Trabecular Osteoblasts Research Articles

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

MiR-196b-5p Regulates Osteoblast and Osteoclast Differentiation and Bone Homeostasis by Targeting SEMA3A.

miR-196b-5p plays a role in various malignancies. We have recently reported its function in regulating adipogenesis. However, it remains to be clarified whether and how miR-196b-5p affects bone cells and bone homeostasis. In this study, in vitro functional experiments showed an inhibitory effect of miR-196b-5p on osteoblast differentiation. Mechanistic explorations revealed that miR-196b-5p directly targeted semaphorin 3a (Sema3a) and inhibited Wnt/β-catenin signaling. SEMA3A attenuated the impaired osteogenesis induced by miR-196b-5p. Osteoblast-specific miR-196b transgenic mice showed significant reduction of bone mass. Trabecular osteoblasts were reduced and bone formation was suppressed, whereas osteoclasts, marrow adipocytes, and serum levels of bone resorption markers were increased in the transgenic mice. The osteoblastic progenitor cells from the transgenic mice had decreased SEMA3A levels and exhibited retarded osteogenic differentiation, whereas those marrow osteoclastic progenitors exhibited enhanced osteoclastogenic differentiation. miR-196b-5p and SEMA3A oppositely regulated the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin. The calvarial osteoblastic cells expressing the transgene promoted osteoclastogenesis, whereas the osteoblasts overexpressing Sema3a inhibited it. Finally, in vivo transfection of miR-196b-5p inhibitor to the marrow reduced ovariectomy-induced bone loss in mice. Our study has identified that miR-196b-5p plays a key role in osteoblast and osteoclast differentiation and regulates bone homeostasis. Inhibition of miR-196b-5p may be beneficial for amelioration of osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

Atypical Fracture of Radial Diaphysis After Sauvé-Kapandji Procedure: A Retrospective Radiographic Evaluation of Fracture Progression.

The Sauvé-Kapandji procedure (SK), which combines distal radioulnar joint arthrodesis with creation of an ulnar pseudarthrosis, achieves good outcomes with few complications for patients with distal radioulnar joint instability or arthritis. The authors describe a case of atypical fracture of the radial diaphysis that occurred in a 79-year-old woman with rheumatoid arthritis who had undergone SK 18 years earlier. The patient had taken alendronate for osteoporosis for 13 years but had discontinued treatment 2 years before onset of symptoms. Retrospective review of serial radiographs revealed focal cortical thickening at the fracture site beginning 18 months before the onset of prodromal pain and becoming more evident over time. The patient underwent surgical repair of the fracture, which had occurred at the attachment of the pronator teres. Histopathological examination of bone excised from the fracture site and from the iliac graft used for fracture repair revealed markedly fewer trabecular osteoblasts than normal and no osteoclasts, indicating severe suppression of bone turnover. It is important to evaluate the radial diaphysis on radiographs taken after SK for early signs of atypical fracture, especially in patients with rheumatoid arthritis and a history of bisphosphonate use. Given the patient's history of bisphosphonate use and the similar radiographic appearance to atypical femoral fractures, the authors applied the term atypical radial fracture to this case. [Orthopedics. 2023;46(5):e317-e320.].

Metastasis suppressor 1 controls osteoblast differentiation and bone homeostasis through regulating Src-Wnt/β-catenin signaling.

Metastasis suppressor 1 (MTSS1) plays an inhibitory role in tumorigenesis and metastasis of a variety of cancers. To date, the function of MTSS1 in the differentiation of marrow stromal progenitor cells remains to be explored. In the current study, we investigated whether and how MTSS1 has a role in osteoblast differentiation and bone homeostasis. Our data showed that MTSS1 mRNA was upregulated during osteoblast differentiation and downregulated in the osteoblastic lineage cells of ovariectomized and aged mice. Functional studies revealed that MTSS1 promoted the osteogenic differentiation from marrow stromal progenitor cells. Mechanistic explorations uncovered that the inactivation of Src and afterward activation of canonical Wnt signaling were involved in osteoblast differentiation induced by MTSS1. The enhanced osteogenic differentiation induced by MTSS1 overexpression was attenuated when Src was simultaneously overexpressed, and conversely, the inhibition of osteogenic differentiation by MTSS1 siRNA was rescued when the Src inhibitor was supplemented to the culture. Finally, the in vivo transfection of MTSS1 siRNA to the marrow of mice significantly reduced the trabecular bone mass, along with the reduction of trabecular osteoblasts, the accumulation of marrow adipocytes, and the increase of phospho-Src-positive cells on the trabeculae. No change in the number of osteoclasts was observed. This study has unraveled that MTSS1 contributes to osteoblast differentiation and bone homeostasis through regulating Src-Wnt/β-catenin signaling. It also suggests the potential of MTSS1 as a new target for the treatment of osteoporosis.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Lactobacillus reuteri alleviates Glucocorticoid induced Avascular Necrosis of Femoral Head.

Glucocorticoids (GC) are well‐known anti‐inflammatory drugs that are widely used to manage many diseases including autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. Chronic GC use can have significant side effects including 1) dysbiosis (an imbalance of gut microbiota composition), 2) increased intestinal permeability which is linked to increased serum endotoxin levels, 3) trabecular bone loss and 4) osteonecrosis (i.e., avascular necrosis of the femur head). Especially, patients with GC‐induced osteonecrosis of femur head suffer from pain and stiffness, resulting in long‐term disability. Our lab and others have shown that the gut (microbiome and barrier function) regulates bone density. Recently, we have also shown that the probiotic Lactobacillus reuteri 6475 (LR) prevented GC‐induced trabecular bone loss in male and female mice. Based on these findings, we examined if LR treatment can prevent GC‐induced osteonecrosis (GCON) of the femur head. Male C57BL/6J (16‐weeks‐old) mice were treated for 8‐weeks with GC (2.5 mg/kg/day, subcutaneously slow release pellet) and supplemented with or without 3.3×108 cfu/ml LR in their drinking water. Femur immunohistochemistry examining von Willebrand factor positive blood vessels revealed that GC‐induced avascularization of the femur head was prevented by LR supplementation. Specifically, compared to controls, GC treatment caused a 50% reduction in the number of blood vessels in the femur head – this was completely prevented by LR treatment. Similarly, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses determined that LR treatment prevented GC‐induced apoptosis of trabecular osteoblast and osteocytes. In addition, LR treatment prevented GC‐induced marrow adiposity. Consistent with these findings, microcomputed tomography indicated that femoral head bone loss was also prevented by LR treatment. Gene expression analysis of the femur head identified that GC‐induction of tumor necrosis factor alpha and Bcl‐2‐associated x protein (pro‐apoptotic) was prevented by LR. Together, these data indicate that probiotic LR supplementation prevents the pathological bone effects of GCs (avascularization, cell apoptosis, adiposity, bone loss) and may be a straightforward cost‐effective way to mitigate avascular necrosis of femoral head.Support or Funding InformationThese studies were supported by funding from the National Institute of Health, grants RO1 DK101050, AT007695 and by Michigan State University Foundation.

Abstract P5-04-29: Osteoblasts regulate mobilization of the myeloid-derived suppressor cells from the bone marrow of breast cancer patients

Abstract MDSCs are T cell suppressive immature myeloid-lineage cells in the tumor microenvironent (TME). MDSCs originate in the bone marrow, circulate and accumulate in the spleen and tumor tissue. MDSCs consist of two morphologically and functionally distinct subpopulations, i.e. monocytic (M-) and polymorphonuclear (PMN-) MDSC. While PMN-MDSCs are more abundant and suppress T cells in an antigen-specific manner, M-MDSCs are earlier-stage MDSCs and suppress T cells in both antigen-specific and nonspecific manners. In contrast to clear functions in TME, how MDSCs are regulated in the bone marrow of tumor hosts remains unclear. We have previously reported that tumor-derived parathyroid hormone-related peptide (PTHrP) increased MDSCs in TME. In this study, we further investigated cellular and molecular mechanisms of PTHrP in mobilizing MDSCs from the bone marrow. Continuous infusion of PTHrP(1-34) in female Balb/C mice using subcutaneous osmotic pumps (80μg/kg, 3 weeks) increased M-MDSC, but not PMN-MDSC, in the circulation and bone marrow. Interestingly, a single administration of PTHrP(1-34) or PTH(1-34) in mice also rapidly increased M-MDSC in the circulation, suggesting that M-MDSC are mobilized from the bone marrow by PTH1R activation.RT-PCR analysis showed that PTH1R is expressed in osteoblasts, but not in human or mouse MDSC. In vitro cell binding assays measuring fluorescently labeled-human or mouse MDSC co-cultured with MC3T3E1 or hFOB osteoblasts were performed to determine the molecular mechanism of MDSC mobilization. We found that PTHrP(1-34) and PTH(1-34), but not PTHrP(7-34), released MDSC-osteoblast binding, and also that anti-VCAM 1 and/or -β1 integrin neutralizing antibodies blocked MDSC-osteoblast binding. Inhibitors of PKA, a downstream mediator of PTH1R, inhibited MDSC-osteoblast binding, while forskolin, a PKA activator, showed the opposite effects. Immunohistochemical staining of the bones of tumor-bearing mice showed that M-MDSCs localize adjacent to trabecular osteoblasts via VCAM-1 (expressed by osteoblasts) and β1 integrin (expressed by M-MDSC). Subsequent experiments demonstrated that PTH/PTHrP-stimulated osteoblasts release VEGF-A and IL-6 to stimulate M-MDSCs via phosphorylation of Src family kinase. Administration of dasatinib, a Src selective inhibitor, significantly suppressed PTHrP-mediated M-MDSC mobilization in vivo. Collectively, our data demonstrate that tumor-derived PTHrP circulate and activates osteoblasts, leading to release of M-MDSCs via disruption of VCAM-1 and β1 integrin retention axis by upregulation of VEGF-A and IL-6 in bone and by Src family kinase phosphorylation in MDCS. In conclusion, this study explains how MDSC, an essential bone marrow-derived cells in TME, are regulated in the bones of cancer patients. Further studies will provide a therapeutic approach to uncouple the partnership between tumor and bone. Citation Format: Serkin Park, Kyoung Jin Lee, Eun Jung Lee, Young Mi Whang, Sun Wook Cho. Osteoblasts regulate mobilization of the myeloid-derived suppressor cells from the bone marrow of breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-29.

Abaloparatide improves cortical geometry and trabecular microarchitecture and increases vertebral and femoral neck strength in a rat model of male osteoporosis.

Androgen deficiency is a leading cause of male osteoporosis, with bone loss driven by an inadequate level of bone formation relative to the extent of bone resorption. Abaloparatide, an osteoanabolic PTH receptor agonist used to treat women with postmenopausal osteoporosis at high risk for fracture, increases bone formation and bone strength in estrogen-deficient animals without increasing bone resorption. This study examined the effects of abaloparatide on bone formation, bone mass, and bone strength in androgen-deficient orchiectomized (ORX) rats, a male osteoporosis model. Four-month-old Sprague-Dawley rats underwent ORX or sham surgery. Eight weeks later, sham-operated rats received vehicle (saline; n = 10) while ORX rats (n = 10/group) received vehicle (Veh) or abaloparatide at 5 or 25 μg/kg (ABL5 or ABL25) by daily s.c. injection for 8 weeks, followed by sacrifice. Dynamic bone histomorphometry indicated that the tibial diaphysis of one or both abaloparatide groups had higher periosteal mineralizing surface, intracortical bone formation rate (BFR), endocortical BFR, and cortical thickness vs Veh controls. Vertebral trabecular BFR was also higher in both abaloparatide groups vs Veh, and the ABL25 group had higher trabecular osteoblast surface without increased osteoclast surface. By micro-CT, the vertebra and distal femur of both abaloparatide-groups had improved trabecular bone volume and micro-architecture, and the femur diaphysis of the ABL25 group had greater cortical thickness with no increase in porosity vs Veh. Biomechanical testing indicated that both abaloparatide-groups had stronger vertebrae and femoral necks vs Veh controls. These findings provide preclinical support for evaluating abaloparatide as an investigational treatment for male osteoporosis.

2. The Effect Of Sipatah-patah (Cissus quadrangularis Salisb) Extract On The Femur Bone Density of White Rat (Rattus norvegicus) with Model Ovariectomy

This research aimed to determine the effect of giving Sipatah-patah extract toward the histopathological and femur bone growth of white rat that was ovariectomized. The experimental animals that were used were 12 white rats divided into 4 treatment group with 3 repetitions. K0 was the ovariectomized rat without giving Sipatah-patah extract (ESP); K1, K2 and K3 were ovariectomized rats which were given Sipatah-patah extract with multilevel doses of 500 mg/kg BW, 700 mg/kg BW and 900 mg/kg BW for 30 days. On the 31st day, rats were euthanized using chloroform and os femur that was taken to being made into histological preparation. There was the decrease in the bones density of the K0 group which is characterized by thinning of trabecular structure, there were lots of osteoclast cells on the edge of the trabecular and lower density of active osteoblasts and passive osteoblasts. The rat of group K1 and K2 showed an improvement on the trabecular structure and lower osteoclast than group K0. The rat of group K3 had a visible improvement of the most congested trabecular structures, cohesive with the most density cell of active osteoblasts than the other groups. The result of this research concluded that the giving of Sipatah-patah extract doses 900 mg/kg BW showed a higher density of trabecular and active osteoblasts than the control group, K1 and K2 on the white rat bones that were ovariectomized.

P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton

Histone deacetylase 4 (Hdac4) regulates chondrocyte hypertrophy. Hdac4−/− mice are runted in size and do not survive to weaning. This phenotype is primarily due to the acceleration of onset of chondrocyte hypertrophy and, as a consequence, inappropriate endochondral mineralization. Previously, we reported that Hdac4 is a repressor of matrix metalloproteinase-13 (Mmp13) transcription, and the absence of Hdac4 leads to increased expression of MMP-13 both in vitro (osteoblastic cells) and in vivo (hypertrophic chondrocytes and trabecular osteoblasts). MMP-13 is thought to be involved in endochondral ossification and bone remodeling. To identify whether the phenotype of Hdac4−/− mice is due to up-regulation of MMP-13, we generated Hdac4/Mmp13 double knockout mice and determined the ability of deletion of MMP-13 to rescue the Hdac4−/− mouse phenotype. Mmp13−/− mice have normal body size. Hdac4−/−/Mmp13−/− double knockout mice are significantly heavier and larger than Hdac4−/− mice, they survive longer, and they recover the thickness of their growth plate zones. In Hdac4−/−/Mmp13−/− double knockout mice, alkaline phosphatase (ALP) expression and TRAP-positive osteoclasts were restored (together with an increase in Mmp9 expression) but osteocalcin (OCN) was not. Micro-CT analysis of the tibiae revealed that Hdac4−/− mice have significantly decreased cortical bone area compared with the wild type mice. In addition, the bone architectural parameter, bone porosity, was significantly decreased in Hdac4−/− mice. Hdac4−/−/Mmp13−/− double knockout mice recover these cortical parameters. Likewise, Hdac4−/− mice exhibit significantly increased Tb.Th and bone mineral density (BMD) while the Hdac4−/−/Mmp13−/− mice significantly recovered these parameters toward normal for this age. Taken together, our findings indicate that the phenotype seen in the Hdac4−/− mice is partially derived from elevation in MMP-13 and may be due to a bone remodeling disorder caused by overexpression of this enzyme.

Glycoprotein130 (Gp130)/interleukin-6 (IL-6) signalling in osteoclasts promotes bone formation in periosteal and trabecular bone

Interleukin-6 (IL-6) and interleukin-11 (IL-11) receptors (IL-6R and IL-11R, respectively) are both expressed in osteoclasts and transduce signal via the glycoprotein130 (gp130) co-receptor, but the physiological role of this pathway is unclear. To determine the critical roles of gp130 signalling in the osteoclast, we generated mice using cathepsin K Cre (CtskCre) to disrupt gp130 signalling in osteoclasts. Bone marrow macrophages from CtskCre.gp130f/f mice generated more osteoclasts in vitro than cells from CtskCre.gp130w/w mice; these osteoclasts were also larger and had more nuclei than controls. While no increase in osteoclast numbers was observed in vivo, osteoclasts on trabecular bone surfaces of CtskCre.gp130f/f mice were more spread out than in control mice, but had no functional defect detectable by serum CTX1 levels or trabecular bone cartilage remnants. However, trabecular osteoblast number and mineralising surfaces were significantly lower in male CtskCre.gp130f/f mice compared to controls, and this was associated with a significantly lower trabecular bone volume at 12weeks of age. Furthermore, CtskCre.gp130f/f mice exhibited greatly suppressed periosteal bone formation at this age, indicated by significant reductions in both double-labelled surface and mineral apposition rate. By 26weeks of age, CtskCre.gp130f/f mice exhibited narrower femora, with lower periosteal and endocortical perimeters than CtskCre.gp130w/w controls. Since IL-6 and IL-11R global knockout mice exhibited a similar reduction in femoral width, we also assessed periosteal bone formation in those strains, and found bone forming surfaces were also reduced in male IL-6 null mice. These data suggest that IL-6/gp130 signalling in the osteoclast is not essential for normal bone resorption in vivo, but maintains both trabecular and periosteal bone formation in male mice by promoting osteoblast activity through the stimulation of osteoclast-derived "coupling factors" and "osteotransmitters", respectively.

Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage.

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond “terminal” differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates – depending on the developmental stage – substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate.

Local origins impart conserved bone type-related differences in human osteoblast behaviour.

Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κβ ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro.

Progressive Increases in Bone Mass and Bone Strength in an Ovariectomized Rat Model of Osteoporosis After 26 Weeks of Treatment With a Sclerostin Antibody

The effects of up to 26 weeks of sclerostin antibody (Scl-Ab) treatment were investigated in ovariectomized (OVX) rats. Two months after surgery, 6-month-old osteopenic OVX rats were treated with vehicle or Scl-Ab (25 mg/kg, sc, one time per week) for 6, 12, or 26 weeks. In vivo dual-energy x-ray absorptiometry analysis demonstrated that the bone mineral density of lumbar vertebrae and femur-tibia increased progressively through 26 weeks of Scl-Ab treatment along with progressive increases in trabecular and cortical bone mass and bone strength at multiple sites. There was a strong correlation between bone mass and maximum load at lumbar vertebra, femoral neck, and diaphysis at weeks 6 and 26. Dynamic histomorphometric analysis showed that lumbar trabecular and tibial shaft endocortical and periosteal bone formation rates (BFR/BS) increased and peaked at week 6 with Scl-Ab-treatment; thereafter trabecular and endocortical BFR/BS gradually declined but remained significantly greater than OVX controls at week 26, whereas periosteal BFR/BS returned to OVX control levels at week 26. In the tibia metaphysis, trabecular BFR/BS in the Scl-Ab treated group remained elevated from week 6 to week 26. The osteoclast surface and eroded surface were significantly lower in Scl-Ab-treated rats than in OVX controls at all times. In summary, bone mass and strength increased progressively over 26 weeks of Scl-Ab treatment in adult OVX rats. The early gains were accompanied by increased cortical and trabecular bone formation and reduced osteoclast activity, whereas later gains were attributed to residual endocortical and trabecular osteoblast stimulation and persistently low osteoclast activity.

Treatment with N- and C-Terminal Peptides of Parathyroid Hormone-Related Protein Partly Compensate the Skeletal Abnormalities in IGF-I Deficient Mice

Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1–36) and PTHrP (107–111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a , cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1–36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1–36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone.

Defective Endochondral Ossification-Derived Matrix and Bone Cells Alter the Lymphopoietic Niche in Collagen X Mouse Models

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders.

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time(1). The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2). Both trabecular osteoblasts(2,3) and sinusoidal endothelium(4) constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin(+) osteoblasts(3) and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1(5) concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity(6). Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands(7) and interferon-α(6) can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin(- )c-Kit(+ )Sca-1(+ )CD150(+ )CD48(- )CD34(-) population has been described(8). Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs(9). A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described(10). In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

Krüppel-like factor 4 regulates membranous and endochondral ossification

Krüppel-like factor 4 (KLF4/GKLF/EZF) is a zinc finger type of transcription factor highly expressed in the skin, intestine, testis, lung and bone. The role played by Klf4 has been studied extensively in normal epithelial development and maintenance; however, its role in bone cells is unknown. Previous reports showed that Klf4 is expressed in the developing flat bones but its expression diminishes postnatally. We now show that in the developing long bones, Klf4 is expressed in the perichondrium, trabecular osteoblasts and prehypertrophic chondrocytes. In contrast, osteoblasts lining at the surface of the bone collar showed extremely low levels of Klf4 expression. To investigate the possible roles played by Klf4 during skeletal development, we generated transgenic mice expressing Klf4 under mouse type I collagen regulatory sequence. Transgenic mice exhibited severe skeletal deformities and died soon after birth. Transgenic mice showed delayed formation of the calvarial bones; and over-expressing Klf4 in primary mouse calvarial osteoblasts in culture resulted in strong repression of mineralization indicating that this regulation of Klf4 is through an osteoblast-autonomous effect. Surprisingly, long bones of the transgenic mice exhibited delayed marrow cavity formation. Even at E18.5, the presumptive marrow space was occupied by cartilage anlage and invasion of the vascular endothelial cells and osteoclasts were seldom observed. Instead of entering the cartilage anlage, osteoclasts accumulated at the periosteum in the transgenic mice. Significantly, osteocalcin, which is known to chemotact osteoclasts, was up-regulated at the perichindrium as early as E14.5 in the mutants. In vitro studies showed that this induction of osteocalcin by Klf4 was regulated at its transcriptional level. Our results demonstrate that Klf4 regulates normal skeletal development through coordinating the differentiation and migration of osteoblasts, chondrocytes, vascular endothelial cells and osteoclasts.

Prevention of osteoradionecrosis of the mandibule in canine with low intensity ultrasound

Objective To investigate the repair effect of low intensity ultrasound on radioactive mandibule bone to provide new idea for prevention of osteoradionecrosis of the mandibule after irradiation.Methods The animal models of radioactive mandibular injury were made and treated with low intensity ultrasound.Low intensity ultrasound treatment group ( Group Ⅱ ) and not with low intensity ultrasound treatment group (Group Ⅰ ) were observed by microvessel density detection,micro-CT,ponceau trichrome stain and so on,and compared with control group.Then,the obtained data were statistically analyzed.Results The animal models of radioactive mandibular injury were successfully established.The microvessel density in Group Ⅱ was greater than that in Group Ⅰ.The bone volume fraction,trabecular thickness,bone surface/bone volume and the trabecular number in Group Ⅱ were significantly larger than those in Group Ⅰ (P ＜ 0.05).The ponceau trichrome staining showed that the osteocytes largely disappeared and that the cancellous bone trabecular atrophied in Group Ⅰ,while new bone formation,trabecular and numerous osteoblasts around trabecular were largely observed in Group Ⅱ.Conclusion Low intensity ultrasound has a good recovery effect on bone tissue after irradiation. Key words: Mandible; Osteoradionecrosis; Ultrasonic therapy; Microvessel density