Significant cases of central cusps, enamel pits, and oral fibromas in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder in which benign nodular tumors form in the cerebral cortex, cerebellum, and throughout the body causing various symptoms. In this study, we summarized the incidence of dental findings in patients with TSC at our hospital and its association with diseases in various organs. Patients diagnosed with TSC at our hospital between January 2013 and September 2017, and who were examined in the dental and oral surgery department were included in this study. The presence of intraoral manifestations (central cusps, enamel pits, oral fibromas) was examined by means of visual inspection, intraoral photography, and X-ray photography. In addition, the relationship with associated diseases (neurological, cutaneous, cardiac, renal, and pulmonary) according to organ and disease severity was examined. The mean age (± SD) of the 42 TSC patients (19 men and 23 women) was 27.8 ± 14.6 years, of which 24 patients (11 men and 13 women) presented with oral manifestations. Of these patients, seven had central cusps, 10 had enamel pits, and 17 had oral fibromas. The group with central cusps had significantly higher neurological issues in the relationship between intraoral manifestations and associated disease based on the involved organ. The prevalence of central cusps in TSC was 16.7%, which is significantly higher than the 2.6% reported in healthy Japanese subjects. The central cusp is a diagnostic factor alongside the presence of enamel pits and oral fibromas, which can aid in the early diagnosis of TSC by dentists.

Antimicrobial susceptibility surveillance of bacterial isolates recovered in Japan from odontogenic infections in 2013.

We report on the findings of the first antimicrobial susceptibility surveillance study in Japan of isolates recovered from odontogenic infections. Of the 38 facilities where patients representing the 4 groups of odontogenic infections were seen, 102 samples were collected from cases of periodontitis (group 1), 6 samples from pericoronitis (group 2), 84 samples from jaw inflammation (group 3) and 54 samples from phlegmon of the jaw bone area (group 4) for a total of 246 samples. The positivity rates of bacterial growth on culture were 85.3%, 100%, 84% and 88.9%, respectively, for groups 1, 2, 3 and 4. Streptococcus spp. isolation rates according to odontogenic infection group were 22% (group 1), 17.7% (group 3) and 20.7% (group 4). Anaerobic isolation rates were 66.9% (group 1), 71.8% (group 3) and 68.2% (group 4). Drug susceptibility tests were performed on 726 strains excluding 121 strains that were undergrown. The breakdown of the strains subjected to testing was 186 Streptococcus spp., 179 anaerobic gram-positive cocci, 246 Prevotella spp., 27 Porphyromonas spp., and 88 Fusobacterium spp. The isolates were tested against 30 antimicrobial agents. Sensitivities to penicillins and cephems were good except for Prevotella spp. The low sensitivities of Prevotella spp is due to ß-lactamase production. Prevotella strains resistant to macrolides, quinolones, and clindamycin were found. No strains resistant to carbapenems or penems were found among all strains tested. No anaerobic bacterial strain was resistant to metronidazole. Antimicrobial susceptibility testing performed on the S. anginosus group and anaerobic bacteria, which are the major pathogens associated with odontogenic infections, showed low MIC90 values to the penicillins which are the first-line antimicrobial agents for odontogenic infections; however, for Prevotella spp., penicillins combined with ß-lactamase inhibitor showed low MIC90 values.

Tracheal cartilage growth promotion by intra-tracheal administration of basic FGF.

PURPOSE: This study aimed to investigate whether intra-tracheal administration of basic fibroblast growth factor (b-FGF) promotes the growth of tracheal cartilage. METHODS: Trachea of 4-week old mice were intubated and 2.5 µg b-FGF administered (Group 4) for periods from 1 to 5 days. Cervical tracheal outer diameter and tracheal ring length were compared in Group 1 (no intervention), Group 2 (tracheal intubation), Group 3 (intra-tracheal administration of distilled water) and Group 4, at 8 weeks of age. Outer diameter and tracheal ring length in Group 4 were also compared with that in Group 1 at 12 and 16 weeks of age. RESULTS: At 8 weeks of age, tracheal ring length with b-FGF administration for more than 4 days in Group 4 was significantly increased over that following 1-day administration. At 8 weeks of age, mean outer diameter and the mean tracheal ring length in Group 4 were significantly greater than in the other groups. Mean outer diameter and mean tracheal ring length were significantly greater in Group 4 than in Group 1 at 12 and 16 weeks of age. CONCLUSION: This study has shown that intra-tracheal administration of b-FGF enlarges the tracheal lumen.

Roles of macrophage migration inhibitory factor in cartilage tissue engineering.

To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif+/+) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif+/+ constructs deteriorated by 8 weeks. Since the Mif+/+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term.

Clinical Findings of a Cantilever Iliac Bone Graft for Secondary Correction of Cleft Lip-Nose Deformities.

The authors performed a cantilever iliac bone graft for the secondary correction of severe cleft lip-nose deformities after the completion of growth. For the purpose of clarifying effects of the cantilever iliac bone grafts and the adverse events with regard to their time course changes after this procedure, the authors retrospectively surveyed long-term morphologic changes in 65 cleft lip, alveolus, and palate patients in whom cleft lip-nose deformities were treated with a cantilever iliac bone graft (age at surgery: 14-45 years old). All postsurgical documents of facial photographs and radiologic images were reviewed to evaluate the effects and adverse events. The main adverse events were deviations of the apex of the nose, excess resorption of the grafted iliac bone, protruding deformations of the grafted iliac bone at the root of the nose, and fracture of the grafted iliac bone. Additional surgery was necessary in 10.7% of patients. Postsurgical changes in facial profiles became favorable, measured on lateral view of cephalometric radiography, achieving morphologic improvements. A cantilever iliac bone graft was effective for improving nasal deformities in cleft lip, alveolus, and palate patients, although the counter measures should be taken to these adverse events.

Biological roles of glial fibrillary acidic protein as a biomarker in cartilage regenerative medicine.

Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology.

Local administration of a hedgehog agonist accelerates fracture healing in a mouse model.

Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showed that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures.

High-risk oral leukoplakia is associated with aberrant promoter methylation of multiple genes.

BACKGROUND: Early detection of oral squamous cell carcinomas (OSCCs) is urgently needed to improve the prognosis and quality of life (QOL) of patients. Oral leukoplakias (OLs), known as the most common premalignant lesions in the oral cavity, often precede OSCCs. Especially, OLs with dysplasia are known to have a high risk of malignant transformation. Here, we searched for the promoter methylation characteristic of high-risk OLs. METHODS: To identify methylation-silenced genes, a combined analysis of methylated DNA immunoprecipitation (MeDIP) - CpG island (CGI) microarray analysis and expression microarray analysis after treatment with a demethylating agent was performed in two OSCC cell lines (Ca9-22 and HSC-2). The methylation statuses of each gene were examined by methylation-specific PCR. RESULTS: A total of 52 genes were identified as candidates for methylation-silenced genes in Ca9-22 or HSC-2. The promoter regions of 13 genes among the 15 genes randomly selected for further analysis were confirmed to be methylated in one or more of five cell lines. In OSCC tissues (n = 26), 8 of the 13 genes, TSPYL5, EGFLAM, CLDN11, NKX2-3, RBP4, CMTM3, TRPC4, and MAP6, were methylated. In OL tissues (n = 24), seven of the eight genes, except for EGFLAM, were found to be methylated in their promoter regions. There were significantly greater numbers of methylated genes in OLs with dysplasia than in those without dysplasia (p < 0.0001). CONCLUSIONS: OLs at high risk for malignant transformation were associated with aberrant promoter methylation of multiple genes.

Computer-assisted preoperative simulation for positioning of plate fixation in Lefort I osteotomy: A case report.

Computed tomography images are used for three-dimensional planning in orthognathic surgery. This facilitates the actual surgery by simulating the surgical scenario. We performed a computer-assisted virtual orthognathic surgical procedure using optically scanned three-dimensional (3D) data and real computed tomography data on a personal computer. It helped maxillary bone movement and positioning and the titanium plate temporary fixation and positioning. This simulated the surgical procedure, which made the procedure easy, and we could perform precise actual surgery and could forecast the postsurgery outcome. This simulation method promises great potential in orthognathic surgery to help surgeons plan and perform operative procedures more precisely.

A 2-Stage Reconstruction of the Jaw Using Vascularized Bone and Secondary Alveolar Ridge Augmentation With Particulate Cancellous Bone and Marrow.

PURPOSE: To present the novel technique for reconstruction of the jaw, that facilitates occlusal restoration using dental implants, in cases with wide bony defects from tumor resection. MATERIALS AND METHODS: After alveolar ridge is augmented using titanium mesh tray and particulate cancellous bone and marrow (PCBM) from iliac bone on reconstructed bone, by way of improvement of maxillomandibular relationship for dental implants. RESULTS: This 2-stage surgery underwent successfully in 3 cases. After 2-stage surgery and occlusal reconstruction using dental implant, the patients experienced no complications, and received satisfaction with results functionally and aesthetically. CONCLUSIONS: Our results suggest that, in cases where bone defect is over a wide area, in addition to vascularized bone grafts, secondary alveolar ridge augmentation using a titanium mesh tray and PCBM on grafted bone can provide satisfactory occlusion further to improvement of facial form.

Macrophage-inducing FasL on chondrocytes forms immune privilege in cartilage tissue engineering, enhancing in vivo regeneration.

To obtain stable outcomes in regenerative medicine, controlling inflammatory reactions is a requirement. Previously, auricular chondrocytes in tissue-engineered cartilage have been shown to express factors related to immune privilege including Fas ligand (FasL) in mice. Since elucidation of mechanism on immune privilege formed in cartilage regeneration may contribute to suppression of excessive inflammation, in this study, we investigated the function of FasL and induction of immune privilege in tissue-engineered cartilage using a mouse subcutaneous model. When cocultured, auricular chondrocytes of FasL-dysfunctional mice, C57BL/6JSlc-gld/gld (gld), induced less cell death and apoptosis of macrophage-like cells, RAW264, compared with chondrocytes of C57BL/6 mice (wild), suggesting that FasL on chondrocytes could induce the apoptosis of macrophages. Meanwhile, the viability of chondrocytes was hardly affected by cocultured RAW264, although the expression of type II collagen was decreased, indicating that macrophages could hamper the maturation of chondrocytes. Tissue-engineered cartilage containing gld chondrocytes exhibited greater infiltration of macrophages, with less accumulation of proteoglycan than did wild constructs. Analysis of the coculture medium identified G-CSF as an inducer of FasL on chondrocytes, and G-CSF-treated tissue-engineered cartilage showed less infiltration of macrophages, with increased formation of cartilage after transplantation. The interactions between chondrocytes and macrophages may increase G-CSF secretion in macrophages and induce FasL on chondrocytes, which in turn induce the apoptosis of macrophages and suppress tissue reactions, promoting the maturation of tissue-engineered cartilage. These findings provide scientific insight into the mechanism of autologous chondrocyte transplantation, which could be applied as a novel strategy for cartilage tissue engineering.

Vision-based markerless registration using stereo vision and an augmented reality surgical navigation system: a pilot study.

BACKGROUND: This study evaluated the use of an augmented reality navigation system that provides a markerless registration system using stereo vision in oral and maxillofacial surgery. METHOD: A feasibility study was performed on a subject, wherein a stereo camera was used for tracking and markerless registration. The computed tomography data obtained from the volunteer was used to create an integral videography image and a 3-dimensional rapid prototype model of the jaw. The overlay of the subject's anatomic site and its 3D-IV image were displayed in real space using a 3D-AR display. Extraction of characteristic points and teeth matching were done using parallax images from two stereo cameras for patient-image registration. RESULTS: Accurate registration of the volunteer's anatomy with IV stereoscopic images via image matching was done using the fully automated markerless system, which recognized the incisal edges of the teeth and captured information pertaining to their position with an average target registration error of < 1 mm. These 3D-CT images were then displayed in real space with high accuracy using AR. Even when the viewing position was changed, the 3D images could be observed as if they were floating in real space without using special glasses. CONCLUSION: Teeth were successfully used for registration via 3D image (contour) matching. This system, without using references or fiducial markers, displayed 3D-CT images in real space with high accuracy. The system provided real-time markerless registration and 3D image matching via stereo vision, which, combined with AR, could have significant clinical applications.

Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids.

Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, ß-tricalcium phosphate (ß-TCP) granules, or ß-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60% those of native bones, respectively). All tests showed poor bone regeneration in the ß-TCP and ß-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

Short Lingual Osteotomy Using a Piezosurgery Ultrasonic Bone-Cutting Device During Sagittal Split Ramus Osteotomy.

Short lingual osteotomy is a useful method for the performance of sagittal split ramus osteotomy involving interference between the proximal and distal bone fragments when lateral differences exist in the setback distance. However, this procedure occasionally results in abnormal fracture and nerve injury; expert surgical skill is thus required. We herein describe a novel technique involving the use of an ultrasonic bone-cutting device (Piezosurgery; Mectron Medical Technology, Carasco, Italy) for vertical osteotomy posterior to the mandibular foramen. Successful short lingual osteotomy was performed using this technique with avoidance of abnormal fracture and neurovascular bundle damage.

Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1(-/-) fetuses, and the phenotype was more severe in Gli1(-/-);Gli2(-/-) newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.

An in vivo murine model for screening cranial bone regenerative materials: testing of a novel synthetic collagen gel.

Rapid and efficient animal models are needed for evaluating the effectiveness of many new candidate bone regenerative materials. We developed an in vivo model screening for calvarial bone regeneration in lipopolysaccharide (LPS)-treated mice, in which materials were overlaid on the periosteum of the calvaria in a 20 min surgery and results were detectable in 1 week. Intraperitoneal LPS injection reduced spontaneous bone formation, and local application of basic fibroblast growth factor (bFGF) increased the bone-forming activities of osteoblasts. A novel synthetic collagen gel, alkali-treated collagen (AlCol) cross-linked with trisuccinimidyl citrate (TSC), acted as a reservoir for basic substances such as bFGF. The AlCol-TSC gel in conjunction with bFGF activated osteoblast activity without the delay in osteoid maturation caused by bFGF administration alone. The AlCol-TSC gel may slow the release of bFGF to improve the imbalance between osteoid formation and bone mineralization. These findings suggest that our model is suitable for screening bone regenerative materials and that the AlCOl-TSC gel functions as a candidate reservoir for the slow release of bFGF.

Correction of upturned nasal tip with a costal cartilage graft in bilateral cleft lip patients.

We used a piece of costal cartilage as a cartilaginous strut to correct the upturned nasal tip in patients with bilateral cleft lip. The grafted cartilage provides more definition of the tip and improves the obtuse nasolabial angle. Neither the septal cartilage nor the ear cartilage has enough strength to shape the tip. This method of correction has consistently produced favorable, long-lasting results in adults and has improved the contour of the nasal tip in younger patients.

Maxillary protraction in patients with cleft lip and palate in mixed dentition: cephalometric evaluation after completion of growth.

Objective : To clarify the short- and long-term effects of maxillary protraction (MP) in mixed dentition in patients with unilateral cleft lip and palate (UCLP). Design : Retrospective study. Setting : University of Tokyo Hospital. Patients and Intervention : Eleven Japanese patients with UCLP in mixed dentition were treated with MP and followed up until the completion of growth. Multibracket treatment had been performed after MP treatment in all patients. Main Outcome Measure : Lateral cephalograms taken before and after MP and after completion of growth were used. Posterior and anterior vertical reference lines (PV, AV) were used to measure the horizontal movements of point A, pogonion, and maxillary first molar (U6). SNA, SNB, ANB, maxillary and mandibular length, mandibular plane angle, Wits value, upper incisor inclination, overjet, and overbite were also measured. Results : Large variation was found in the effects of MP, and five patients eventually required orthognathic surgery. In average change with MP, the maxilla showed favorable forward growth. Point A had moved forward from PV but not AV. The mandible rotated backward. However, ANB and the Wits value did not improve. U6 moved forward, and the overjet improved. After MP, the skeletal Class III relationship became severe. Conclusions : MP was effective as an early treatment for UCLP patients. However, its effects showed large variation and were in conflict with facial growth. Conscientious explanation of the expected effects and associated problems should be given to the patients/parents before its application.

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-ß1 (TGF-ß1) administration suppresses osteoblast differentiation.

TGF-ß1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-ß1, although single low dose TGF-ß1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-ß1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-ß1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-ß1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-ß1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-ß1 administration. This study showed that persistence of TGF-ß1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.

Gli1 protein participates in Hedgehog-mediated specification of osteoblast lineage during endochondral ossification.

With regard to Hedgehog signaling in mammalian development, the majority of research has focused on Gli2 and Gli3 rather than Gli1. This is because Gli1(-/-) mice do not show any gross abnormalities in adulthood, and no detailed analyses of fetal Gli1(-/-) mice are available. In this study, we investigated the physiological role of Gli1 in osteogenesis. Histological analyses revealed that bone formation was impaired in Gli1(-/-) fetuses compared with WT fetuses. Gli1(-/-) perichondrial cells expressed neither runt-related transcription factor 2 (Runx2) nor osterix, master regulators of osteogenesis, in contrast to WT cells. In vitro analyses showed that overexpression of Gli1 up-regulated early osteogenesis-related genes in both WT and Runx2(-/-) perichondrial cells, and Gli1 activated transcription of those genes via its association with their 5'-regulatory regions, underlying the function of Gli1 in the perichondrium. Moreover, Gli1(-/-);Gli2(-/-) mice showed more severe phenotypes of impaired bone formation than either Gli1(-/-) or Gli2(-/-) mice, and osteoblast differentiation was impaired in Gli1(-/-);Gli3(-/-) perichondrial cells compared with Gli3(-/-) cells in vitro. These data suggest that Gli1 itself can induce early osteoblast differentiation, at least to some extent, in a Runx2-independent manner. It also plays a redundant role with Gli2 and is involved in the repressor function of Gli3 in osteogenesis. On the basis of these findings, we propose that upon Hedgehog input, Gli1 functions collectively with Gli2 and Gli3 in osteogenesis.