The Majority of CD45- Ter119- CD31- Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors.

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45- Ter119- CD31- (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51- TNCs. Ly6D+ CD44+ CD51- TNCs phenotypically and functionally resembled CD45+ pro-B lymphoid cells, whereas Ly6D- CD44+ CD51- TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44+ CD51- TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44+ CD51- TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45- hematopoietic progenitors.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

INTRODUCTION: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. MATERIALS AND METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. CONCLUSION: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

p53 deficiency promotes bone regeneration by functional regulation of mesenchymal stromal cells and osteoblasts.

INTRODUCTION: The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing. MATERIALS AND METHODS: Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies. RESULTS: KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing. CONCLUSION: p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts.

Arteriolar niches maintain haematopoietic stem cell quiescence.

Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant three-dimensional associations in the mouse bone marrow among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal bone marrow. These arterioles are ensheathed exclusively by rare NG2 (also known as CSPG4)(+) pericytes, distinct from sinusoid-associated leptin receptor (LEPR)(+) cells. Pharmacological or genetic activation of the HSC cell cycle alters the distribution of HSCs from NG2(+) periarteriolar niches to LEPR(+) perisinusoidal niches. Conditional depletion of NG2(+) cells induces HSC cycling and reduces functional long-term repopulating HSCs in the bone marrow. These results thus indicate that arteriolar niches are indispensable for maintaining HSC quiescence.

[Characterization of bone marrow mesenchymal stem cells.]

Bones support the body as part of the human musculoskeletal system. They also contain bone marrow, which is a site of hematopoiesis. Bone marrow mesenchymal stem cells play a vital role by regulating skeletal tissue formation and maintaining hematopoiesis. While the presence of bone marrow-derived mesenchymal stem cells has been indicated, they have yet to be fully understood in vivo. Recent studies using genetic mouse models revealed that perivascular stromal cells function as mesenchymal stem cells, and their differentiation status may vary during the early stage of life to adulthood. Furthermore, studies have investigated the underlying mechanisms that regulate the cell fate decision of mesenchymal stem cells. These findings could lead to the design of new therapeutic approaches for metabolic bone disease and hematopoietic disease.

The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts.

The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents.

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice.

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.

[Bone Cell Biology Assessed by Microscopic Approach. Mechanisms of in vivo osteoclastogenesis].

Many new findings about osteoclastogenesis have been provided by in vitro osteoclast culture methods. However, it is necessary to identify genuine in vivo osteoclast precursors and analyze their dynamics in order to completely understand in vivo osteoclastogenesis. Previously, we identified an in vivo osteoclast precursor (qOP : quiescent osteoclast precursors). In this review, the differentiation and movement of qOP will be described based on recent experimental data. Moreover, I will show evidence that the expression level of RANK in qOP is increased in bone tissue, which is an important event for in vivo osteoclastogenesis.

Fos plays an essential role in the upregulation of RANK expression in osteoclast precursors within the bone microenvironment.

Fos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL(-/-)) mice and Fos(-/-) mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-positive osteoclast precursors are present in bone of RANKL(-/-) mice but not Fos(-/-) mice. Here we report the role of Fos in RANK expression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shown to express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not in osteoclast precursors in Fos(-/-) mice. RANK mRNA and protein levels in bone were lower in Fos(-/-) mice than RANKL(-/-) mice, suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos(-/-) mice, RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing with RANKL(-/-) osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblastic cells in bone in Fos(-/-) mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos(-/-) macrophages. Overexpression of Fos in Fos(-/-) macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos(-/-) macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest that Fos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.

Tetracyclines convert the osteoclastic-differentiation pathway of progenitor cells to produce dendritic cell-like cells.

Tetracyclines, such as doxycycline and minocycline, are used to suppress the growth of bacteria in patients with inflammatory diseases. Tetracyclines have been shown to prevent bone loss, but the mechanism involved is unknown. Osteoclasts and dendritic cells (DCs) are derived from common progenitors, such as bone marrow-derived macrophages (BMMs). In this article, we show that tetracyclines convert the differentiation pathway, resulting in DC-like cells not osteoclasts. Doxycycline and minocycline inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis of BMMs, but they had no effects on cell growth and phagocytic activity. They influenced neither the proliferation nor the differentiation of bone-forming osteoblasts. Surprisingly, doxycycline and minocycline induced the expression of DC markers, CD11c and CD86, in BMMs in the presence of RANKL. STAT5 is involved in DC differentiation induced by GM-CSF. Midostaurin, a STAT5-signaling inhibitor, and an anti-GM-CSF-neutralizing Ab suppressed the differentiation induced by GM-CSF but not by tetracyclines. In vivo, the injection of tetracyclines into RANKL-injected mice and RANKL-transgenic mice suppressed RANKL-induced osteoclastogenesis and promoted the concomitant appearance of CD11c(+) cells. These results suggested that tetracyclines prevent bone loss induced by local inflammation, including rheumatoid arthritis and periodontitis, through osteoclast-DC-like cell conversion.

In vivo dynamics of hard tissue-forming cell origins: Insights from Cre/loxP-based cell lineage tracing studies.

Bone tissue provides structural support for our bodies, with the inner bone marrow (BM) acting as a hematopoietic organ. Within the BM tissue, two types of stem cells play crucial roles: mesenchymal stem cells (MSCs) (or skeletal stem cells) and hematopoietic stem cells (HSCs). These stem cells are intricately connected, where BM-MSCs give rise to bone-forming osteoblasts and serve as essential components in the BM microenvironment for sustaining HSCs. Despite the mid-20th century proposal of BM-MSCs, their in vivo identification remained elusive owing to a lack of tools for analyzing stemness, specifically self-renewal and multipotency. To address this challenge, Cre/loxP-based cell lineage tracing analyses are being employed. This technology facilitated the in vivo labeling of specific cells, enabling the tracking of their lineage, determining their stemness, and providing a deeper understanding of the in vivo dynamics governing stem cell populations responsible for maintaining hard tissues. This review delves into cell lineage tracing studies conducted using commonly employed genetically modified mice expressing Cre under the influence of LepR, Gli1, and Axin2 genes. These studies focus on research fields spanning long bones and oral/maxillofacial hard tissues, offering insights into the in vivo dynamics of stem cell populations crucial for hard tissue homeostasis.

Osteoblast differentiation of Gli1⁺ cells via Wnt and BMP signaling pathways during orthodontic tooth movement.

OBJECTIVES: Factors that induce bone formation during orthodontic tooth movement (OTM) remain unclear. Gli1 was recently identified as a stem cell marker in the periodontal ligament (PDL). Therefore, we evaluated the mechanism of differentiation of Cre/LoxP-mediated Gli1/Tomato+ cells into osteoblasts during OTM. METHODS: After the final administration of tamoxifen to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato mice for 2 days, nickel-titanium closed coil springs were attached between the upper anterior alveolar bone and the first molar. Immunohistochemical localizations of ß-catenin, Smad4, and Runx2 were observed in the PDL on 2, 5, and 10 days after OTM initiation. RESULTS: In the untreated tooth, few Gli1/Tomato+ cells were detected in the PDL. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased in the PDL on the tension side. On this side, 49.3 ± 7.0% of ß-catenin+ and 48.7 ± 5.7% of Smad4+ cells were found in the PDL, and Runx2 expression was detected in some Gli1/Tomato+ cells apart from the alveolar bone. The number of positive cells in the PDL reached a maximum on day 5. In contrast, on the compression side, ß-catenin and Smad4 exhibited less immunoreactivity. On day 10, Gli1/Tomato+ cells were aligned on the alveolar bone on the tension side, with some expressing Runx2. CONCLUSIONS: Gli1+ cells in the PDL differentiated into osteoblasts during OTM. Wnt and bone morphogenetic proteins signaling pathways may be involved in this differentiation.

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation.

OBJECTIVE: Leptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DESIGN: Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. RESULTS: Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. CONCLUSION: LRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Sclerostin modulates mineralization degree and stiffness profile in the fibrocartilaginous enthesis for mechanical tissue integrity.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.

Bone formation ability of Gli1+ cells in the periodontal ligament after tooth extraction.

During the process of socket healing after tooth extraction, osteoblasts appear in the tooth socket and form alveolar bone; however, the source of these osteoblasts is still uncertain. Recently, it has been demonstrated that cells expressing Gli1, a downstream factor of sonic hedgehog signaling, exhibit stem cell properties in the periodontal ligament (PDL). Therefore, in the present study, the differentiation ability of Gli1+-PDL cells after tooth extraction was analyzed using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. After the final administration of tamoxifen to iGli1/Tomato mice, Gli1/Tomato+ cells were rarely detected in the PDL. One day after the tooth extraction, although inflammatory cells appeared in the tooth socket, Periostin+ PDL-like tissues having a few Gli1/Tomato+ cells remained near the alveolar bone. Three days after the extraction, the number of Gli1/Tomato+ cells increased as evidenced by numerous PCNA+ cells in the socket. Some of these Gli1/Tomato+ cells expressed BMP4 and Phosphorylated (P)-Smad1/5/8. After seven days, the Osteopontin+ bone matrix was formed in the tooth socket apart from the alveolar bone. Many Gli1/Tomato+ osteoblasts that were positive for Runx2+ were arranged on the surface of the newly formed bone matrix. In the absence of Gli1+-PDL cells in Gli1-CreERT2/Rosa26-loxP-stop-loxP-tdDTA (iGli1/DTA) mice, the amount of newly formed bone matrix was significantly reduced in the tooth socket. Therefore, these results collectively suggest that Gli1+-PDL cells differentiate into osteoblasts to form the bone matrix in the tooth socket; thus, this differentiation might be regulated, at least in part, by bone morphogenetic protein (BMP) signaling.

Differentiation ability of Gli1+ cells during orthodontic tooth movement.

Orthodontic tooth movement (OTM) induces bone formation on the alveolar bone of the tension side; however, the mechanism of osteoblast differentiation is not fully understood. Gli1 is an essential transcription factor for hedgehog signaling and functions in undifferentiated cells during embryogenesis. In this study, we examined the differentiation of Gli1+ cells in the periodontal ligament (PDL) during OTM using a lineage-tracing analysis. After the final administration of tamoxifen for 2 days to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice, Gli1/Tomato+ cells were rarely observed near endomucin+ blood vessels in the PDL. Osteoblasts lining the alveolar bone did not exhibit Gli1/Tomato fluorescence. To move the first molar of iGli1/Tomato mice medially, nickel-titanium closed-coil springs were attached between the upper anterior alveolar bone and the first molar. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased along with numerous PCNA+ cells in the PDL of the tension side. As some Gli1/Tomato+ cells exhibited positive expression of osterix, an osteoblast differentiation marker, Gli1+ cells probably differentiated into osteoblast progenitor cells. On day 10, the newly formed bone labeled by calcein administration during OTM was detected on the surface of the original alveolar bone of the tension side. Gli1/Tomato+ cells expressing osterix localized to the surface of the newly formed bone. In contrast, in the PDL of the compression side, Gli1/Tomato+ cells proliferated before day 10 and expressed type I collagen, suggesting that the Gli1+ cells also differentiated into fibroblasts. Collectively, these results demonstrate that Gli1+ cells in the PDL can differentiate into osteoblasts at the tension side and may function in bone remodeling as well as fibril formation in the PDL during OTM.

Mesenchymal cell TRPM7 expression is required for bone formation via the regulation of chondrogenesis.

Transient receptor potential melastatin-subfamily member 7 (TRPM7) is a bifunctional protein containing a kinase fused to an ion channel permeated with cations, including Ca2+ and Mg2+. Trpm7-null mice show embryonic lethality. Paired related homeobox 1 (Prx1) is expressed in undifferentiated mesenchymal cells such as the progenitor cells of both chondrocytes and osteoblasts involved in limb skeleton formation. Prx1-Cre-dependent Trpm7 mesenchymal-deleted mice were generated to examine the role of TRPM7 in bone development. We found that Prx1-Cre;Trpm7fl/fl mice had shortened bones and impaired trabecular bone formation. Trabecular bone parameters, such as the bone volume (BV/TV), and trabecular number (Tb.N), were decreased in Prx1-Cre;Trpm7fl/fl mice. The cortical bone parameters of cortical bone area (Ct.Ar) and cortical bone thickness (Ct.Th) were also down-regulated in these mice. The bone formation rate in Prx1-Cre;Trpm7fl/fl mice was unchanged, but the hypertrophic area and cell size of the zone were smaller, and the expression of Col2a1, Col10a1 and Mmp13 was downregulated compared with control mice. These findings suggest impaired chondrogenesis in Prx1-Cre;Trpm7fl/fl mice compared to control mice. The receptor activator of nuclear factor-kappa B ligand (RANKL) expression was increased, and RANKL-positive cells and osteoclasts were markedly accumulated in the boundary region between the growth plate and trabecular bone. In contrast, TRPM7 KR mice, which are kinase-dead mutants in which the TRPM7 ion channel function has not been altered, showed no marked differences in trabecular or cortical bone parameters compared to wild-type mice. These findings suggest that TRPM7 is critical as a cation channel rather than as a kinase in bone development via the regulation of chondrogenesis.

Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells.

The lineage of periodontal ligament (PDL) stem cells contributes to alveolar bone (AB) and cementum formation, which are essential for tooth-jawbone attachment. Leptin receptor (LepR), a skeletal stem cell marker, is expressed in PDL; however, the stem cell capacity of LepR+ PDL cells remains unclear. We used a Cre/LoxP-based approach and detected LepR-cre-labeled cells in the perivascular around the root apex; their number increased with age. In the juvenile stage, LepR+ PDL cells differentiated into AB-embedded osteocytes rather than cementocytes, but their contribution to both increased with age. The frequency of LepR+ PDL cell-derived lineages in hard tissue was < 20% per total cells at 1-year-old. Similarly, LepR+ PDL cells differentiated into osteocytes following tooth extraction, but their frequency was < 9%. Additionally, both LepR+ and LepR- PDL cells demonstrated spheroid-forming capacity, which is an indicator of self-renewal. These results indicate that both LepR+ and LepR- PDL populations contributed to hard tissue formation. LepR- PDL cells increased the expression of LepR during spheroid formation, suggesting that the LepR- PDL cells may hierarchically sit upstream of LepR+ PDL cells. Collectively, the origin of hard tissue-forming cells in the PDL is heterogeneous, some of which express LepR.

Differentiation of committed osteoblast progenitors by octacalcium phosphate compared to calcium-deficient hydroxyapatite in Lepr-cre/Tomato mouse tibia.

This study aimed to investigate the accumulation and differentiation of mesenchymal stem cells (MSCs) around octacalcium phosphate (OCP) compared with those around calcium-deficient hydroxyapatite (CDHA), a material obtained through hydrolysis of the original OCP. Leptin receptor (Lepr)-expressing bone marrow-derived MSCs around the OCP and CDHA were pursued utilizing genetically modified Lepr-cre/Tomato mice. OCP and CDHA granules were implanted into the tibia defect of the mice for 10 weeks and subjected to histomorphometric and immunohistochemical analyses. The structural properties of OCP and CDHA after inoculation into mouse subcutaneous tissue (until 4 weeks) or culture mediums (14 days) were analyzed using physicochemical techniques. In vitro osteoblastic differentiation of primary MSCs was examined with the materials for 14 days. While Lepr-cre/Tomato positive cells (red) accumulated around both OCP and CDHA, Lepr and osteocalcin double-positive osteoblastic cells (yellow) were significantly more abundant around OCP than around CDHA in the early implantation period. OCP enhanced the osteoblastic differentiation of MSCs more than CDHA in vitro. Physicochemical and structual analyses provided evidence that OCP tended to convert to the apatitic phase in the tested physiological environments. The higher osteoconductivity of OCP originated from a capacity-enhancing osteoblastic differentiation of committed osteoblast progenitors in bone marrow accompanied by OCP hydrolysis. STATEMENT OF SIGNIFICANCE: MSCs play a key role in bone regeneration through osteoblastic differentiation. Calcium phosphates have been widely applied as bone substitute materials, and OCP has a better ability to promote osteoblast differentiation of MSCs than that of HA in vitro. However, it is not clear how MSCs accumulate in the bone marrow and differentiate into osteoblasts during bone regeneration in vivo. In this study, we focused on the leptin receptor, a marker of bone marrow-derived MSCs. Using genetically modified mice labeled with the red fluorescent protein Tomato, we observed the accumulation of MSCs around calcium phosphates implanted in tibia bone defects and their differentiation into osteoblasts.