Axin2-expressing cells in the periodontal ligament are regulated by bone morphogenetic protein signalling and play a pivotal role in periodontium development.

AIM: To date, controversies still exist regarding the exact cellular origin and regulatory mechanisms of periodontium development, which hinders efforts to achieve ideal periodontal tissue regeneration. Axin2-expressing cells in the periodontal ligament (PDL) have been shown to be a novel progenitor cell population that is essential for periodontal homeostasis. In the current study, we aimed to elucidate the regulatory role of bone morphogenetic protein receptor type 1A (BMPR1A)-mediated BMP signalling in Axin2-expressing cells during periodontium development. MATERIALS AND METHODS: Two strains of Axin2 gene reporter mice, Axin2lacZ/+ and Axin2CreERT2/+ ; R26RtdTomato/+ mice, were used. We next generated Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice to genetically ablate of Axin2-lineage cells. Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ mice were established to conditionally knock out Bmpr1a in Axin2-lineage cells. Multiple approaches, including micro-computed tomography, calcein green, and alizarin red double-labelling, scanning electron microscopy, and histological and immunostaining assays, were used to analyse periodontal phenotypes and molecular mechanisms. RESULTS: X-gal staining revealed that Axin2-expressing cells in the PDL were mainly distributed along the alveolar bone and cementum surface. Cell lineage tracing and cell ablation assays further demonstrated the indispensable role of Axin2-expressing cells in periodontium development. Next, we found that conditional knockout of Bmpr1a in Axin2-lineage cells led to periodontal defects, which were characterized by alveolar bone loss, impaired cementogenesis, and abnormal Sharpey's fibres. CONCLUSIONS: Our findings suggest that Axin2-expressing cells in the PDL are essential for periodontium development, which is regulated by BMP signalling.

The identification of critical time windows of postnatal root elongation in response to Wnt/ß-catenin signaling.

OBJECTIVES: In this study, we attempted to define the precise window of time for molar root elongation using a gain-of-function mutation of ß-catenin model. MATERIALS AND METHODS: Both the control and constitutively activated ß-catenin (CA-ß-cat) mice received a one-time tamoxifen administration (for activation of ß-catenin at newborn, postnatal day 3, or 5, or 7, or 9) and were harvested at the same stage of P21. Multiple approaches were used to define the window of time of postnatal tooth root formation. RESULTS: In the early activation groups (tamoxifen induction at newborn, or P3 or P5), there was a lack of molar root elongation in the CA-ß-cat mice. When induced at P7, the root length was slightly reduced at P21. However, the root length was essentially the same as that in the control when ß-cat activated at P9. This study indicates that root elongation occurs in a narrow time of window, which is highly sensitive to a change of ß-catenin levels. Molecular studies showed a drastic decrease in the levels of nuclear factor I-C (NFIC) and osterix (OSX), plus sharp reductions of odontoblast differentiation markers, including Nestin, dentin sialoprotein (DSP), and dentin matrix protein 1 (DMP1) at both mRNA and protein levels. CONCLUSIONS: Murine molar root elongation is precisely regulated by the Wnt/ß-catenin signaling within a narrow window of time (newborn to day 5).

The Emerging Role of Cell Transdifferentiation in Skeletal Development and Diseases.

The vertebrate musculoskeletal system is known to be formed by mesenchymal stem cells condensing into tissue elements, which then differentiate into cartilage, bone, tendon/ligament, and muscle cells. These lineage-committed cells mature into end-stage differentiated cells, like hypertrophic chondrocytes and osteocytes, which are expected to expire and to be replaced by newly differentiated cells arising from the same lineage pathway. However, there is emerging evidence of the role of cell transdifferentiation in bone development and disease. Although the concept of cell transdifferentiation is not new, a breakthrough in cell lineage tracing allowed scientists to trace cell fates in vivo. Using this powerful tool, new theories have been established: (1) hypertrophic chondrocytes can transdifferentiate into bone cells during endochondral bone formation, fracture repair, and some bone diseases, and (2) tendon cells, beyond their conventional role in joint movement, directly participate in normal bone and cartilage formation, and ectopic ossification. The goal of this review is to obtain a better understanding of the key roles of cell transdifferentiation in skeletal development and diseases. We will first review the transdifferentiation of chondrocytes to bone cells during endochondral bone formation. Specifically, we will include the history of the debate on the fate of chondrocytes during bone formation, the key findings obtained in recent years on the critical factors and molecules that regulate this cell fate change, and the role of chondrocyte transdifferentiation in skeletal trauma and diseases. In addition, we will also summarize the latest discoveries on the novel roles of tendon cells and adipocytes on skeletal formation and diseases.

The vital role of Gli1+ mesenchymal stem cells in tissue development and homeostasis.

The hedgehog (Hh) signaling pathway plays an essential role in both tissue development and homeostasis. Glioma-associated oncogene homolog 1 (Gli1) is one of the vital transcriptional factors as well as the direct target gene in the Hh signaling pathway. The cells expressing the Gli1 gene (Gli1+ cells) have been identified as mesenchymal stem cells (MSCs) that are responsible for various tissue developments, homeostasis, and injury repair. This review outlines some recent discoveries on the crucial roles of Gli1+ MSCs in the development and homeostasis of varieties of hard and soft tissues.

FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.

BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.

Abnormal lacuno-canalicular network and negative correlation between serum osteocalcin and Cobb angle indicate abnormal osteocyte function in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a prevalent spinal deformity occurring during peripubertal growth period that affects 1-4% of adolescents globally without clear etiopathogenetic mechanism. Low bone mineral density is an independent and significant prognostic factor for curve progression. Currently, the cause underlying low bone mass in AIS remains elusive. Osteocytes play an important role in bone metabolism and mineral homeostasis, but its role in AIS has not been studied. In the present study, iliac bone tissues were harvested from 21 patients with AIS (mean age of 14.3 ± 2.20 yr old) with a mean Cobb angle of 55.6 ± 10.61° and 13 non-AIS controls (mean age of 16.5 ± 4.79 yr old) intraoperatively. Acid-etched scanning electron microscopy (SEM) images of AIS demonstrated abnormal osteocytes that were more rounded and cobblestone-like in shape and were aligned in irregular clusters with shorter and disorganized canaliculi. Further quantitative analysis with FITC-Imaris technique showed a significant reduction in the canalicular number and length as well as an increase in lacunar volume and area in AIS. SEM with energy-dispersive X-ray spectroscopy analysis demonstrated a lower calcium-to-phosphorus ratio at the perilacunar/canalicular region. Moreover, microindentaion results revealed lower values of Vickers hardness and elastic modulus in AIS when compared with controls. In addition, in the parallel study of 99 AIS (27 with severe Cobb angle of 65.8 ± 14.1° and 72 with mild Cobb angle of 26.6 ± 9.1°) with different curve severity, the serum osteocalcin level was found to be significantly and negatively associated with the Cobb angle. In summary, the findings in this series of studies demonstrated the potential link of abnormal osteocyte lacuno-canalicular network structure and function to the observed abnormal bone mineralization in AIS, which may shed light on etiopathogenesis of AIS.-Chen, H., Zhang, J., Wang, Y., Cheuk, K.-Y., Hung, A. L. H., Lam, T.-P., Qiu, Y., Feng, J. Q., Lee, W. Y. W., Cheng, J. C. Y. Abnormal lacuno-canalicular network and negative correlation between serum osteocalcin and Cobb angle indicate abnormal osteocyte function in adolescent idiopathic scoliosis.

Blockade to pathological remodeling of infarcted heart tissue using a porcupine antagonist.

The secreted Wnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate the Wnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury.

Effects of transverse bodily movements of maxillary premolars on the surrounding hard tissue.

INTRODUCTION: This experimental study was designed to (1) produce buccal translation of maxillary premolars and (2) evaluate the effects on the buccal alveolar bone. METHODS: A randomized split-mouth study was designed based on 7 adult male beagle dogs. The experimental side received a custom cantilever appliance fabricated to produce a translatory force through the maxillary second premolar's center of resistance. The contralateral second premolar received no appliance and served as the control. The premolars underwent 6-7 weeks of buccal translation, followed by 3 weeks of fixed retention. Biweekly tooth movements were evaluated using intraoral and radiographic measurements. Pretreatment and posttreatment models were measured to assess tipping. Three-dimensional microscopic tomography was used to quantify the amount and density of buccal bone. Bone formation and turnover were assessed using fluorescent labeling, hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and bone sialoprotein immunostaining. RESULTS: The applied force (100 g of force) translated (1.4 mm) and minimally tipped (4°) the experimental teeth. Lateral translation produced dehiscences at the mesial and distal roots, with 2.0 mm and 2.2 mm loss of vertical bone height, respectively. Bone thickness decreased significantly (P < 0.05) at the apical (∼0.4 mm), midroot (∼0.4 mm), and coronal (∼0.2 mm) levels. Fluorescent imaging, hematoxylin and eosin staining, and immunostaining for bone sialoprotein all showed new bone formation extending along the entire periosteal surface of the second premolar's buccal plate. Tartrate-resistant acid phosphatase staining demonstrated greater osteoclastic activity on the experimental than that of control sections. CONCLUSIONS: New buccal bone forms on the periosteal surface during and after tooth translation, but the amount of bone that forms is less than the amount of bone loss, resulting in a net decrease in buccal bone thickness and a loss of crestal bone.

Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo.

Osteoporosis, osteopenia, and pathological bone fractures are frequent complications of iron-overload conditions such as hereditary hemochromatosis, thalassemia, and sickle cell disease. Moreover, animal models of iron overload have revealed increased bone resorption and decreased bone formation. Although systemic iron overload affects multiple organs and tissues, leading to significant changes on bone modeling and remodeling, the cell autonomous effects of excessive iron on bone cells remain unknown. Here, to elucidate the role of cellular iron homeostasis in osteoclasts, we generated two mouse strains in which solute carrier family 40 member 1 (Slc40a1), a gene encoding ferroportin (FPN), the sole iron exporter in mammalian cells, was specifically deleted in myeloid osteoclast precursors or mature cells. The FPN deletion mildly increased iron levels in both precursor and mature osteoclasts, and its loss in precursors, but not in mature cells, increased osteoclastogenesis and decreased bone mass in vivo Of note, these phenotypes were more pronounced in female than in male mice. In vitro studies revealed that the elevated intracellular iron promoted macrophage proliferation and amplified expression of nuclear factor of activated T cells 1 (Nfatc1) and PPARG coactivator 1ß (Pgc-1ß), two transcription factors critical for osteoclast differentiation. However, the iron excess did not affect osteoclast survival. While increased iron stimulated global mitochondrial metabolism in osteoclast precursors, it had little influence on mitochondrial mass and reactive oxygen species production. These results indicate that FPN-regulated intracellular iron levels are critical for mitochondrial metabolism, osteoclastogenesis, and skeletal homeostasis in mice.

Aberrant miR-145-5p/ß-catenin signal impairs osteocyte function in adolescent idiopathic scoliosis.

Recently, noncoding RNAs have been thought to play important roles in the sporadic occurrence of spinal deformity of adolescent idiopathic scoliosis (AIS). As a prognostic factor for curve progression, low bone mass has been hypothesized to crosstalk with AIS pathogenesis. Abnormal osteoblasts activities are reported in AIS without a clear mechanism. In this study, bone biopsies from patients with AIS and control subjects and the primary osteoblasts derived from those samples were used to identify the potential microRNA (miRNA) candidates that interfere with osteoblasts and osteocytes function. Microarray analysis identified miRNA-145-5p (miR-145) as a potential upstream regulator. miR-145 and ß-catenin mRNA ( CTNNB1) were overexpressed in AIS bone tissues and primary osteoblasts, and their expression correlated positively in AIS. Knockdown of miR-145 restored impaired osteocyte activity through the down-regulation of active ß-catenin expression and its transcriptional activity. Significant negative correlations between circulating miR-145 and serum sclerostin, osteopontin, and osteoprotegerin were noted in patients with AIS, which was in line with our cellular findings. This is the first study to demonstrate the effect of aberrant miRNA expression and its effect on osteocyte function in AIS, which may contribute to the low bone mass. Our findings also provide insight into the development of circulating microRNAs as a bone quality biomarker or even a prognostic biomarker for AIS.-Zhang, J., Chen, H., Leung, R. K. K., Choy, K. W., Lam, T. P., Ng, B. K. W., Qiu,Y., Feng, J. Q., Cheng, J. C. Y., Lee, W. Y. W. Aberrant miR-145-5p/ß-catenin signal impairs osteocyte function in adolescent idiopathic scoliosis.

Sclerostin Antibody Reverses the Severe Sublesional Bone Loss in Rats After Chronic Spinal Cord Injury.

To date, no efficacious therapy exists that will prevent or treat the severe osteoporosis in individuals with neurologically motor-complete spinal cord injury (SCI). Recent preclinical studies have demonstrated that sclerostin antibody (Scl-Ab) can prevent sublesional bone loss after acute SCI in rats. However, it remains unknown whether sclerostin inhibition reverses substantial bone loss in the vast majority of the SCI population who have been injured for several years. This preclinical study tested the efficacy of Scl-Ab to reverse the bone loss that has occurred in a rodent model after chronic motor-complete SCI. Male Wistar rats underwent either complete spinal cord transection or only laminectomy. Twelve weeks after SCI, the rats were treated with Scl-Ab at 25 mg/kg/week or vehicle for 8 weeks. In the SCI group that did not receive Scl-Ab, 20 weeks of SCI resulted in a significant reduction of bone mineral density (BMD) and estimated bone strength, and deterioration of bone structure at the distal femoral metaphysis. Treatment with Scl-Ab largely restored BMD, bone structure, and bone mechanical strength. Histomorphometric analysis showed that Scl-Ab increased bone formation in animals with chronic SCI. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increased Tcf7, ENC1, and the OPG/RANKL ratio expression, and decreased SOST expression. Our findings demonstrate for the first time that Scl-Ab reverses the sublesional bone loss when therapy is begun after relatively prolonged spinal cord transection. The study suggests that, in addition to being a treatment option to prevent bone loss after acute SCI, sclerostin antagonism may be a valid clinical approach to reverse the severe bone loss that invariably occurs in patients with chronic SCI.

Wnt3α and transforming growth factor-ß induce myofibroblast differentiation from periodontal ligament cells via different pathways.

Myofibroblasts are specialized cells that play a key role in connective tissue remodeling and reconstruction. Alpha-smooth muscle actin (α-SMA), vimentin and tenascin-C are myofibroblast phenotype, while α-SMA is the phenotypic marker. The observation that human periodontal ligament cells (hPDLCs) differentiate into myofibroblasts under orthodontic force has provided a new perspective for understanding of the biological and biomechanical mechanisms involved in orthodontic tooth movement. However, the cell-specific molecular mechanisms leading to myofibroblast differentiation in the periodontal ligament (PDL) remain unclear. In this study, we found that expression of Wnt3α, transforming growth factor-ß1 (TGF-ß1), α-SMA and tenascin-C increased in both tension and compression regions of the PDL under orthodontic load compared with unloaded control, suggesting that upregulated Wnt3α and TGF-ß1 signaling might have roles in myofibroblast differentiation in response to orthodontic force. We reveal in vitro that both Wnt3α and TGF-ß1 promote myofibroblast differentiation from hPDLCs. Dickkopf-1 (DKK1) impairs Wnt3α-induced myofibroblast differentiation in a ß-catenin-dependent manner. TGF-ß1 stimulates myofibroblast differentiation via a JNK-dependent mechanism. DKK1 has no significant effect on TGF-ß1-induced myofibroblastic phenotype.

Elevation of a full-thickness mucoperiosteal flap alone accelerates orthodontic tooth movement.

INTRODUCTION: Our objective was to determine whether the elevation of a full-thickness mucoperiosteal flap alone, without cortical cuts, decreases the amount of bone around teeth and accelerates mesial tooth movements. METHODS: The mandibular second premolars of 7 beagle dogs were extracted, and on a randomly selected side, a full-thickness mucoperiosteal buccal flap extending from the distal aspect of the third premolar to the mesial aspect of the first premolar was elevated. The other side did not receive flap surgery. The mandibular third premolars were protracted with orthodontic appliances. Tooth movements were analyzed biweekly over an 8-week period with calipers and radiographs. The amount and density of bone were analyzed using microcomputed tomography; bone remodeling was evaluated with histologic sections. RESULTS: Experimental tooth movements measured intraorally between cusp tips were significantly greater (25.3%) than control tooth movements. The approximate center of resistance measured radiographically also moved significantly more (about 31%) on the experimental than on the control side. The experimental premolar tipped more than the control premolar (10.5° vs 8.7°), but the difference was not statistically significant. The medullary bone volume fraction mesial to the third premolar was significantly less (9.1%) and the bone was significantly less dense (9%) on the experimental side than on the control side. Histology showed no apparent side differences in the numbers of osteoclasts and osteoblasts evident in the medullary bone. CONCLUSIONS: Elevation of a full-thickness mucoperiosteal flap alone (ie, without injury to bone) decreases the amount and density of medullary bone surrounding the tooth and accelerates tooth movement. Due to its limited effects, elevation of a flap alone to increase tooth movements may not be justified.

Induced ablation of Bmp1 and Tll1 produces osteogenesis imperfecta in mice.

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.

Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and ß-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study.

Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model.

Understanding periodontal ligament (PDL) biology and developing an effective treatment for bone and PDL damage due to periodontitis have been long-standing aims in dental medicine. Here, we first demonstrated by cell lineage tracing and mineral double-labeling approaches that murine PDL progenitor cells display a 2- and 3-fold higher mineral deposition rate than the periosteum and endosteum at the age of 4 weeks, respectively. We next proved that the pathologic changes in osteocytes (Ocys; changes from a spindle shape to round shape with a >50% reduction in the dendrite number/length, and an increase in SOST) are the key pathologic factors responsible for bone and PDL damage in periostin-null mice (a periodontitis animal model) using a newly developed 3-dimensional FITC-Imaris technique. Importantly, we proved that deleting the Sost gene (a potent inhibitor of WNT signaling) or blocking sclerostin function by using the mAb in this periodontitis model significantly restores bone and PDL defects (n = 4-5; P < 0.05). Together, identification of the key contribution of the PDL in normal alveolar bone formation, the pathologic changes of the Ocys in periodontitis bone loss, and the novel link between sclerostin and Wnt signaling in the PDL will aid future drug development in the treatment of patients with periodontitis.

Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice.

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bone-sparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα(flox/flox) mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα(-/-)). Male Dmp1-ERα(-/-) mice displayed a substantial reduction in trabecular bone volume (-20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (-45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα(-/-) mice. The male Dmp1-ERα(-/-) mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα(-/-) female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα(-/-) female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα(-/-) mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.

Critical roles of periostin in the process of orthodontic tooth movement.

AIM: The process of orthodontic tooth movement (OTM) involves multiple mechanisms of action including bone and extracellular matrix remodelling, although the role of periodontal ligament (PDL) in this process is largely unknown. Periostin, which is highly expressed in the PDL, is known to be responsible for mechanical stimulation in maintaining the integrity of periodontal tissues. We hypothesize that this protein plays an important role during OTM. MATERIAL AND METHODS: By using spring in 4-week-old wild-type (WT) and periostin null mice, the rate of tooth movement and mineralization were evaluated. For the evaluation, double labelling, expression of sclerostin (SOST), number of TRAP-positive cells, and quality of collagen fibrils by Sirius red were analysed and compared between these two groups. RESULTS: Our findings showed that the distance of the tooth movement and mineral deposition rates were significantly reduced in periostin null mice (P < 0.05), with a lack of expression changes in SOST as observed in the WT group. The arrangement, digestion, and integrity of collagen fibrils were impaired in periostin null mice. The number of osteoclasts reflected by expressions of TRAP (tartrate-resistant acid phosphatase) in the null mice was also significantly lower than the WT control (P < 0.05). CONCLUSION: Periostin plays a stimulatory role in both SOST and TRAP responses to OTM in the compassion site, although it is not clear if this role is direct or indirect during orthodontic loading.

Constitutive nuclear expression of dentin matrix protein 1 fails to rescue the Dmp1-null phenotype.

Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvß3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as (NLS)DMP1) or to the extracellular matrix as the WT type (referred to as (SP)DMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted (NLS)DMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the (SP)DMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes.

Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling.

Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF­neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.