Ivermectin represses Wnt/ß-catenin signaling by binding to TELO2, a regulator of phosphatidylinositol 3-kinase-related kinases.

Ivermectin (IVM), an avermectin-derivative anthelmintic, specifically binds to glutamate-gated chloride ion channels (GluCls), causing paralysis in invertebrates. IVM also exhibits other biological activities such as Wnt/ß-catenin pathway inhibition in vertebrates that do not possess GluCls. This study showed that affinity purification using immobilized IVM B1a isolated TELO2, a cofactor of phosphatidylinositol 3-kinase-related kinases (PIKKs), as a specific IVM B1a-binding protein. TELO2 knockdown reduced cytoplasmic ß-catenin and the transcriptional activation of ß-catenin/TCF. IVM B1a bound to TELO2 through the C-terminal α-helix, in which mutations conferred IVM resistance. IVM reduced the TELO2 and PIKK protein levels and the AKT and S6 kinase phosphorylation levels. The inhibition of mTOR kinase reduced the cytoplasmic ß-catenin level. Therefore, IVM binds to TELO2, inhibiting PIKKs and reducing the cytoplasmic ß-catenin level. In conclusion, our data indicate TELO2 as a druggable target for human diseases involving abnormalities of the Wnt/ß-catenin pathway and PIKKs, including mTOR.

Orthodontic correction of severe Class II malocclusion in a patient with Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a complex disorder that affects multiple systems and may cause craniofacial and dentofacial abnormalities. However, there is still a lack of evidence in the literature regarding the progress of orthodontic treatment in patients with PWS. This case report describes the successful orthodontic treatment of a patient with PWS. A girl, 9 years 0 months of age, who had been diagnosed with PWS had protruding maxillary incisors and a convex profile. Her malocclusion was due to the posteriorly positioned mandible. Screening tests for sleep apnea syndrome showed that she had sleep-disordered breathing, including obstructive sleep apnea and bruxism. We also observed an excessive overjet of 10.0 mm, a deep overbite of 6.8 mm, and the congenital absence of the mandibular second premolars. The patient was diagnosed with an Angle Class II malocclusion and a skeletal Class II jaw-base relationship with a deep overbite. Functional appliance therapy with mandibular advancement, which can enlarge the upper airway and increase the upper airspace, was performed to prevent further deterioration of the patient's obstructive sleep apnea. An acceptable occlusion with a proper facial profile and functional excursion were achieved without interference after comprehensive 2-stage treatment that incorporated orthodontic therapy for the patient's excessive overjet and deep overbite. The resulting occlusion was stable, and the occlusal force and the contact area gradually increased over a 2-year retention period. These results suggest that orthodontic treatment offers the opportunity to greatly improve the health and quality of life of people with PWS.

Augmentation of the therapeutic efficacy of WEE1 kinase inhibitor AZD1775 by inhibiting the YAP-E2F1-DNA damage response pathway axis.

The main reasons for failure of cancer chemotherapy are intrinsic and acquired drug resistance. The Hippo pathway effector Yes-associated protein (YAP) is associated with resistance to both cytotoxic and molecular targeted drugs. Several lines of evidence indicate that YAP activates transcriptional programmes to promote cell cycle progression and DNA damage responses. Therefore, we hypothesised that YAP is involved in the sensitivity of cancer cells to small-molecule agents targeting cell cycle-related proteins. Here, we report that the inactivation of YAP sensitises the OVCAR-8 ovarian cancer cell line to AZD1775, a small-molecule WEE1 kinase inhibitor. The accumulation of DNA damage and mitotic failures induced by AZD1775-based therapy were further enhanced by YAP depletion. YAP depletion reduced the expression of the Fanconi anaemia (FA) pathway components required for DNA repair and their transcriptional regulator E2F1. These results suggest that YAP activates the DNA damage response pathway, exemplified by the FA pathway and E2F1. Furthermore, we aimed to apply this finding to combination chemotherapy against ovarian cancers. The regimen containing dasatinib, which inhibits the nuclear localisation of YAP, improved the response to AZD1775-based therapy in the OVCAR-8 ovarian cancer cell line. We propose that dasatinib acts as a chemosensitiser for a subset of molecular targeted drugs, including AZD1775, by targeting YAP.

[Indirect usage of miniscrew anchorage to intrude overerupted mandibular incisors in a Class II patient with a deep overbite]. / Intérêts de l'utilisation des mini-vis orthodontiques en tant qu'ancrage indirect dans la correction de l'égression d'incisives mandibulaires pour un patient présentant une classe II hypodivergente∗.

Vertical dentoalveolar discrepancies are a common problem in orthodontic patients but are often difficult to treat with traditional mechanics. This case report illustrates the successful treatment of overerupted mandibular incisors via the indirect use of miniscrew anchorage. A woman (age, 22 years 9 months) had chief complaints of maxillary incisor protrusion and crooked teeth. An excessive curve of Spee caused by elongation of the mandibular incisors was also found. The patient was diagnosed with a severe Class II Division 1 malocclusion and a deep overbite. After extraction of the mandibular first premolars and the subsequent leveling phase, the elongated incisors were intruded with a novel method, which involved the combined use of sectional archwires and miniscrews placed in the premolar areas. After the procedure, the mandibular incisors had been intruded by 6.5 mm with no undesirable side effects. The total active treatment period was 42 months. The resultant occlusion and satisfactory facial profile were maintained after 30 months of retention. Our novel intrusion approach shows potential for correcting a deep overbite.

Orthodontic treatment of a patient with unilateral orofacial muscle dysfunction: The efficacy of myofunctional therapy on the treatment outcome.

The orofacial muscle is an important factor in the harmony of the occlusion, and its dysfunction significantly influences a patient's occlusion after craniofacial growth and development. In this case report, we describe the successful orthodontic treatment of a patient with unilateral orofacial muscle dysfunction. A boy, 10 years 0 months of age, with a chief complaint of anterior open bite, was diagnosed with a Class III malocclusion with facial musculoskeletal asymmetry. His maxillomandibular relationships were unstable, and he was unable to lift the right corner of his mouth upon smiling because of weak right orofacial muscles. A satisfactory occlusion and a balanced smile were achieved after orthodontic treatment combined with orofacial myofunctional therapy, including muscle exercises. An acceptable occlusion and facial proportion were maintained after a 2-year retention period. These results suggest that orthodontic treatment with orofacial myofunctional therapy is an effective option for a patient with orofacial muscle dysfunction.

Outcome assessment of lingual and labial appliances compared with cephalometric analysis, peer assessment rating, and objective grading system in Angle Class II extraction cases.

OBJECTIVE: To validate our hypothesis that there would be significant differences in treatment outcomes, including cephalometric values, degree of root resorption, occlusal indices, and functional aspect, between cases treated with labial and lingual appliances. MATERIALS AND METHODS: Twenty-four consecutively treated Class II cases with extractions and lingual appliance were compared with 25 matched cases treated with extraction and labial appliance. Orthodontic treatment outcomes were evaluated by cephalometric analysis, peer assessment rating, and an objective grading system (OGS). Additionally, functional analysis was also performed in both groups after orthodontic treatment. Statistical comparison was performed using the Wilcoxon signed rank test within the groups, and the Mann-Whitney U-test was used to compare between the labial and lingual groups. RESULTS: The only significant difference between the groups was that the interincisal angle was larger in the lingual group than in the labial group. OGS evaluation showed that control over root angulation was significantly worse in the lingual group than in the labial group. There was no significant difference between groups in the amount of root resorption or in functional evaluation. CONCLUSIONS: Generally, lingual appliances offer comparable treatment results to those obtained with labial appliances. However, care should be taken with lingual appliances because they are more prone to produce uprighted incisors and root angulation.

Long-term stability of implant-anchored orthodontics in an adult patient with a Class II Division 2 malocclusion and a unilateral molar scissors-bite.

This article reports the successful treatment using miniscrew anchorage of an adult patient with a severe deep overbite and a unilateral scissors-bite. A 23-year-old woman had chief complaints of maxillary incisal crowding and difficulty chewing. She was diagnosed with a severe Class II Division 2 malocclusion with anterior crowding and a unilateral scissors-bite caused by buccal elongation of the maxillary left second molar. The maxillary first premolars were extracted, and 3 miniscrews were implanted as skeletal anchorage to resolve the functional and esthetic problems. The total active treatment period was 41 months. As a result of the implant-anchored orthodontic treatment, both the patient's facial profile and occlusion significantly improved. The asymmetric movements of the incisor paths and bilateral condyles during lateral excursions disappeared. The satisfactory facial profile and resultant occlusion were maintained throughout a 49-month retention period. The patient was satisfied with the treatment results.

Indirect usage of miniscrew anchorage to intrude overerupted mandibular incisors in a Class II patient with a deep overbite.

Vertical dentoalveolar discrepancies are a common problem in orthodontic patients but are often difficult to treat with traditional mechanics. This case report illustrates the successful treatment of overerupted mandibular incisors via the indirect use of miniscrew anchorage. A woman (age, 22 years 9 months) had chief complaints of maxillary incisor protrusion and crooked teeth. An excessive curve of Spee caused by elongation of the mandibular incisors was also found. The patient was diagnosed with a severe Class II Division 1 malocclusion and a deep overbite. After extraction of the mandibular first premolars and the subsequent leveling phase, the elongated incisors were intruded with a novel method, which involved the combined use of sectional archwires and miniscrews placed in the premolar areas. After the procedure, the mandibular incisors had been intruded by 6.5 mm with no undesirable side effects. The total active treatment period was 42 months. The resultant occlusion and satisfactory facial profile were maintained after 30 months of retention. Our novel intrusion approach shows potential for correcting a deep overbite.

The early mouse 3D osteocyte network in the presence and absence of mechanical loading.

Osteocytes are considered to act as mechanosensory cells in bone. They form a functional synctia in which their processes become interconnected to constitute a three-dimensional (3D) network. Previous studies reported that in mice, the two-dimensional osteocyte network becomes progressively more regular as they grow, although the key factors governing the arrangement of the osteocyte network during bone growth remain unknown. In this study, we characterized the 3D formation of the osteocyte network during bone growth. Morphological skeletal changes have been reported to occur in response to mechanical loading and unloading. In order to evaluate the effect of mechanical unloading on osteocyte network formation, we subjected newborn mice to sciatic neurectomy in order to immobilize their left hind limb as an unloading model. The osteocyte network was visualized by staining osteocyte cell bodies and processes with fluorescently labeled phalloidin. First, we compared the osteocyte network in the femora of embryonic and 6-week-old mice in order to understand the morphological changes that occur with normal growth and mechanical loading. In embryonic mice, the osteocyte network in the femur cortical bone displayed a random cell body distribution, non-directional orientation of cell processes, and irregularly shaped cells. In 6-week-old mice, the 3D network contained spindle-shaped osteocytes, which were arranged parallel to the longitudinal axis of the femur. In addition, more and longer cell processes radiated from each osteocyte. Second, we compared the cortical osteocyte networks of 6-week-old mice that had or had not undergone sciatic neurectomy in order to evaluate the effect of unloading on osteocyte network formation. The osteocyte network formation in both cortical bone and cancellous bone was affected by mechanical loading. However, there were differences in the extent of network formation between cortical bone and cancellous bone in response to mechanical loading with regard to the orientation, nuclear shape and branch formation.

Ex vivo real-time observation of Ca(2+) signaling in living bone in response to shear stress applied on the bone surface.

Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca(2+)](i)) mobilization. Our recently developed ex vivo live [Ca(2+)](i) imaging system revealed that bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca(2+)](i) signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca(2+)](i) changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 α-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca(2+)](i) responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd(3+), is less affected in the flow-induced [Ca(2+)](i) response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca(2+)](i) oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca(2+)](i) mobilization could be involved as an early signaling event in mechanotransduction.

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress.

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.

In situ imaging of the autonomous intracellular Ca(2+) oscillations of osteoblasts and osteocytes in bone.

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca(2+) acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca(2+) dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca(2+) imaging system that allows the dynamic intracellular Ca(2+) concentration ([Ca(2+)](i)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca(2+)](i) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca(2+) stores, abolished these [Ca(2+)](i) responses in both osteoblasts and osteocytes, indicating that Ca(2+) release from intracellular stores plays a key role in the [Ca(2+)](i) oscillations of these bone cells in intact bone explants. Another possible [Ca(2+)](i) transient system to be considered is gap junctional communication through which Ca(2+) and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca(2+)](i) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca(2+)](i) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations that required the release of intracellular Ca(2+) stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca(2+)](i) oscillations of bone cells.

The three-dimensional morphometry and cell-cell communication of the osteocyte network in chick and mouse embryonic calvaria.

The study of osteocytes has progressed in chicks. We examined whether chick osteocyte data can be applied to other species. We used mice for comparison because they are common clinical tools in biomedical research and useful for future study. We analyzed the three-dimensional (3D) osteocyte network and gap junctional intercellular communication (GJIC) in living embryonic calvaria for the anatomical features. Embryonic parietal bones were stained with fluorescently labeled phalloidin and observed using confocal laser scanning microscopy. GJIC between osteocytes in chick and mouse parietal bone was assessed using fluorescence recovery after photobleaching (FRAP). The values for one chick and mouse osteocyte, respectively, were calculated as follows: cell processes 1,131 ± 139 µm, 2,668 ± 596 µm; surface area 1,128 ± 358 µm(2), 2,654 ± 659 µm(2); and cell volume 455 ± 90 µm(3), 1,328 ± 210 µm(3). The density of 3D osteocyte processes in the bone matrix was not significantly different. FRAP analysis showed dye coupling among osteocytes in chick and mouse bone. The fluorescence intensity recovered to 49.0 ± 2.4% in chicks and 39.9 ± 2.4% in mice after 5 minutes. Fluorescence recovery was similar within 4 minutes. The difference in osteocyte size between the two species might have affected their functions. Osteocyte processes in the two species may sense similarly changes in the exterior environment. We successfully conducted morphological and functional analyses of the osteocyte network in chicks and mice. The size of the osteocytes in bone differed between the two species.

A method for observing silver-stained osteocytes in situ in 3-microm sections using ultra-high voltage electron microscopy tomography.

Osteocytes are surrounded by hard bone matrix, and it has not been possible previously to directly observe the in situ architecture of osteocyte morphology in bone. Electron microscope tomography, however, is a technique that has the unique potential to provide three-dimensional (3D) visualization of cellular ultrastructure. This approach is based on reconstruction of 3D volumes from a tilt series of electron micrographs of cells, and resolution at the nanometer level has been achieved. We applied electron microscope tomography to thick sections of silver-stained osteocytes in bone using a Hitachi H-3000 ultra-high voltage electron microscope equipped with a 360 degrees tilt specimen holder, at an accelerating voltage of 2 MeV. Osteocytes with numerous processes and branches were clearly seen in the serial tilt series acquired from 3-microm-thick sections. Reconstruction of young osteocytes showed the 3D topographic morphology of the cell body and processes at high resolution. This morphological data on osteocytes should provide useful information to those who study osteocyte physiology and the several models used to explain their mechanosensory properties.

Use of palatal miniscrew anchorage and lingual multi-bracket appliances to enhance efficiency of molar scissors-bite correction.

This article reports the successful treatment method of scissors-bite correction using miniscrew anchorage and a lingual multi-bracket appliance. A female patient, 17 years and 4 months old, had a chief complaint of crowding of anterior teeth. The patient was given the diagnosis of Angle Class I malocclusion with bimaxillary protrusion and incisor crowding. She also showed a scissors-bite of the second molar on the right side. Miniscrews were inserted into the palatal region of the upper second molar to reinforce the anchorage, and a lingual multi-bracket appliance was placed into the maxilla. Miniscrews inserted palatally were used to correct the scissors-bite in the first 3 months; afterward, they were used to retract the six anterior teeth. The total active treatment period was 26 months. Because of the bite-plane effect, the upper and lower molars were separated in occlusion, and the scissors-bite was corrected effectively within a short time. The combined use of palatal miniscrew anchorage and lingual multi-bracket appliances enhances efficiency of molar scissors-bite correction.

The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

Adult patient with mandibular protrusion and unstable occlusion treated with titanium screw anchorage.

Orthodontic treatment for adults can be difficult because they often have several problems, including some associated with aging. This article reports the successful use of miniscrews to treat a 51-year-old woman with Class III malocclusion, unstable occlusion, periodontal disease, and temporomandibular disorder. The patient had a skeletal Class III jaw-base relationship, anterior and unilateral posterior crossbites, and a deviated midline. It was therefore necessary to confirm her mandibular position with a stabilization splint. Miniscrews were implanted in the retromolar area and used to move the mandibular teeth en masse distally. After orthodontic treatment, the mandibular molars and anterior teeth were moved distally, and the patient had a stable occlusion. Acceptable occlusion and periodontal health were maintained after 1 year of retention. Our results suggest that miniscrews are useful for en-masse distal movement of mandibular teeth in aging patients.

Clinical evaluation of a low-friction attachment device during canine retraction.

OBJECTIVE: The present study used a split-mouth design to compare the amount of canine movement and the retraction time between brackets with Clear Snap and brackets with stainless steel ligature wires for three different levels of retraction force. MATERIALS AND METHODS: A sample of 30 patients was used. After initial leveling, the canine was retracted using a 50-g (n = 10), 100-g (n = 10), or 150-g (n = 10) closed-coil spring. The canine on one side was chosen at random, and Clear Snap was attached to the bracket during the retraction period. The other side was used as a control. The amount of canine retraction was measured with a digital vernier caliper. Statistical analysis was performed by analysis of variance. RESULTS: The average canine retraction time was approximately 2 to 3 months less in all experimental groups (50, 100, and 150 g) compared to the control group. In the control group, 150 g resulted in a shorter duration of canine retraction compared to 50 g. There was no significant difference in the duration of canine retraction among the experimental groups. A greater amount of mean total canine movement was observed in all experimental groups compared to the control groups. CONCLUSION: A shorter duration of canine retraction time was observed with Clear Snap attached when compared with the control. The authors suggest that with the use of Clear Snap, less than 50 g of force may effectively retract a canine.

Anterior open bite with temporomandibular disorder treated with titanium screw anchorage: evaluation of morphological and functional improvement.

Skeletal anterior open bite is one of the most challenging malocclusions to correct because it is difficult to establish absolute anchorage for molar intrusion with traditional orthodontic mechanics. In addition, patients with anterior open bite sometimes have signs and symptoms of temporomandibular disorders (TMD). In this article, we report the successful treatment of a patient with severe skeletal anterior open bite and TMD; we used titanium screw anchorage. The patient, a woman, age 19 years 11 months, had an open bite of -4.0 mm and increased anterior lower facial height. The titanium screws were implanted in the mandible, and intrusion force was provided with elastic chains for 6 months. After active treatment for 36 months, her mandibular first molars were intruded about 3.0 mm, and good occlusion was achieved. Her retrognathic chin and convex profile were improved both by upward rotation of the mandible and advancement genioplasty with vertical reduction. After treatment, the TMD signs and symptoms were reduced, and improvements of both function and occlusion were achieved. Molar intrusion with titanium screw anchorage might be a useful treatment option to improve function, occlusion, and facial esthetics in patients with severe anterior open bite and TMD.

Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes.

Cell shape is the most critical determinant of cell function and is potentially influenced by the organization of a cell's cytoskeletal components. It has been reported that three-dimensionally cultured osteoblasts have a morphology that closely resembles that of osteocytes, most notably including formation of processes. We have previously shown the critical differences between cytoskeletal components in osteoblasts and osteocytes in two-dimensional culture. We have now extended that investigation to the cytoskeletal components of 3D-cultured osteoblasts and osteocytes using 3D cultures of the osteoblast cell line, MC3T3-E1, and primary osteocytes grown in collagen gel. Three-dimensional fluorescent image reconstructions for actin, fimbrin, alpha-actinin, myosin, tropomyosin, and microtubules were made using IMARIS software. Actin, fimbrin, alpha-actinin, myosin, and tropomyosin all appeared in the processes of both cell types, but fimbrin and myosin showed differences in their distribution patterns between cell types. Microtubules were limited in distribution to the proximal region of osteocyte processes but extended the entire length of MC3T3-E1 cell processes. Microtubules were essential for the integrity and formation of MC3T3-E1 cell processes, but osteocyte processes were dependent on actin. These results showed that there are significant differences between the actin and microtubule cytoskeletons in the processes of 3D-cultured MC3T3-E1 cells and in the processes of 3D-cultured primary osteocytes. These differences in the cytoskeleton of the processes of 3D-cultured osteoblasts and of osteocyte dendrites suggest that osteoblast processes may have a different functional role than the osteocyte dendritic network.