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Appropriate regeneration of jawbone after dental or surgical procedures relies on the recruitment of osteoprogenitor cells able to differentiate into matrix-producing osteoblasts. In this context, photobiomodulation (PBM) has emerged as promising therapy to improve tissue regeneration and to facilitate wound healing processes. The aim of this study was to determine the effect of PBM on human osteoprogenitor cells isolated from mandibular trabecular bone. Bone marrow stromal cell cultures were established from 4 donors and induced toward osteogenic differentiation for 14 days in a standard osteogenic assay. Cells were irradiated with a combined red/near-infrared (NIR) laser following different schedules and expression of osteogenic, matrix-related, osteoclastogenic and inflammatory genes was analyzed by quantitative PCR. Gene expression analysis revealed no overall effects of PBM on osteogenic differentiation. However, a statistically significant reduction was observed in the transcripts of COL1A1 and MMP13, two important genes involved in the bone matrix homeostasis. Most important, PBM significantly downregulated the expression of RANKL, IL6 and IL1B, three genes that are involved in both osteoclastogenesis and inflammation. In conclusion, PBM with a red/NIR laser did not modulate the osteogenic phenotype of mandibular osteoprogenitors but markedly reduced their expression of matrix-related genes and their pro-osteoclastogenic and pro-inflammatory profile. [ABSTRACT FROM AUTHOR]

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. Key messages: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is chronic, autoimmune joint inflammation characterized by irreversible joint destruction. Besides increased resorption, destruction is a result of decreased bone formation, due to suppressed differentiation and function of the mesenchymal lineage-derived osteoblasts in inflammatory milieu. In this study, we analyzed the cellular composition of synovial tissue from 11 RA and 10 control patients harvested during planned surgeries in order to characterize resident synovial progenitor populations. Synovial cells were released by collagenase, and labeled for flow cytometry by two antibody panels: 1. CD3-FITC, CD14-PE, 7-AAD, CD11b-PECy7, CD235a-APC, CD19-APCeF780; and 2. 7-AAD, CD105-PECy7, CD45/CD31/CD235a-APC, and CD200-APCeF780. The proportions of lymphocytes (CD3+, CD19+) and myeloid (CD11b+, CD14+) cells were higher in synovial tissue from the patients with RA than in the controls. Among non-hematopoietic (CD45−CD31−CD235a−) cells, there was a decrease in the proportion of CD200+CD105− and increase in the proportion of CD200−CD105+ cells in synovial tissue from the patients with RA in comparison to the control patients. The proportions of both populations were associated with inflammatory activity and could discriminate between the RA and the controls. [ABSTRACT FROM AUTHOR]

Despite the best treatment, approximately 10% of fractures still face undesirable repair and result in delayed unions or non‐unions. Dynamic mechanical stimulation promotes bone formation, when applied at the correct time frame, with optimal loading magnitude, frequency, and repetition. Controlled mechanical loading significantly increases osteogenic cells during the matrix deposition phase of bone repair. In the bone defect, the blood vessel network guides the initial bone formation activities. A unique blood vessel subtype (Type H) exists in bone, which expresses high levels of CD31 and endomucin, and functions to couple angiogenesis and osteogenesis. However, how this form of controlled mechanical loading regulates the Type H vessels and promotes bone formation is still not clear. Sphingosine 1‐phosphate (S1P) participates in the bone anabolic process and is a key regulator of the blood vessel. Its receptor, sphingosine 1‐phosphate receptor 1 (S1Pr1), is a mechanosensitive protein that regulates vascular integrity. Therefore, we hypothesis that controlled anabolic mechanical loading promotes bone repair by acting on Type H vessels. To study the effect of S1Pr1 on loading induced‐bone repair, we utilized a stabilized tibial defect model, which allows for the application of anabolic mechanical loading. Mechanical loading upregulated S1Pr1 within the entire defect, with up to 80% expressed in blood vessels, as observed by deep tissue imaging. Additionally, S1Pr1 antagonism by W146 inhibited the anabolic effects of mechanical loading. We showed that mechanical loading or activating S1Pr1 could induce YAP nuclear translocation, a key regulator in the cell's mechanical response, in endothelial cells (ECs) in vitro. Inhibition of S1Pr1 in endothelial cells by siRNA reduced loading‐induced YAP nuclear translocation and expressions of angiogenic genes. In vivo, YAP nuclear translocation in Type H vessels was up‐regulated after mechanical loading but was inhibited by antagonizing S1Pr1. S1Pr1 agonist, FTY720, increased bone volume and Type H vessel volume, similar to that of mechanical stimulation. In conclusion, controlled anabolic mechanical loading enhanced bone formation mainly through Type H vessels in a S1Pr1‐dependent manner. [ABSTRACT FROM AUTHOR]

Biomaterials are used as implants for bone and dental disabilities. However, wear particles from the implants cause osteolysis following total joint arthroplasty (TJA). Ceramic implants are considered safe and elicit a minimal response to cause periprosthetic osteolysis. However, few reports have highlighted the adverse effect of ceramic particles such as alumina (Al2O3) on various cell types. Hence, we aimed to investigate the effect of Al2O3 particles on osteoprogenitors. A comparative treatment of Al2O3, Ti, and UHMWPE particles to osteoprogenitors at a similar concentration of 200 μg/mL showed that only Al2O3 particles were able to suppress the early and late differentiation markers of osteoprogenitors, including collagen synthesis, alkaline phosphatase (ALP) activity and mRNA expression of Runx2, OSX, Col1α, and OCN. Al2O3 particles even induced inflammation and activated the NFkB signaling pathway in osteoprogenitors. Moreover, bone-forming signals such as the WNT/β-catenin signaling pathway were inhibited by the Al2O3 particles. Al2O3 particles were found to induce the mRNA expression of WNT/β-catenin signaling antagonists such as DKK2, WIF, and sFRP1 several times in osteoprogenitors. Taken together, this study highlights a mechanistic view of the effect of Al2O3 particles on osteoprogenitors and suggests therapeutic targets such as NFĸB and WNT signaling pathways for ceramic particle-induced osteolysis. [ABSTRACT FROM AUTHOR]

HDAC4 is a class II histone deacetylation protein with a well-characterized role in chondrocyte differentiation and skeletal development, and dysregulated expression or haploinsufficiency of Hdac4 leads to skeletal formation and malformation disorders. The early lethality of hdac4 ablation mice hindered further investigation of its role in postnatal bone growth and development. Therefore, this study aims to investigate the significant role of Hdac4 in postnatal endochondral bone development using two mouse models with conditional deletion of Hdac4 in Sp7-expressing osteoprogenitors or chondrocytes and monitored postnatal bone development. The phenotype of Acan-CreERT2; Hdac4 fl/fl mice largely resembled that of conventional Hdac4 -/- mice. But phenotypic characterizations of mice with Hdac4 inactivation in Sp7-expressing osteoprogenitors (Sp7-Cre; Hdac4 fl/fl ) showed dwarfism with body and limb shortening and remarkable skeletal defects. Micro-computed tomography analysis of tibias further demonstrated that loss of Hdac4 expression impaired bone formation and microarchitecture, mainly characterized by dysplasia of trabecular and cortical bone in young mice. Our in vivo and in vitro data support a crucial role for Hdac4 in regulating osteoblast proliferation and differentiation, bone matrix protein production, angiogenesis, and ultimately trabecular and cortical bone formation. Moreover, RNA-seq analysis implicated Hdac4 in the regulation of key genes and pathways necessary to affect the accumulation of extracellular matrix, biological processes related to signal transduction, and skeletal growth. Collectively, our data show that postnatal expression of Hdac4 in Sp7-expressing osteoprogenitors provides essential regulatory oversight of endochondral bone formation, bone morphology, and homeostasis., Competing Interests: Conflict of interest All authors state no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1 -GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts. • Absence of CaMKK2 in early osteoblasts stimulates trabecular bone formation in both sexes. • Deletion of CaMKK2 in osteoprogenitors enhances trabecular bone mass but the increase is sustained only in females. • Periosteal bone formation is enhanced only in males following the conditional loss of CaMKK2 in osteoprogenitors. • The loss of CaMKK2 in early osteoblasts has a sex-divergent effect on osteoblast function. [ABSTRACT FROM AUTHOR]

Abstract Background Ankylosing spondylitis (AS) is characteristically male-predominant, and progressive spinal ankylosis affects male patients more severely; however, the hormonal effects in males with AS are poorly understood. Methods In the present study, the regulatory effects of dutasteride, a 5-α reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), were examined in curdlan-administered male SKG mice to determine spinal bone formation, bone metabolism-related markers, and interleukin (IL)-17A cytokine and T cell populations. In addition, the effects of DHT on primary osteoprogenitors from the facet joints of AS patients were assessed based on osteoblast-related parameters. DHT level was measured, and the correlation with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) was analyzed in AS patients. Results In curdlan-administered SKG mice, dutasteride treatment resulted in an increased accumulation of hydroxyapatite in the spine which was positively correlated with serum IL-17A levels. In the analysis of bone metabolism-related molecules, a decrease in sclerostin levels was observed in the sera in the dutasteride group. Continuous exposure to DHT resulted in fewer calcium deposits in AS osteoprogenitors during osteoblast differentiation. DHT-treated AS osteoprogenitors showed decreased osteocalcin and increased DKK1 and SOST1 mRNA expression, supporting the results of the in vivo experiments. Treatment with dutasteride upregulated bone formation in the spine of curdlan-administered SKG mice and DHT treatment downregulated osteoblast differentiation in vitro. Conclusions Treatment with dutasteride affected the bone formation in the spine of curdlan-treated SKG mice, and DHT treatment attenuated osteoblast differentiation in vitro. Therefore, contrary to what could be expected if osteoblasts contributed to spinal ankylosis, DHT inhibition might increase rather than decrease the progression of spinal ankylosis despite the higher levels of DHT observed in many AS patients.

Circulating osteogenic precursor (COP) cells are a population of progenitor cells in the peripheral blood with the capacity to form bone in vitro and in vivo. They have characteristics of the mesenchymal stem and progenitor pool found in the bone marrow; however, more recently, a population of COP cells has been identified with markers of the hematopoietic lineage such as CD45 and CD34. While this population has been associated with several bone pathologies, a lack of cell culture models and inconsistent characterization has limited mechanistic research into their behavior and physiology. In this study, we describe a method for the isolation of CD45+/CD34+/alkaline phosphatase (ALP) + COP cells via fluorescence-activated cell sorting, as well as their expansion and differentiation in culture. Hematopoietic COP cells are a discreet population within the monocyte fraction of the peripheral blood mononuclear cells, which form proliferative, fibroblastoid colonies in culture. Their expression of hematopoietic markers decreases with time in culture, but they express markers of osteogenesis and deposit calcium with differentiation. It is hoped that this will provide a standard for their isolation, for consistency in future research efforts, to allow for the translation of COP cells into clinical settings.

Rheumatoid arthritis (RA) is chronic, autoimmune joint inflammation characterized by irreversible joint destruction. Besides increased resorption, destruction is a result of decreased bone formation, due to suppressed differentiation and function of the mesenchymal lineage-derived osteoblasts in inflammatory milieu. In this study, we analyzed the cellular composition of synovial tissue from 11 RA and 10 control patients harvested during planned surgeries in order to characterize resident synovial progenitor populations. Synovial cells were released by collagenase, and labeled for flow cytometry by two antibody panels: 1. CD3-FITC, CD14-PE, 7-AAD, CD11b-PECy7, CD235a-APC, CD19-APCeF780; and 2. 7-AAD, CD105-PECy7, CD45/CD31/CD235a-APC, and CD200-APCeF780. The proportions of lymphocytes (CD3+, CD19+) and myeloid (CD11b+, CD14+) cells were higher in synovial tissue from the patients with RA than in the controls. Among non-hematopoietic (CD45−CD31−CD235a−) cells, there was a decrease in the proportion of CD200+CD105− and increase in the proportion of CD200−CD105+ cells in synovial tissue from the patients with RA in comparison to the control patients. The proportions of both populations were associated with inflammatory activity and could discriminate between the RA and the controls.

Biomaterials are used as implants for bone and dental disabilities. However, wear particles from the implants cause osteolysis following total joint arthroplasty (TJA). Ceramic implants are considered safe and elicit a minimal response to cause periprosthetic osteolysis. However, few reports have highlighted the adverse effect of ceramic particles such as alumina (Al2O3) on various cell types. Hence, we aimed to investigate the effect of Al2O3 particles on osteoprogenitors. A comparative treatment of Al2O3, Ti, and UHMWPE particles to osteoprogenitors at a similar concentration of 200 μg/mL showed that only Al2O3 particles were able to suppress the early and late differentiation markers of osteoprogenitors, including collagen synthesis, alkaline phosphatase (ALP) activity and mRNA expression of Runx2, OSX, Col1α, and OCN. Al2O3 particles even induced inflammation and activated the NFkB signaling pathway in osteoprogenitors. Moreover, bone-forming signals such as the WNT/β-catenin signaling pathway were inhibited by the Al2O3 particles. Al2O3 particles were found to induce the mRNA expression of WNT/β-catenin signaling antagonists such as DKK2, WIF, and sFRP1 several times in osteoprogenitors. Taken together, this study highlights a mechanistic view of the effect of Al2O3 particles on osteoprogenitors and suggests therapeutic targets such as NFĸB and WNT signaling pathways for ceramic particle-induced osteolysis.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (−37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Purpose: The aim of this study was to evaluate the bone regeneration potential of concentrated bone marrow aspirate (BMA)-coated hydroxyapatite (HA) for reconstruction of mandibular defects caused by the removal of benign pathologies. Patients and Methods: This prospective clinical study included ten patients with histopathologically proven benign pathologies of the mandible measuring

Recruitment and proliferation of osteoprogenitors during the reversal-resorption phase, and their differentiation into mature bone-forming osteoblasts is crucial for initiation of bone formation during bone remodeling. This study investigates the osteoprogenitors' gradual recruitment, proliferation, and differentiation into bone-forming osteoblasts within intracortical remodeling events of healthy adolescent humans. The study was conducted on cortical bone specimens from 11 adolescent human controls – patients undergoing surgery due to coxa valga. The osteoprogenitor recruitment route and differentiation into osteoblasts were backtracked using immunostainings and in situ hybridizations with osteoblastic markers (CD271/ NGFR , osterix/ SP7 , COL3A1 and COL1A1). The osteoblastic cell populations were defined based on the pore surfaces, and their proliferation index (Ki67), density and number/circumference were estimated in multiplex-immunofluorescence (Ki67, TRAcP, CD34) stained sections. During the reversal-resorption phase, osteoclasts are intermixed with (COL3A1 + NFGR +) osteoblastic reversal cells, which are considered to be osteoprogenitors of (COL1A1 + SP7 +) bone-forming osteoblasts. Initiation of bone formation requires a critical density of these osteoprogenitors (43 ± 9 cells/mm), which is reached though proliferation (4.4 ± 0.5 % proliferative) and even more so through recruitment of osteoprogenitors, but challenged by the ongoing expansion of the canal circumference. These osteoprogenitors most likely originate from osteoblastic bone lining cells and mainly lumen osteoprogenitors, which expand their population though proliferation (4.6 ± 0.3 %) and vascular recruitment. These lumen osteoprogenitors resemble canopy cells above trabecular remodeling sites, and like canopy cells they extend above bone-forming osteoblasts where they may rejuvenate the osteoblast population during bone formation. Initiation of bone formation during intracortical remodeling requires a critical density of osteoprogenitors on eroded surfaces, which is reached though proliferation and recruitment of local osteoprogenitors: bone lining cells and lumen osteoprogenitors. • Osteoblastic reversal cells are identical to osteoprogenitors • Initiation of bone formation requires a critical density of osteoprogenitors on eroded surfaces • Osteoprogenitor expansion on eroded surfaces results from recruitment and some internal proliferation. • Bone lining cells may also be a source of osteoprogenitors during remodeling of existing canals [ABSTRACT FROM AUTHOR]

Elucidating the effects of mechanical stimulation on bone repair is crucial for optimization of the healing process. Specifically, the regulatory role that mechanical loading exerts on the osteogenic stem cell pool and vascular morphology during healing is incompletely understood. Because dynamic loading has been shown to enhance osteogenesis and repair, we hypothesized that loading induces the expansion of the osteoprogenitor cell population within a healing bone defect, leading to an increased presence of osteogenic cells. We further hypothesized that loading during the repair process regulates vascular and collagen matrix morphology and spatial interactions between vessels and osteogenic cells. To address these hypotheses, we used a mechanobiological bone repair model, which produces a consistent and reproducible intramembranous repair response confined in time and space. Bilateral tibial defects were created in adult C57BL/6 mice, which were subjected to axial compressive dynamic loading either during the early cellular invasion phase on postsurgical days (PSDs) 2 to 5 or during the matrix deposition phase on PSD 5 to 8. Confocal and two‐photon microscopy was used to generate high‐resolution three‐dimensional (3D) renderings of longitudinal thick sections of the defect on PSD 10. Endomucin (EMCN)‐positive vessels, Paired related homeobox 1 (Prrx1+) stem cell antigen‐1 positive (Sca‐1+) primitive osteoprogenitors (OPCs), and osterix positive (Osx+) preosteoblasts were visualized and quantified using deep tissue immunohistochemistry. New bone matrix was visualized with second harmonic generation autofluorescence of collagen fibers. We found that mechanical loading during the matrix deposition phase (PSD 5 to 8) increased vessel volume and number, and aligned vessels and collagen fibers to the load‐bearing direction of bone. Furthermore, loading led to a significant increase in the proliferation and number of Prrx1+ Sca‐1+ primitive OPCs, but not Osx+ preosteoblasts within the defect. Together, these data illustrate the adaptation of both collagen matrix and vascular morphology to better withstand mechanical load during bone repair, and that the mechanoresponsive cell population consists of the primitive osteogenic progenitors. © 2019 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an inflammatory joint disease that eventually leads to permanent bone and cartilage destruction. Fas has already been established as the regulator of inflammation in RA, but its role in bone formation under arthritic conditions is not completely defined. The aim of this study was to assess the effect of Fas inactivation on the bone damage during murine antigen-induced arthritis. Subchondral bone of wild-type (WT) and Fas-knockout (Fas-/-) mice was evaluated by histomorphometry and microcomputerized tomography. Proportions of synovial bone and cartilage progenitors were assessed by flow cytometry. Synovial bone and cartilage progenitors were purified by fluorescence-activated cell sorting and expression of Fas and Fas-induced apoptosis were analyzed in vitro. Results showed that Fas-/- mice developed attenuated arthritis characterized by preserved epiphyseal bone and cartilage. A proportion of the earliest CD200+ bone and cartilage progenitors was reduced in WT mice with arthritis and was unaltered in Fas-/- mice. During osteoblastic differentiation in vitro, CD200+ cells express the highest levels of Fas and are removed by Fas ligation. These results suggest that Fas-induced apoptosis of early CD200+ osteoprogenitor population represents potential mechanism underlying the impaired bone formation in arthritis, so their preservation may represent the bone-protective mechanism during arthritis. [ABSTRACT FROM AUTHOR]

Objective: Bone morphogenetic protein receptor type 2 (BMPR2) has been associated with radiographic changes in ankylosing spondylitis (AS), but further characterization of the cellular signaling pathway in osteoprogenitor (OP) is not clearly understood. The aim of this study was to investigate the expression of BMPR2 and bone morphogenetic protein 2 (BMP2)-mediated responsibility in AS., Methods: We collected 10 healthy control (HC) and 14 AS-OPs derived from facet joints. Subsequently, we then conducted RNA sequencing with two samples per group and selected BMP-related genes. Facet joint tissues and derived primary OPs were evaluated by validation of selected RNA sequencing data, immunohistochemistry, and comparison of osteogenic differentiation potential., Results: Based on RNA-sequencing analysis, we found that BMPR2 expression is higher in AS-OPs compared to in HC-OPs. We also validated the increased BMPR2 expression in facet joint tissues with AS and its derived OPs in messenger RNA and protein levels. Additionally, primary AS-OPs showed much greater response to osteogenic differentiation induced by BMP2 and a higher capacity for smad1/5/8-induced RUNX2 expression compared to HCs., Conclusion: The expression of BMPR2 was found to be significantly increased in facet joint tissues of patients with AS. These findings suggest that BMPR2 may play a role in the BMP2-mediated progression of AS., Competing Interests: CONFLICT OF INTEREST No potential conflict of interest relevant to this article was reported., (Copyright © 2023 by The Korean College of Rheumatology. All rights reserved.)

Changes in bone marrow niches can lead to the occurrence of myelodysplastic syndrome (MDS). As an important part of the bone marrow niche, osteoblasts serve a key role in the progression of MDS. The present study investigated the quantity and function of osteoblasts and, through in vitro assays, detected changes in signaling pathways and the association with progression in MDS patients. The ratios of osteoprogenitors (CD34+OCN+) and OCN+CD34‑Lin‑ osteoblasts in MDS patients were significantly less than those of normal controls. The results of this study demonstrated that the quantity and activity of osteoblasts in MDS patients were lower than those in normal controls. Furthermore the activity of osteoblasts in patients correlated with the severity of MDS. The quantity of osteoblasts cultured in vitro from high‑risk and very high‑risk MDS patients (WHO Classification‑Based Prognostic Scoring System score 3‑6) was decreased. The levels of T‑cell immunoglobulin and mucin domain‑containing 3 (TIM3) and Jagged 1 were also increased in the osteoblasts in vitro. These results indicated that osteoblasts are abnormally altered in MDS patients, and that there are associations between abnormal changes of osteoblasts and the severity of MDS. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to evaluate the bone regeneration potential of concentrated bone marrow aspirate (BMA)-coated hydroxyapatite (HA) for reconstruction of mandibular defects caused by the removal of benign pathologies. Patients and Methods: This prospective clinical study included ten patients with histopathologically proven benign pathologies of the mandible measuring <5 cm anteroposteriorly, who were treated with enucleation or marginal resection, followed by autologous concentrated BMA-coated synthetic biphasic HA (HA and beta-tricalcium phosphate) graft placement. Clinical and radiological evaluations of grafted sites of the mandible were done at 1 week, 1, 3, and 6 months postoperatively using Irwin's radiologic staging and grayscale histogram. Results: All patients (10/10, 100%) had proper incorporation of the graft with the normal adjacent bone. Grayscale histogram revealed the initial stages of graft resorption, followed by formation of new bone-grafted sites. No complications such as infection and total graft loss were encountered except for one patient who had partial wound dehiscence that responded well to local wound care and resuturing. Conclusion: Concentrated BMA-coated synthetic HA effectively promotes bone regeneration in small-to-moderate-sized defects of the mandible. [ABSTRACT FROM AUTHOR]

Backgroud Osteoporosis is one of the bone-metabolic diseases associated with decreased bone renewal and bone mineral density. β-aminoisobutyric acid (BAIBA), a natural thymine catabolite, can reduce inflammation in skeletal muscle and alleviate hepatic endoplasmic reticulum stress. However, the roles of BAIBA in osteoblast proliferation and differentiation remain largely unknown. Methods The cultured MC3T3-E1 cells received various treatments in this study, including BAIBA alone, H 2 O 2 alone, BAIBA plus N -acetyl- l -cysteine and BAIBA plus apocynin. Cell proliferation was determined by CCK-8 assay and 3 H-Thymidine incorporation. Cell differentiation was evaluated by determining mRNA level of differentiation makers and ALP, and ALP activity. Reactive oxygen species (ROS) were determined by DHE staining while superoxide anion level and NAD(P)H oxidase activity were determined by the lucigenin-derived chemiluminescence method. The content of hydrogen peroxide (H 2 O 2 ) was detected using a commercial kit. The level of NOX1, NOX2 and NOX4 was determined by Western-blot or qRT-PCR. Results We show that treatment of BAIBA stimulated the proliferation of MC3T3-E1 osteoprogenitor cells and enhanced the gene expression of osteoblast differentiation markers. Incubation of MC3T3-E1 cells with BAIBA evoked increases in NAD(P)H oxidase-derived reactive oxygen species (ROS). Scavenging of reactive oxygen species ( N -acetyl- l -cysteine) or inhibition of NAD(P)H oxidase (apocynin) abolished the BAIBA-elicited proliferation and differentiation of MC3T3-E1 cells. Conclusion Our results provide the first evidence that BAIBA stimulates proliferation and differentiation of osteoprogenitor cells via activation of NAD(P)H oxidase/ROS signaling. [ABSTRACT FROM AUTHOR]

Subchondral bone tissue plays a key role in the initiation and progression of human and experimental osteoarthritis and has received considerable interest as a treatment target. Elevated bone turnover and remodeling leads to subchondral bone sclerosis that is characterized by an increase in bone material that is less mineralized. The aim of this study was to investigate whether perturbations in subchondral bone-resident progenitor cells might play a role in aberrant bone formation in osteoarthritis. Colony formation assays indicated similar clonogenicity of progenitor cells from non-sclerotic and sclerotic subchondral trabecular bone tissues of osteoarthritic knee and hip joints compared with controls from iliac crest bone. However, the osteogenic potential at the clonal level was approximately two-fold higher in osteoarthritis than controls. An osteogenic differentiation assay indicated an efficient induction of alkaline phosphatase activity but blunted in vitro matrix mineralization irrespective of the presence of sclerosis. Micro-computed tomography and histology demonstrated the formation of de novo calcified tissues by osteoblast-like cells in an ectopic implantation model. The expression of bone sialoprotein, a marker for osteoblast maturation and mineralization, was significantly less in sclerotic progenitor cells. Perturbation of resident progenitor cell function is associated with subchondral bone sclerosis and may be a treatment target for osteoarthritis. [ABSTRACT FROM AUTHOR]

Aim: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. Methods: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. Results: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. Conclusion: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair. [ABSTRACT FROM AUTHOR]

Periprosthetic osteolysis remains the leading obstacle for total joint replacements. Primarily, it was thought that aseptic loosening is mainly caused by macrophage mediated inflammatory process arising from production of wear debris. The role of osteoclasts and its sequential bone resorption ability has been extensively studied, but little is known about impaired osteogenesis during osteolysis. In the current study, we have tried to delineate the regulatory mechanism of osteogenic signals by Ti particles in osteoprogenitor cells as well its participatory role in wear debris induced osteolysis. Implantation of Ti particles on mice calvaria induced pro-inflammatory response, elevated expression of COX2 and reduced the expression of Osterix. Treatment of Ti particles to MC3T3 E-1 cells displayed decreased osteogenic activity including ALP activity, mineralization and mRNA levels several osteogenic genes. Moreover, the basal activity of WNT and BMP signaling pathways was suppressed in MC3T3 E-1 cells treated with Ti particles. As an early response to Ti particles, MC3T3 E-1 cells showed activation of ERK and JNK. Co-inhibition of ERK and JNK with their specific inhibitors resulted in partial recovery of WNT and BMP signaling activity as well as ALP activity and collagen synthesis. Finally, LiCl mediated activation of WNT signaling pathway demonstrated rescue of Ti particle facilitated suppression of Osterix expression in mice calvaria. Our results provide evidences that WNT signaling pathway is regulated by ERK, JNK, and BMP signaling pathway during wear debris induced inflammatory osteolysis and may be considered as suitable therapeutic targets for the treatment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 912-926, 2017. [ABSTRACT FROM AUTHOR]

Endochondral ossification, the mechanism responsible for the development of the long bones, is dependent on an extremely stringent coordination between the processes of chondrocyte maturation in the growth plate, vascular expansion in the surrounding tissues, and osteoblast differentiation and osteogenesis in the perichondrium and the developing bone center. The synchronization of these processes occurring in adjacent tissues is regulated through vigorous crosstalk between chondrocytes, endothelial cells and osteoblast lineage cells. Our knowledge about the molecular constituents of these bidirectional communications is undoubtedly incomplete, but certainly some signaling pathways effective in cartilage have been recognized to play key roles in steering vascularization and osteogenesis in the perichondrial tissues. These include hypoxia-driven signaling pathways, governed by the hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF), which are absolutely essential for the survival and functioning of chondrocytes in the avascular growth plate, at least in part by regulating the oxygenation of developing cartilage through the stimulation of angiogenesis in the surrounding tissues. A second coordinating signal emanating from cartilage and regulating developmental processes in the adjacent perichondrium is Indian Hedgehog (IHH). IHH, produced by pre-hypertrophic and early hypertrophic chondrocytes in the growth plate, induces the differentiation of adjacent perichondrial progenitor cells into osteoblasts, thereby harmonizing the site and time of bone formation with the developmental progression of chondrogenesis. Both signaling pathways represent vital mediators of the tightly organized conversion of avascular cartilage into vascularized and mineralized bone during endochondral ossification. [ABSTRACT FROM AUTHOR]

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 ( EfnB1 fl/fl ) in osteogenic progenitor cells, under the control of the osterix ( Osx:Cre ) promoter. The Osx:EfnB1 −/− mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1 −/− mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1 −/− mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification. [ABSTRACT FROM AUTHOR]

We have found that the previously uncharacterized bone morphogeneticprotein-9 (BMP9) is one of the most osteogenic factors.However, it is unclear if BMP9 cross-talks with TGFβ1 during osteogenicdifferentiation. Using the recombinant BMP9 adenovirus,we find that low concentration of rhTGFβ1 synergistically inducesalkaline phosphatase activity in BMP9-transduced C3H10T1/2cells and produces more pronounced matrix mineralization.However, higher concentrations of TGFβ1 inhibit BMP9-inducedosteogenic activity. Real-time PCR and Western blotting indicatethat BMP9 in combination with low dose of TGFβ1 potentiates theexpression of later osteogenic markers osteopontin, osteocalcinand collagen type 1 (COL1a2), while higher concentrations ofTGFβ1 decrease the expression of osteopontin and osteocalcin butnot COL1a2. Cell cycle analysis reveals that TGFβ1 inhibitsC3H10T1/2 proliferation in BMP9-induced osteogenesis and restrictsthe cells in G0/G1 phase. Our findings strongly suggest thatTGFβ1 may exert a biphasic effect on BMP9-induced osteogenicdifferentiation of mesenchymal stem cells.

Subchondral bone tissue plays a key role in the initiation and progression of human and experimental osteoarthritis and has received considerable interest as a treatment target. Elevated bone turnover and remodeling leads to subchondral bone sclerosis that is characterized by an increase in bone material that is less mineralized. The aim of this study was to investigate whether perturbations in subchondral bone-resident progenitor cells might play a role in aberrant bone formation in osteoarthritis. Colony formation assays indicated similar clonogenicity of progenitor cells from non-sclerotic and sclerotic subchondral trabecular bone tissues of osteoarthritic knee and hip joints compared with controls from iliac crest bone. However, the osteogenic potential at the clonal level was approximately two-fold higher in osteoarthritis than controls. An osteogenic differentiation assay indicated an efficient induction of alkaline phosphatase activity but blunted in vitro matrix mineralization irrespective of the presence of sclerosis. Micro-computed tomography and histology demonstrated the formation of de novo calcified tissues by osteoblast-like cells in an ectopic implantation model. The expression of bone sialoprotein, a marker for osteoblast maturation and mineralization, was significantly less in sclerotic progenitor cells. Perturbation of resident progenitor cell function is associated with subchondral bone sclerosis and may be a treatment target for osteoarthritis.

Aggrecan (Acan) is a large proteoglycan molecule constituting the extracellular matrix of cartilage, secreted by chondrocytes. To specifically target the chondrocyte lineage, researchers have widely used the AcanCreER mouse model. Evaluation of specificity and efficiency of recombination, requires Cre animals to be crossed with reporter mice. In order to accurately interpret data from Cre models, it is imperative to consider A) the amount of recombination occurring in cells/tissues that are not intended for targeting (i.e. , non-specific expression), B) the efficiency of Cre recombination, which can depend on dose and duration of tamoxifen treatment, and C) the activation of CreER without tamoxifen induction, known as "Cre leakage." Using a highly sensitive reporter mouse (Ai9, tdTomato), we performed a comprehensive analysis of the AcanCreER system. Surprisingly, we observed expression in cells within the periosteum. These cells expand at a stage when chondrocytes are not yet present within the forming callus tissue (Acan/Ai9+ cells). In pulse-chase experiments, we confirmed that fibroblastic Acan/Ai9+ cells within the periosteum can directly give rise to osteoblasts. Our results show that Acan/Ai9+ is not specific for the chondrocyte lineage in the fracture callus or with the tibial holes. The expression of AcanCreER in periosteal progenitor cells complicates the interpretation of studies evaluating the transition of chondrocytes to osteoblasts (termed transdifferentiation). Awareness of these issues and the limitations of the system will lead to better data interpretation. • Aggrecan-CreER is not specifically expressed in the chondrocyte lineage. • AcanCreER is activated in periosteal fibroblasts that expands during fracture healing. • AcanCreER cells in bone injury can directly give rise to osteoblasts. [ABSTRACT FROM AUTHOR]

Aim Circulating osteoprogenitors and receptor activator of nuclear factor kappa-B ligand ( RANKL) expression in immune cells have been implicated in the pathogenesis of osteoporosis and vascular calcification. The role played by statin therapy in the bone-vascular axis is unknown. Methods Twenty naïve postmenopausal osteoporotic hypercholesterolemic women were treated with Atorvastatin 40 mg/day for 3 months. Gene expression analysis was performed to assess modification in osteoprotegerin ( OPG)/ RANK/ RANKL expression in isolated T cells and monocytes. A flow cytometry analysis was used to study changes in the levels of circulating osteoprogenitor cells. Results After 3 months of treatment, Atorvastatin significantly reduced total cholesterol and LDL-C, without affecting HDL-C and triglycerides. Among circulating bone and phosphocalcium homeostasis markers, we found a significant increase in OPG levels ( P < 0.01) and a modest reduction in osteocalcin ( OCN) ( P < 0.05). We also observed a significant reduction in RANKL expression in T cells ( P < 0.05). No differences were found in the expression of RANK in T cells and RANKL and RANK in monocytes. OPG expression was low in both immune cell types and was not affected by the treatment. As for circulating osteoprogenitors, we found a significant reduction of CD34+ BAP+ ( P < 0.05) and CD34+ OCN+ BAP+ ( P < 0.05) cells. In vitro studies showed that Atorvastatin reduced RANKL expression in activated human T-lymphoblastoid cells (Jurkat cell line). Conclusions Three-month Atorvastatin treatment leads to a reduction in circulating osteoprogenitor cells and RANKL expression in T cells, as well as increase in OPG serum levels. These data suggest that statins could have protective effects in the bone-vascular axis. [ABSTRACT FROM AUTHOR]

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1bright/Glycophorin A− phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis. S tem C ells 2016;34:418-430 [ABSTRACT FROM AUTHOR]

Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD+ B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1. [ABSTRACT FROM AUTHOR]

Purpose: The cellular component of subchondral bone is thought to be responsible for aberrant bone remodeling in osteoarthritis (OA). Direct phenotypical analysis of the cellular compartment is critical to better understand the OA disease process. This study provides proof-of-principle for flow cytometry-based phenotyping of isolated subchondral trabecular bone (STB) marrow cells without prior use of cell culture techniques.Methods: Tibial plateaus were obtained from OA patients undergoing total knee arthroplasty. Subchondral bone chips were digested with collagenase IA and single cell suspensions were directly phenotyped using flow cytometry. Cells were analyzed for the expression of alkaline phosphatase (ALP) as osteoblast/osteoprogenitor marker and monocyte/macrophage markers (CD14, CD68, HLA-DR, CD115).Results: MTT staining revealed abundant viable cells in the bone marrow compartment of STB prior to digestion, which were efficiently released by collagenase. Within the CD45-negative cell fraction, approximately 20% of the cells were positive for the early osteoblast/osteoprogenitor marker ALP. Within the CD45+ hematopoietic cell fraction, the majority of cells were of monocytic origin (>80%) displaying strong surface expression of CD14. Discreet macrophage populations (CD14+/HLA-DR+/CD68+) and putative osteoclast progenitors (CD45+/HLA-DR-/CD115+) were unequivocally identified. Osteoblast, macrophage and osteoclast progenitor presence in the subchondral bone unit (SBU) was confirmed by (immuno)histochemical staining for osteocalcin, CD68 and tartrate-resistant acid phosphatase, respectively.Conclusions: Flow cytometric analysis is a valuable methodology to study the cellular compartment of STB marrow. This method provides a proof of principle that the whole resident cell population can be directly phenotypically characterized without the prior use of cell culture techniques. [ABSTRACT FROM AUTHOR]

The success of a clinically-applicable bone tissue engineering construct for large area bone defects depends on its ability to allow for homogeneous bone regeneration throughout the construct. Insufficient vascularization, and consequently inadequate oxygen tension, throughout constructs has been largely cited as the most significant obstacle facing successful bone regeneration in large area defects. The development of constructs that support bone and vessel-forming cell growth and function throughout the scaffold structure are desired for large-area bone defect repair. Here, we developed oxygen tension-controlled matrices that support more homogenous oxygen levels throughout the constructs. Specifically, we examined polylactic co-glycolic acid (PLGA) scaffolds with optimized pore distribution and the percent pore volumes, and demonstrated significantly decreased oxygen and pH gradient from the exterior of the construct to the interior after long-term cell culture in vitro. We confirmed the ability of these optimized constructs to support the cellular survival via live/dead assay. In addition, we examined their ability to support the maintenance of two clinically relevant progenitor cell populations for bone tissue engineering and vascularization, namely mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs), and confirmed the expression of key bone and vascular markers via immunofluorescence. [ABSTRACT FROM AUTHOR]

Highlights: [•] Coupling of bone resorption to formation occurs at the osteoclast-canopy interface. [•] The reversal phase is prolonged when the osteoclast-canopy interface is reduced. [•] Alendronate reduces the osteoclast-canopy interface whereas odanacatib increases it. [Copyright &y& Elsevier]

Aim Sinus augmentation procedures commonly employ osteoconductive scaffolding materials to stimulate and support bone formation. The aim of this study was to develop a simple screening methodology for the evaluation of the osteoconductive potential of various bone graft materials prior to clinical use. Materials and methods Materials tested were Bio-Oss, Bi-Ostetic, OraGraft, and ProOsteon. These Simple and composite bone substitutes were embedded with osteoprogenitor cells derived from either the human maxillary sinus schneiderian membrane (h MSSM) or from maxillary tuberosity bone marrow and then monitored both in vitro and in vivo. Results Cell adherence and proliferation was most pronounced in OraGraft, followed by ProOsteon. In vivo bone formation, within the bone graft, was also observed, with most marked results in OraGraft and ProOsteon grafts. Conclusions The proposed osteoconductivity testing method proved simple, informative, and reliable for the purpose of screening candidate biomaterials for sinus lifting or sinus augmentation. [ABSTRACT FROM AUTHOR]

Bone repair is a complicated process that includes many types of cells, signaling molecules, and growth factors. Fracture healing involves a temporally and spatially regulated biologic process that involves recruitment of stem cells to the injury site, tissue specific differentiation, angiogenesis, and remodeling. In light of its proximity to bone and abundant vascularity, muscle is an important potential source of cells and signals for bone healing. More complete understanding of the role of muscle in bone formation and repair will provide new therapeutic approaches to enhance fracture healing. Recent studies establish that muscle-derived stem cells are able to differentiate into cartilage and bone and can directly participate in fracture healing. The role of muscle-derived stem cells is particularly important in fractures associated with more severe injury to the periosteum. Sarcopenia is a serious consequence of aging, and studies show a strong association between bone mass and lean muscle mass. Muscle anabolic agents may improve function and reduce the incidence of fracture with aging. [ABSTRACT FROM AUTHOR]

The skeleton is a high-renewal organ that undergoes ongoing cycles of remodeling. The regenerative bone formation arm ultimately declines in the aging, postmenopausal skeleton, but current therapies do not adequately address this deficit. Bone marrow is the primary source of the skeletal anabolic response and the mesenchymal stem cells (MSCs), which give rise to bone matrix-producing osteoblasts. The identity of these stem cells is emerging, but it now appears that the term 'MSC' has often been misapplied to the bone marrow stromal cell (BMSC), a progeny of the MSC. Nevertheless, the changes in BMSC phenotype associated with age and estrogen depletion likely contribute to the attenuated regenerative competence of the marrow and may reflect alterations in MSC phenotype. Here we summarize current concepts in bone marrow MSC identity, and within this context, review recent observations on changes in bone marrow population dynamics associated with aging and menopause. [ABSTRACT FROM AUTHOR]

Background. Recent approaches have sought to harness the potential of stem cells to regenerate bone. that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an atitologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use. Methods. Femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency. Results. BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate. Conclusions. These studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healing. [ABSTRACT FROM AUTHOR]

Objective and design: The activity of immune cells affects the balance between bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts, respectively. This study was aimed at determining the possible interaction between inflammatory conditions and collagen type I degrading MMP (mainly MMP-2 and MMP-9) synthesis and secretion in rat osteoprogenitors. Materials and methods: The study was performed using primary rat bone marrow-derived osteoprogenitors during their advanced osteogenesis. Biochemical, immunohistochemical, and molecular biology techniques were used to investigate the influence of pro-inflammatory cytokines on MMP-2 and MMP-9 synthesis and secretion in osteoprogenitors. Results: Results indicated that both synthesis and secretion of MMPs (MMP-1, -2, -8, -9, and -13) were significantly induced after pro-inflammatory cytokine treatments, except MMP-2, whose levels remained unchanged. NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) inhibition assays showed that induced MMP-9 secretion by inflammatory cytokines was mediated by activation of NF-κB via the classical pathway and that oxidants play a significant role in this signal transduction pathway. In contrast, no such effect was observed for synthesis of MMP-2. Conclusions: These results indicate the possibility that inflammatory processes may trigger osteoblasts to absorb bone by secreting elevated levels of MMPs capable of degrading collagen type I, especially MMP-9 which is upregulated due to increased NF-κB transcription activity. [ABSTRACT FROM AUTHOR]

Abstract: Bone formation and remodeling require generation of osteoprogenitors from bone marrow stem cells (MSC), which are regulated by growth factors and hormones, with putative roles in mesenchymal cell differentiation. Hepatocyte growth factor (HGF) is a pleiotropic growth factor, and together with its high affinity receptor cMet are widely expressed in normal tissues. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D; produced mainly in the kidney, but also by osteoblasts. We previously reported that HGF and 1,25OHD act together to increase osteogenic differentiation of human MSC (hMSC) potentially through increasing p53. Although p53 does not induce the vitamin D receptor (VDR), p63, a member of the p53 family of transcription factors has been reported to up-regulate VDR expression in some tumor cell lines, and thus might play a part in HGF-regulated VDR expression. Our hypothesis is that the combination of HGF and 1,25OHD can induce hMSC differentiation by up-regulation of 1,25OHD and/or VDR expression to increase cell response(s) to 1,25OHD. Using real-time RT-qPCR, Western blots, luciferase reporter assays, and siRNAs, as well as antibodies to specific signaling molecules we showed that HGF induced VDR gene expression, as well as up-regulated p63 gene expression. p63 gene knockdown by siRNA eliminated the effects of HGF on VDR gene expression as measured by RT-qPCR, Western blots and luciferase reporter assay, and downstream on osteogenic differentiation markers, including alkaline phosphatase staining. Differentiation is a coordinated process of cell cycle exit and tissue-specific gene expression. These results suggest HGF might be a good candidate to coordinate the regulation of these two processes during hMSC osteogenic differentiation. p63 could be a key connecting molecule on the pathway of HGF-induced VDR expression. Understanding the role of these factors and their actions could have important clinical implications for the use of hMSC in the development of novel stem cell therapies. [Copyright &y& Elsevier]

Abstract: Human bone marrow-derived stem cells (hMSCs) are a major source of osteoprogenitors. Hepatocyte growth factor (HGF), a glycoprotein constitutively produced by hMSCs, is reported to act on differentiated osteoblasts and also osteoclasts. Moreover, HGF has been shown by us and others to enhance osteoblastic differentiation from hMSCs. Typically, the pro-differentiation effects of HGF have required cooperative action with regulatory factors such as vitamin D or bone matrix material. Here, we have pursued the molecular mechanisms underlying the osteogenic effect of HGF on hMSCs, the principal precursors to bone forming cells. HGF treatment of hMSCs reduced the cell number over time and increased G1/S cell-cycle arrest compared to control (non-treated) cells. RT-qPCR showed treatment with HGF increased gene expression of the cell-cycle inhibitors p53, p21, and p27, possibly explaining the cell growth inhibition and G1 arrest, a step critical to phenotypic differentiation. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect on hMSCs and the HGF-mediated increase in p53, p21, and p27, strongly supporting a role for these cell-cycle inhibitors in HGF''s regulation of hMSCs. HGF in combination with a known inducer of osteogenic differentiation, 1,25-dihydroxyvitamin D, significantly increased cell maturation/differentiation as indicated by an increase in several osteoblast markers. Taken together these results demonstrate that HGF significantly enhances hMSC osteoblast differentiation by 1,25-dihydroxyvitamin D. [Copyright &y& Elsevier]