAL AND METHODS: Defects, 10 mm wide and 3 mm deep, were prepared at both lateral aspects of the mandible in 12 rabbits. One implant was installed in the center of the defect, and bovine xenografts produced either at low (Bio-Oss®; Low-T) or high (Cerabone®; High-T) temperatures were used to fill the defects. A collagen membrane was placed to cover the sites. Healing was evaluated 10 weeks after surgery. RESULTS: In both groups, most sites showed optimal healing with closure of the coronal entrance of the defects. However, residual defects occupied by soft tissues and biomaterial particles were observed, even though generally limited to some regions of the defect. Osseointegration of the implant surface in the region of the defect was poor in both groups. CONCLUSIONS: Circumferential marginal critical-size defects around implants filled with bovine xenografts presented regions with a complete healing in both groups. However, the healing was not complete at all regions in most defects; therefore, a complete optimal healing of critical-size marginal defects cannot be predicted.

Implants Placed with a Ring Technique Using Inlay and Onlay Block Xenografts in the Mandible of Rabbits.

BACKGROUND: Xenogenous bone has been proposed as an alternative to overcome the disadvantages of autogenous grafting. The aim of the present study was to study bone dynamics at inlay and onlay xenografts used for bone augmentation applying a ring technique. METHODS: The bone at the lateral surface of the mandibular angle of 12 adult male New Zealand White rabbits was exposed bilaterally. The cortical layer received multiple perforations on one side of the mandible, and a xenograft block of collagenated cancellous equine bone, 7 mm in diameter and 3 mm in width, was fixed on the prepared surface using an implant (onlay group). On the opposite side, a defect 7 mm in diameter and 3 mm in depth was prepared, and the xenograft block was adapted to the defect and fixed with an implant (inlay group). RESULTS: After ten weeks of healing, in the onlay grafts, new bone was mainly formed on the trabeculae surface, reaching in some specimens the most coronal regions of the block. In the inlay grafts, new bone was found arranged on the trabecular surfaces but also occupying the spaces among the trabeculae. The entrance of the defect was often found close to the top of the block by newly formed bone. A higher percentage of new bone was found in the inlay (19.0 ± 9.3%) compared to the onlay (10.4 ± 7.4%) groups (p = 0.031). The mean gain in osseointegration at the implant in relation to the base of the original 3 mm deep defect was 0.95 ± 1.05% in the onlay group and 0.78 ± 0.71% in the inlay group (p = 0.603). CONCLUSION: The inlay grafts exhibited a higher new bone percentage than the onlay block grafts possibly due to the defect conformation that presented more sources for bone growth. The trabecular conformation and the composition of the grafts made possible the expression of the osteoconductive properties of the material used. This resulted, in several specimens, in the growth of bone on the graft trabeculae toward the most superior regions in both groups and in the closure of the coronal entrance of the defects in the inlay group. The clinical relevance of this experiment is that the ring technique applied as an inlay method could be suitable for bone augmentation.

Histological Comparison of Collagenated Cancellous Equine Bone Blocks Used as Inlay or Onlay for Lateral Bone Augmentation in Rabbits.

BACKGROUND: The conformation of the recipient site for an inlay graft presents an increased contact with the parent bone compared to an onlay graft. This might favor bone growth within the inlay compared to onlay grafts. Hence, the objective of this study was to compare the bone incorporation and remodeling processes of xenogeneic en bloc grafts placed using two bone grafting techniques, i.e., onlay vs. inlay. METHODS: In this prospective, randomized, split-mouth study (test and control sides in the same animal), two bone grafting techniques were comparatively evaluated. The lateral aspect of the rabbit mandible was used as the recipient site, bilaterally. On one side of the mandible, the cortical bone was perforated with drills to allow a better bone formation from the bone wound and the marrow spaces. A xenogeneic bone block was fixed in the center of the prepared region, representing the onlay site. On the other side of the mandible, a 7 mm wide and 3 mm deep circumferential defect was prepared using trephines and drills. A xenogeneic bone block was fixed in the center of the defect, representing the inlay site. Two healing periods were applied in the study: 2 and 10 weeks, each represented by 10 rabbits (n = 10 for each period). RESULTS: After 2 weeks of healing, the mean percentage of new bone was 10.4% and 23.3% at the onlay and inlay grafts, respectively (p = 0.022). After 10 weeks of healing, new bone increased to 13.2% at the onlay sites and 25.4% at the inlay sites (p = 0.080). In the 10-week period, the inlay grafts presented a homogeneous growth of new bone in all regions, while in the onlay grafts, low percentages of new bone were observed in the external regions. CONCLUSION: The percentage of new bone increased faster and was higher in the inlay grafts than in the onlay grafts. This outcome might be related to the self-contained conformation of the recipient site in the inlay group, which offered more sources for new bone formation compared to the one-wall conformation of the recipient sites in the onlay group. The osteoconductive properties of the biomaterial allowed the newly formed bone to reach the most peripheral regions in both groups. The osteoconductive properties of the biomaterial, together with the protection offered by the collagen membrane, allowed marginal closure of the defects by newly formed bone in the inlay group.

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ.

Introduction: The potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals. Methods: We used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice. Results: Percentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly. Discussion: ZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw.

Design and Synthesis of Cathepsin-K-Activated Osteoadsorptive Fluorogenic Sentinel (OFS) Probes for Detecting Early Osteoclastic Bone Resorption in a Multiple Myeloma Mouse Model.

We describe the design and synthesis of OFS-1, an Osteoadsorptive Fluorogenic Sentinel imaging probe that is adsorbed by hydroxyapatite (HAp) and bone mineral surfaces, where it generates an external fluorescent signal in response to osteoclast-secreted cathepsin K (Ctsk). The probe consists of a bone-anchoring bisphosphonate moiety connected to a Förster resonance energy transfer (FRET) internally quenched fluorescent (IQF) dye pair, linked by a Ctsk peptide substrate, GHPGGPQG. Key structural features contributing to the effectiveness of OFS-1 were defined by structure-activity relationship (SAR) and modeling studies comparing OFS-1 with two cognates, OFS-2 and OFS-3. In solution or when preadsorbed on HAp, OFS-1 exhibited strong fluorescence when exposed to Ctsk (2.5-20 nM). Time-lapse photomicrographs obtained after seeding human osteoclasts onto HAp-coated well plates containing preadsorbed OFS-1 revealed bright fluorescence at the periphery of resorbing cells. OFS-1 administered systemically detected early osteolysis colocalized with orthotopic engraftment of RPMI-8226-Luc human multiple myeloma cells at a metastatic skeletal site in a humanized mouse model. OFS-1 is thus a promising new imaging tool for detecting abnormal bone resorption.

Neuronal PAS Domain 2 (Npas2)-Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction.

The circadian clock, which consists of endogenous self-sustained and cell-autonomous oscillations in mammalian cells, is known to regulate a wide range of peripheral tissues. The unique upregulation of a clock gene, neuronal PAS domain protein 2 (Npas2), observed along with fibroblast aging prompted us to investigate the role of Npas2 in the homeostasis of dermal structure using in vivo and in vitro wound healing models. Time-course healing of a full-thickness skin punched wound exhibited significantly faster wound closure in Npas2-/- mice than wild-type (WT) C57Bl/6J mice. Dorsal skin fibroblasts isolated from WT, Npas2+/-, and Npas2-/- mice exhibited consistent profiles of core clock gene expression except for Npas2 and Per2. In vitro behavioral characterizations of dermal fibroblasts revealed that Npas2-/- mutation was associated with increased proliferation, migration, and cell contraction measured by floating collagen gel contraction and single-cell force contraction assays. Npas2 knockout fibroblasts carrying sustained the high expression level of type XII and XIV FAICT collagens and synthesized dermis-like thick collagen fibers in vitro. Confocal laser scanning microscopy demonstrated the reconstruction of dermis-like collagen architecture in the wound healing area of Npas2-/- mice. This study indicates that the induced Npas2 expression in fibroblasts may interfere with skin homeostasis, wound healing, and dermal tissue reconstruction, providing a basis for novel therapeutic target and strategy. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Rescue bisphosphonate treatment of alveolar bone improves extraction socket healing and reduces osteonecrosis in zoledronate-treated mice.

Bisphosphonate (BP)-related osteonecrosis of the jaw, previously known as BRONJ, now referred to more broadly as medication-related osteonecrosis of the jaw (MRONJ), is a morbid condition that represents a significant risk for oncology patients who have received high dose intravenous (IV) infusion of a potent nitrogen containing BP (N-BP) drug. At present, no clinical procedure is available to prevent or effectively treat MRONJ. Although the pathophysiological basis is not yet fully understood, legacy adsorbed N-BP in jawbone has been proposed to be associated with BRONJ by one or more mechanisms. We hypothesized that removal of the pre-adsorbed N-BP drug common to these pathological mechanisms from alveolar bone could be an effective preventative/therapeutic strategy. This study demonstrates that fluorescently labeled BP pre-adsorbed on the surface of murine maxillo-cranial bone in vivo can be displaced by subsequent application of other BPs. We previously described rodent BRONJ models involving the combination of N-BP treatment such as zoledronate (ZOL) and dental initiating factors such as tooth extraction. We further refined our mouse model by using gel food during the first 7 days of the tooth extraction wound healing period, which decreased confounding food pellet impaction problems in the open boney socket. This refined mouse model does not manifest BRONJ-like severe jawbone exposure, but development of osteonecrosis around the extraction socket and chronic gingival inflammation are clearly exhibited. In this study, we examined the effect of benign BP displacement of legacy N-BP on tooth extraction wound healing in the in vivo model. Systemic IV administration of a low potency BP (lpBP: defined as inactive at 100 µM in a standard protein anti-prenylation assay) did not significantly attenuate jawbone osteonecrosis. We then developed an intra-oral formulation of lpBP, which when injected into the gingiva adjacent to the tooth prior to extraction, dramatically reduced the osteocyte necrosis area. Furthermore, the tooth extraction wound healing pattern was normalized, as evidenced by timely closure of oral soft tissue without epithelial hyperplasia, significantly reduced gingival inflammation and increased new bone filling in the extraction socket. Our results are consistent with the hypothesis that local application of a rescue BP prior to dental surgery can decrease the amount of a legacy N-BP drug in proximate jawbone surfaces below the threshold that promotes osteocyte necrosis. This observation should provide a conceptual basis for a novel strategy to improve socket healing in patients treated with BPs while preserving therapeutic benefit from anti-resorptive N-BP drug in vertebral and appendicular bones.

Neuronal PAS domain 2 (Npas2) facilitated osseointegration of titanium implant with rough surface through a neuroskeletal mechanism.

Titanium (Ti) biomaterials have been applied to a wide range of implantable medical devices. When placed in bone marrow, Ti-biomaterials integrate to the surrounding bone tissue by mechanisms that are not fully understood. We have previously identified an unexpected upregulation of circadian clock molecule neuronal PAS domain 2 (Npas2) in successfully integrated implant with a rough surface. This study aimed to elucidate the molecular mechanism of osseointegration through determining the role of Npas2. Human bone marrow stromal cells (BMSC) that were cultured on a Ti disc with SLA surface exhibited increased NPAS2 expression compared to BMSC cultured on a machined surface. A mouse model was developed in which miniature Ti implants were surgically placed into femur bone marrow. The implant push-out test and bone-to-implant contact measurements demonstrated the establishment of osseointegration in 3 weeks. By contrast, in Npas2 functional knockout (KO) mice, the implant push-out value measured for SLA surface Ti implant was significantly decreased. Npas2 KO mice demonstrated normal femur bone structure surrounding the Ti implant; however, the recovered implants revealed abnormal remnant mineralized tissue, which lacked dense collagen architecture typically found on recovered implants from wild type mice. To explore the mechanisms leading to the induced Npas2 expression, an unbiased chemical genetics analysis was conducted using mouse BMSC carrying an Npas2-reporter gene for high throughput screening of Library of Pharmacologically Active Compounds. Npas2 modulating compounds were found clustered in regulatory networks of the α2-adrenergic receptor and its downstream cAMP/CREB signaling pathway. Mouse primary BMSC exposed to SLA Ti disc significantly increased the expression of α2-adrenergic receptors, but the expression of ß2-adrenergic receptor was unaffected. Our data provides the first evidence that peripheral clock gene component Npas2 plays a role in facilitating the enhanced osseointegration through neuroskeletal regulatory pathways induced by BMSC in contact with rough surface Ti implant.

Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells.

Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing Npas2, Bmal1, and Vdr was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.

Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Human mandibular trabecular bone density correlation with mechanical strength: implications for implant dentistry.

PURPOSE: The prognosis of a dental implant depends on the mechanical properties of the bone. The preoperative diagnosis of bone quality has become more important, because the immediate loading procedure is now widely used, and a firm initial stability is required. Quantitative computed tomography (QCT) is an effective method of determining selectively the bone mineral density (BMD) of the trabecular bone mostly responsible for the survival of the implant. In this project, we investigated the relationship between the BMD measured by QCT and the mechanical strength of the mandible. MATERIALS AND METHODS: The BMD (mg/mL) of 58 trabecular bone specimens from 6 embalmed human cadaver mandibles were obtained using QCT. The compressive strength to break point was performed with a mechanical device. Each specimen was washed and weighed. RESULTS: The mean values of BMD for the anterior, premolar, and molar region were 633.3, 571.0, and 518.3 mg/mL, respectively. The mean values of compressive strength were 0.237, 0.216, and 0.196 kN, and the ash weights were 0.047, 0.044, and 0.039 g, respectively. There was a positive correlation between the BMD and the mechanical strength (R = 0.77) and between the ash weight and the mechanical strength (R = 0.84). CONCLUSION: There was strong correlation of bone strength, bone mineral, and bone density by QCT. QCT is an excellent preoperative diagnostic tool to select the most mechanically appropriate implant for initial stability and improve the survival prognosis.

Students' opinion of a predoctoral implant training program.

Implant treatment today is highly predictable and provides valid restorative options for the completely or partially edentulous patient. In Japan, many dental care facilities have incorporated implant treatment, and such treatment is no longer rare. For predoctoral students, the educational environment related to implants is not always applicable in present clinical settings. In this article, we describe the implant training program developed at our university for predoctoral education, and we report the changes in student opinions regarding implant treatment by comparing pre- and post-training opinions. The newly developed models for implant training were effective in increasing student understanding that implant treatment is one prosthetic option for restoring missing teeth. In a survey of predoctoral students, responses indicating negative opinions toward implant treatment decreased after training, and responses indicating positive opinions increased. These findings indicated that this training was effective in deepening student understanding of implant treatment.

Chronological changes in the ultrastructure of titanium-bone interfaces: analysis by light microscopy, transmission electron microscopy, and micro-computed tomography.

PURPOSES: The objectives of this study were to chronologically examine the titanium-bone interfaces and to clarify the process of osseointegration using light microscopy, transmission electron microscopy (TEM), and micro-computed tomography (CT). MATERIALS AND METHODS: Experimental implants (Ti-coating plastic implants) were placed into tibiae of 8-week-old rats. Animals were sacrificed at 1 to 28 days after implant placement and prepared tissue specimens for a light microscope, a TEM, and micro-CT. RESULTS: New bone formation began 5 days after implant placement, and osseointegration was obtained by 14 days after implant placement. Osseointegration was well developed by 28 days after implant placement. DISCUSSION: TEM and quantitative computer tomography (QCT) results indicated that bone formation in osseointegration of titanium implants did not occur from the surfaces of the implant or preexisting bone, but it was likely that bone formation progressed at a site a small distance away from the surface. The bone formation took place in a scattered manner. Small bone fragments adhered to each other and transformed into reticular-shaped bone, and finally these bones became lamellar bone. CONCLUSION: Comparative analysis of the titanium-bone interfaces using light microscopy, TEM, and QCT by micro-CT revealed the precise process of osseointegration.