On-Bone Fixation of Free Gingival Graft Induces an Osteoinductive Effect in Human Alveolar Bone.

We examined alveolar bone samples in the area of on-bone fixation of a free gingival graft performed during surgery in patients aged 37-55 years with a diagnosis of secondary partial adentia of the upper and lower jaws. Six months after fixation of the graft in the alveolar bone, foci of neoosteogenesis were found in the contact zone. They were characterized by the appearance of appositional lines, cords of basophilic osteoblasts, and growing osteons. An immunohistochemical study revealed an increase in the number of CD44+, CD29+, and osteocalcin+ cells in the layer of the outer circumferential lamellae, primary osteons, and the lining of the Haversian canals. TGF-ß1+ cells were located in the intertrabecular reticular tissue and wall of microvessels. The results indicate activation of mesenchymal stem cells in the area of localization of the graft and differentiating osteoblasts. The observed osteoinductive effect of free gingival graft is associated with its participation in reorganization in MSC and induction of morphogenetic molecules.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells.

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

Activity and morphologic changes in the mandible after mandibular osteotomy.

INTRODUCTION: Orthognathic surgery accelerates orthodontic tooth movement, and tooth movement accelerates with demineralized bone and accelerated bone remodeling. The purpose of this study was to ascertain whether orthognathic surgery induces accelerated bone remodeling. The research design included a human model and an animal model. METHODS: The levels of serum tartrate resistant acid phosphatase-5b (TRAP) and bone alkaline phosphatase (BALP) were measured in 15 patients after sagittal split ramus osteotomy. For the animal study, 18 rabbits were divided into 6 groups: a control group and 5 surgery groups. The rabbits in the surgery groups had osteotomies in the molar regions of the mandible. Changes in bone mass of the anterior mandibles were examined by microcomputed tomography, and changes in osteoblast and osteoclast numbers were analyzed by real-time polymerase chain reaction, hematoxylin and eosin staining, TRAP staining, and alkaline phosphatase staining. RESULTS: In the 15 patients, TRAP-5b increased from 1 to 8 weeks postoperatively, and BALP increased significantly in 2 weeks postoperatively. In the rabbits, the levels of mRNA expression of TRAP were increased at 3 weeks, and matrix metalloproteinase 9 was increased at 4 and 8 weeks, whereas mRNA expression of BALP and bone morphogenetic protein 2 were increased at 4 weeks. Bone loss was detected from 1 week postoperatively and reached the maximum at 3 weeks; and bone mass and mechanical structure did not recoverer to preoperative levels until 8 weeks postoperatively. CONCLUSIONS: These findings show active bone remodeling induced by osteotomy.

Comparison of Intraoral Bone Regeneration with Iliac and Alveolar BMSCs.

This study compared the osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) of iliac and alveolar origins (I-BMSCs and Al-BMSCs, respectively), which were transplanted in combination with ß tricalcium phosphate (ß-TCP) in peri-implant bone defects to investigate the osseointegration between dental implants and tissue-engineered bone in dogs. Specifically, I-BMSCs and Al-BMSCs were cultured, characterized, and seeded on ß-TCP and subjected to immunoblotting analyses and alkaline phosphatase activity assays. Subsequently, these cell-seeded scaffolds were implanted into defects that were freshly generated in the mandibular premolar areas of 4 dogs. The defects were covered with ß-TCP + Al-BMSCs ( n = 6), ß-TCP + I-BMSCs ( n = 6), or ß-TCP ( n = 6) or served as the blank control ( n = 6). After healing for 12 wk, the formation and mineralization of new bones were assessed through micro-computed tomographic, histologic, and histomorphometric analyses, and bone-to-implant contacts were measured in the specimens. It was evident that in this large animal model, I-BMSCs and Al-BMSCs manifested similarly strong osteogenic potential, as significantly more new bone was formed in the Al-BMSC and I-BMSC groups than otherwise ( P < 0.01). Therefore, Al-BMSCs are emerging as an efficient alternative for autologous mesenchymal stem cells in regenerative dental and maxillofacial therapies. I-BMSCs, if not restricted in their bioavailability, can also be of great utility in bone tissue-engineering applications.

Platelet-rich fibrin/aspirin complex promotes alveolar bone regeneration in periodontal defect in rats.

BACKGROUND AND OBJECTIVES: The efficacy and outcomes of aspirin in local defects and the use of platelet-rich fibrin (PRF) in periodontal defects were investigated. Whether the PRF/aspirin complex is a suitable scaffold and delivery system to carry sustained-release aspirin/salicylic acid to promote periodontal bone regeneration was determined. MATERIAL AND METHODS: PRF and PRF/aspirin complex were prepared. The concentrations of aspirin/salicylic acid released from the PRF/aspirin complex were calculated at 37°C. Periodontal ligament mesenchymal cells were cultured on six-well plates with PRF or PRF/aspirin complex gel to analyze proliferation and migration. The alveolar bone between the inferior buccal mesial root and anterior buccal distal root of the first maxillary molar was removed in 15 rats randomly divided into three groups: no treatment, PRF or PRF/aspirin complex. Twelve weeks post-transplantation, 2D/3D micro-computed tomography and histomorphometric technique were used for quantitative analyses. RESULTS: The PRF/aspirin complex provided a sustained-release aspirin/salicylic acid. Peak concentrations occurred 4 hours after transplantation and were sustained to 48 hours at 37°C; the total concentration of released aspirin/salicylic acid was 83.5 mg/mL, respectively. The sustained-release promoted the proliferation and migration of periodontal ligament mesenchymal cells. Micro-computed tomography and histological data showed that both the PRF and PRF/aspirin complex enhanced periodontal bone formation (P<.05). Moreover, the new bone formation was two times greater in the PRF/aspirin complex group than the PRF group. CONCLUSION: Aspirin/salicylic acid could be sustained-released from PRF/aspirin complex, which could inhibit inflammation and improve the function of mesenchymal cells. The data might provide a new safe and easy clinical therapeutic strategy to promote periodontal bone reparation.

Repair of maxillary cystic bone defects with mesenchymal stem cells seeded on a cross-linked serum scaffold.

Tissue engineering combining cross-linked serum scaffolds with bone-derived mesenchymal stem cells has displayed excellent results for repair of maxillofacial bone defects in animal models, but it had not been tested in humans yet. We present here a pilot clinical trial using autologous bone-derived mesenchymal stem cells (H-MSV) grown in a serum cross-linked scaffold (BioMax) for treatment of maxillary cysts in 9 patients. Cells obtained from alveolar bone were seeded in the BioMax scaffold prepared from autologous serum, expanded under GMP conditions, and subjected to osteogenic differentiation for 3-4 weeks before application. Evolution of the cystic cavity was followed by computerized tomography (CT) for 7 months. There was no inflammation or other adverse effects, and the CT density of the cyst interior increased significantly after the treatment. The ratio of the CT values after/before treatment was (mean ± SE) 2.52 ± 0.45; in contrast, the density of the contralateral control area of spongy alveolar bone without treatment did not change (ratio after/before, 0.99 ± 0.14). In conclusion, cell therapy with BioMax could be considered as an alternative therapy for maxillary bone defects and other losses of bone substance. Further research with allogeneic cells would be useful for reducing costs and improving logistics. CLINICAL TRIAL REGISTRATION NUMBERS: EudraCT 2010-024246-30 and NCT01389661.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

TLR expression profile of human alveolar bone proper-derived stem/progenitor cells and osteoblasts.

Alveolar bone proper-derived mesenchymal stem/progenitor cells (AB-MSCs) and alveolar osteoblasts (OBs) are pivotal cells with positive attributes in regenerative medicine. During regenerative approaches, AB-MSCs may interact with their surrounding environment via their expressed toll-like-receptors (TLRs). This study aimed to depict for the first time the TLRs expression profile of AB-MSCs and OBs. Cells were isolated from human alveolar bone proper, and STRO-1-immunomagnetically sorted to segregate AB-MSCs and OBs. Cell populations were separately seeded out to obtain single colony forming units (CFUs), and were characterized for CD14, CD34, CD45, CD73, CD90, CD105, and CD146 expression as well as for their multilineage differentiation potential. Following incubation of AB-MSCs and OBs in basic medium, their TLRs expression profiles were characterized at mRNA and protein levels. In contrast to OBs, AB-MSCs showed all predefined mesenchymal stem/progenitor cell characteristics. At a protein level, AB-MSCs showed a distinctive expression profile of TLRs 1, 2, 3, 4, 5, 6, 7, 8, and 10 in different quantities, without TLR9 expression. According to their median expression values, TLR2 was the highest expressed, followed by TLRs 4, 5, 7, 1, 10, 8, 3, and finally 6. In contrast, OBs did not express TLR3 and TLR9. According to their median expression values they further showed a different sequence of TLRs expression, with TLR2 highest expressed, followed by TLRs 10, 4, 7, 5, 1, 8, and 6. This study describes for the first time the characteristic TLRs expression profile of AB-MSCs as well as OBs, which could impact their specific sensitivity to pathogenic as well as body tissue compounds, and their therapeutic potential in-vivo.

Functions of Periostin in dental tissues and its role in periodontal tissues' regeneration.

The goal of periodontal regenerative therapy is to predictably restore the tooth's supporting periodontal tissues and form a new connective tissue attachment of periodontal ligament (PDL) fibers and new alveolar bone. Periostin is a matricellular protein so named for its expression primarily in the periosteum and PDL of adult mice. Its biological functions have been widely studied in areas such as cardiovascular physiology and oncology. Despite being initially identified in the dental tissues and bone, investigations of Periostin functions in PDL and alveolar-bone-related physiopathology are less abundant. Recently, several studies have suggested that Periostin may be an important regulator of periodontal tissue formation. By promoting collagen fibrillogenesis and the migration of fibroblasts and osteoblasts, Periostin might play a pivotal part in regeneration of the PDL and alveolar bone following periodontal surgery. The aim of this article is to provide an extensive review of the implications of Periostin in periodontal tissue biology and its potential use in periodontal tissue regeneration.

Can stem cells enhance bone formation in the human edentulous alveolar ridge? A systematic review and meta-analysis.

Several non-biological materials are currently being used to increase the alveolar bone volume to support dental implants. Recently, stem cell therapy has emerged as a promising biological substitute or adjuvant to enhance bone healing. In order to determine if stem cell therapy has enough clinical evidence to bone ridge augmentation in humans, a systematic review and meta-analysis were conducted. Two independent investigators searched the Entrez PubMed, SCOPUS and Web of Science databases for eligible randomized clinical trials that describe stem cell therapies for alveolar bone formation. The included studies were evaluated for risk of bias. A random-effects meta-analysis model was used to evaluate the percentage of bone formation in the selected studies. Heterogeneity was evaluated using the Cochrane Chi 2 and I 2. Nine eligible trials were included. These studies presented an overall unclear risk of bias. A comparison between the lower heterogeneity studies and the long term observational outcomes showed a slight tendency to enhance bone formation. High heterogeneity between the included studies was observed. The lack of outcome standardization made a wide-ranging comparison difficult. The application of stem cells in oral surgery and implantology appears to be promising although more standardized study designs, increased samples and long-term observations are needed to strength the clinical evidence that stem cell therapy is effective for alveolar bone formation.

PTHrP Nuclear Localization and Carboxyl Terminus Sequences Modulate Dental and Mandibular Development in Part via the Action of p27.

To determine whether the action of the PTHrP nuclear localization sequence and C terminus is mediated through p27 in modulating dental and mandibular development, compound mutant mice, which are homozygous for both p27 deletion and the PTHrP1-84 knock-in mutation (p27(-/-)Pthrp(KI/KI)), were generated. Their teeth and mandibular phenotypes were compared with those of p27(-/-), Pthrp(KI/KI), and wild-type mice. At 2 weeks of age, the mandibular mineral density, alveolar bone volume, osteoblast numbers, and dental volume, dentin sialoprotein-immunopositive areas in the first molar were increased significantly in p27(-/-) mice and decreased dramatically in both Pthrp(KI/KI) and p27(-/-) Pthrp(KI/KI) mice compared with wild-type mice; however, these parameters were partly rescued in p27(-/-) Pthrp(KI/KI) mice compared with Pthrp(KI/KI) mice. These data demonstrate that the deletion of p27 in Pthrp(KI/KI) mice can partially rescue defects in dental and mandibular development. Furthermore, we found that deletion of p27 in Pthrp(KI/KI) mice partially corrected the dental and mandibular phenotype by modulating cell cyclin-regulating molecules and antioxidant enzymes. This study therefore indicates that the p27 pathway may function downstream in the action of PTHrP nuclear localization sequence to regulate dental and mandibular development.

Periodontal Ligament and Alveolar Bone in Health and Adaptation: Tooth Movement.

The periodontal ligament (PDL) and alveolar bone are two critical tissues for understanding orthodontic tooth movement. The current literature is replete with descriptive studies of multiple cell types and their matrices in the PDL and alveolar bone, but is deficient with how stem/progenitor cells differentiate into PDL and alveolar bone cells. Can one type of orthodontic force with a specific magnitude and frequency activate osteoblasts, whereas another force type activates osteoclasts? This chapter will discuss the biology of not only mature cells and their matrices in the periodontal ligament and alveolar bone, but also stem/progenitor cells that differentiate into fibroblasts, osteoblasts and osteoclasts. Key advances in tooth movement rely on further understanding of osteoblast and fibroblast differentiation from mesenchymal stem/progenitor cells, and osteoclastogenesis from the hematopoietic/monocyte lineage.

Regional Acceleratory Phenomenon.

The regional acceleratory phenomenon (RAP) is a tissue reaction to a noxious stimulus that increases the healing capacities of the affected tissues. It is typical not only of hard tissues such as bone and cartilage, but also of soft tissues. The RAP is characterized by acceleration of the normal cellular activities, as an 'SOS' phenomenon of the body that has to respond to the new perturbation. In the alveolar bone, the RAP is characterized, at a cellular level, by increased activation of the basic multicellular units (BMUs), thereby increasing the remodeling space. At the tissue level, the RAP is characterized by the production of woven bone, with the typical unorganized pattern, that will be reorganized into lamellar bone at a later stage. In the alveolar bone, the RAP occurs typically in the healing process of the alveolar sockets after tooth extraction, in periodontal disease, after surgery and trauma and during orthodontic tooth movement. In relation to orthodontic tooth movement, the RAP can be seen as a tissue response to the mechanical cyclical perturbation that induces the formation of microdamage that has to be removed to avoid their accumulation and the following bone failure. The adaptation to the new orthodontically induced mechanical environment is ensured by an increased activation of the BMU that returns to normal levels after few months.

Sol-gel processing of novel bioactive Mg-containing silicate scaffolds for alveolar bone regeneration.

Periodontal tissue regeneration is an important application area of biomaterials, given the large proportion of the population affected by periodontal diseases like periodontitis. The aim of this study was the synthesis of a novel porous bioceramic scaffold in the SiO2-CaO-MgO system with specific properties targeted for alveolar bone tissue regeneration using a modification of the traditional foam replica technique. Since bioceramic scaffolds are considered brittle, scaffolds were also coated with gelatin in order to increase their mechanical stability. Gelatin was chosen for its biocompatibility, biodegradability, low-cost, and low immunogenicity. However, gelatin degrades very fast in water solutions. For this reason, two different cross-linking agents were evaluated. Genipin, a non-toxic gardenia extract and the chemical compound 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), which is also considered non-toxic. The results of the investigation indicated that all scaffolds presented an open, interconnected porosity and pores' sizes in the range of 300-600 µm, fast apatite-forming ability, biocompatibility, and suitable mechanical stability.

Pilot investigation of the molecular discrimination of human osteoblasts from different bone entities.

In oral and maxillofacial surgery, autologous grafts from the iliac crest remain the 'gold standard' for alveolar ridge reconstruction, whereas intraoral bone grafts are considered in smaller defects. To date, a comparison of the osteogenic potential of osteoblasts with regard to their tissue origin is missing. Primary osteoblasts have proven useful for the investigation of the tissue-specific osteogenic properties. The present study compares primary human alveolar (aHOBs) and iliac osteoblasts (iHOBs) derived from three female patients undergoing routine intraoral bone grafting. Proliferation potential of the osteoblasts was evaluated using real-time impedance monitoring. Relative gene expression of bone specific biomarkers was analyzed and quantified using quantitative polymerase chain reactions (qPCR). Immunohistochemistry and phase contrast microscopy were performed, as well as alkaline phosphatase assay and alizarin red staining to visualize morphology and mineralization capacity. A twofold faster proliferation rate of aHOBs compared with iHOBs (130 h vs. 80 h) was observed. Alkaline phosphatase activity and alizarin red staining in both HOBs indicated similar mineralization capacity. Gene expression of seven genes (BMP1, CSF-1, TGFBR1, ICAM1, VCAM1, SPP1 and DLX5) was significantly higher in iHOB than in aHOB samples. These data suggest a higher osteogenic potential of osteoblasts derived from the iliac crest compared with primary osteoblasts from the alveolar bone and may lead to a better understanding of the molecular impact of bone cells from different bone entities on bone regeneration in alveolar ridge reconstructions.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

Osteoprotegerin expressed by osteoclasts: an autoregulator of osteoclastogenesis.

Osteoprotegerin (OPG) is secreted by stromal and osteoblastic lineage cells and inhibits osteoclastogenesis by preventing the interaction of receptor activator of nuclear factor-κB ligand (RANKL) with receptor activator of nuclear factor-κB (RANK). In this study, the expression of OPG in osteoclasts themselves and its biological functions during osteoclastogenesis were investigated for the first time. OPG expression in vivo in the developing rat maxilla was examined by immunofluorescence analysis. OPG expression in osteoclasts during in vitro osteoclastogenesis was determined by reverse-transcription polymerase chain-reaction (RT-PCR), Western blot, and immunofluorescence staining. We determined the function of OPG produced by osteoclasts during osteoclastogenesis by silencing the OPG gene. The effects of OPG on bone-resorbing activity and apoptosis of mature osteoclasts were examined by the assay of resorptive pit formation on calcium-phosphate-coated plate and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. In the immunofluorescence findings, strong immunoreactivities were unexpectedly seen in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts around the growing and erupting tooth germs in the rat alveolar bone. In vitro, OPG expression was significantly increased during the differentiation of osteoclasts from mouse bone-marrow-derived cells treated with a combination of macrophage colony-stimulating factor (M-CSF) and RANKL. Interestingly, it was found that OPG small interfering (si)RNA treatment during osteoclastogenesis enhanced the sizes of osteoclasts, but attenuated their bone-resorbing activity. Also, the increased chromosomal DNA fragmentation and caspase-3 activity in the late phase of osteoclastogenesis were found to be decreased by treatment with OPG siRNA. Furthermore, effects of OPG siRNA treatment on osteoclastogenesis and bone-resorbing activity were recovered by the treatment of exogenous OPG. These results suggest that OPG, expressed by the osteoclasts themselves, may play an auto-regulatory role in the late phase of osteoclastogenesis through the induction of apoptosis.

Force-induced Adrb2 in periodontal ligament cells promotes tooth movement.

The sympathetic nervous system (SNS) regulates bone resorption through ß-2 adrenergic receptor (Adrb2). In orthodontic tooth movement (OTM), mechanical force induces and regulates alveolar bone remodeling. Compressive force-associated osteoclast differentiation and alveolar bone resorption are the rate-limiting steps of tooth movement. However, whether mechanical force can activate Adrb2 and thus contribute to OTM remains unknown. In this study, orthodontic nickel-titanium springs were applied to the upper first molars of rats and Adrb1/2(-/-) mice to confirm the role of SNS and Adrb2 in OTM. The results showed that blockage of SNS activity in the jawbones of rats by means of superior cervical ganglion ectomy reduced OTM distance from 860 to 540 µm after 14 d of force application. In addition, the injection of nonselective Adrb2 agonist isoproterenol activated the downstream signaling of SNS to accelerate OTM from 300 to 540 µm after 7 d of force application. Adrb1/2(-/-) mice showed significantly reduced OTM distance (19.5 µm) compared with the wild-type mice (107.6 µm) after 7 d of force application. Histopathologic analysis showed that the number of Adrb2-positive cells increased in the compressive region of periodontal ligament after orthodontic force was applied on rats. Mechanistically, mechanical compressive force upregulated Adrb2 expression in primary-cultured human periodontal ligament cells (PDLCs) through the elevation of intracellular Ca(2+) concentration. Activation of Adrb2 in PDLCs increased the RANKL/OPG ratio and promoted the peripheral blood mononuclear cell differentiation to osteoclasts in the cocultured system. Upregulation of Adrb2 in PDLCs promoted osteoclastogenesis, which accelerated OTM through Adrb2-enhanced bone resorption. In summary, this study suggests that mechanical force-induced Adrb2 activation in PDLCs contributes to SNS-regulated OTM.

Histologic investigation of gingival epithelium implantation and the nonincision placement of miniscrews.

PURPOSE: This study investigated whether the nonincision placement of miniscrews could lead to ectopic implantation of epithelium at the bone-implant interface and, if so, whether the epithelial cells could survive. The fate of grafted epithelial cells was also investigated. MATERIALS AND METHODS: Ten healthy male beagle dogs were included in the study and were divided into three groups. Group 1 dogs (n = 3) were implanted with miniscrews without any other procedures. In group 2, epithelial cells were collected, infected by adeno-associated virus-2 containing green fluorescent protein (GFP), and injected into pilot holes drilled before miniscrew implantation in three dogs, and one dog was injected with epithelial cells as a negative control. In group 3, tiny gingival tissue pieces were injected into pilot holes drilled before miniscrew implantation. In group 1, hematoxylin-eosin and fluorescent immunohistochemical (IHC) staining of cytokeratin were performed on both undecalcified and decalcified slices. In groups 2 and 3, IHC staining of cytokeratin and apoptotic testing were performed. RESULTS: No ectopically implanted epithelial cells were found at the bone-implant interface of the miniscrews in group 1. In group 2, fluorescence emitted by cytokeratin antibody and by GFP was observed in specimens at 0 days, had decreased by 3 days, and had disappeared by 7 days. In group 3, areas of fluorescent cytokeratin-positive tissues were much smaller at 7 days than at 0 days, and none were found at 28 days. In groups 2 and 3, there were many apoptotic epithelial cells at the bone-implant interface at 3 days and 7 days, respectively. CONCLUSION: There was little possibility of implantation of epithelium into alveolar bone though placement of miniscrews with a nonincision procedure. Furthermore, even when epithelial cells or tissue was ectopically implanted, it was soon cleared through an apoptotic process.