Bone Lining Cells Research Articles

Tri-Lineage Differentiation Potential of Stem Cells from Human Exfoliated Deciduous Teeth - An In-Vitro Study

ABSTRACT Background: The use of SHED might bring advantages for tissue engineering over the use of stem cells from adult human teeth as SHEDs were reported to have higher proliferation rate and increase cell population doublings as compared with stem cells from permanent teeth. Objective: The study’s objective was to assess the growth kinetics and tri-lineage differentiation capacity of stem cells derived from human exfoliated deciduous teeth. Material and Method: After being propagated through multiple sequential subcultures, adherent fibroblastic cells in the pulp tissue culture from human exfoliation teeth were evaluated to assess their tripotent differentiation potential into bone, cartilage, and adipose cell lineages. Result: The stem cells derived from dental pulp had a comparatively high ability for proliferation and successfully differentiated into chondrocytes and osteocytes while less significant for adipocytes. Conclusion: Stem cells from Human Exfoliated Deciduous teeth are mesenchymal stem cells which are multipotent and can serve as a promising incentive for therapeutic and future research purposes.

Evaluation of romosozumab's effects on bone marrow adiposity in postmenopausal osteoporotic women: results from the FRAME bone biopsy sub-study.

Romosozumab, a humanized monoclonal antibody that binds and inhibits sclerostin, produces a marked increase in bone formation with a concomitant decreased bone resorption. This transient rise in bone formation in the first 2 months of treatment is mainly due to an increased modeling-based bone formation. This requires the recruitment and differentiation of osteoblasts, one possibility being a preferential switch in commitment of precursors to osteoblasts over adipocytes. The purpose of this study was to analyze the marrow adiposity in transiliac bone biopsies at months 2 or 12 from the FRAME biopsy sub-study in patients receiving romosozumab or placebo. The total adipocyte area, number, and density were measured on the total cancellous bone area. The size and shape at the individual adipocyte level were assessed including the mean adipocyte area, perimeter, min and max diameters, and aspect ratio. No significant difference in total adipocyte area, number, or density between placebo and romosozumab groups was observed at months 2 and 12, and no difference was observed between 2 and 12months. After 2 or 12months, romosozumab did not modify the size or shape of the adipocytes. No relationship between the adipocyte parameters and the dynamic parameters of bone formation could be evidenced. In conclusion, based on the analysis of a small number of biopsies, no effect of romosozumab on bone marrow adiposity of iliac crest was identified after 2 and 12months suggesting that the modeling-based formation observed at month 2 was not due to a preferential commitment of the precursor to osteoblast over adipocyte cell lines but may result from a reactivation of bone lining cells and from a progenitor pool independent of the marrow adipocyte population.

Estrogen deficiency in rats alters the expression and localization of redox proteins in the bone and bone marrow

Imbalances in bone formation/bone resorption ratio can occur under diverse conditions and diseases leading to an alteration of bone mineral density and strength. One of the most dramatic changes altering bone’s microarchitecture occurs under estrogen deficient conditions in humans and in other mammals. For instance, estrogen deficiency during menopause in women induces the formation and accumulation of reactive oxygen species (ROS) and a dysregulation of redox signaling. The bone and bone marrow are especially susceptible to the effects of oxidative stress and ROS. In tissues like the bone, cells are equipped with important defense systems to maintain homeostasis and to survive. Redox proteins of the thioredoxin family represent the most important protective system in cells that act against ROS and repair their intracellular effects. Redox proteins of the thioredoxin family comprise thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs). From all these protein classes, Prxs are the only ones that work as classical ROS scavengers being able to reduce hydrogen peroxide (H2O2), peroxynitrites and organic peroxides. Prxs are localized in different cellular compartments such as mitochondria (Prx3), cytosol and nucleus (Prx1 and Prx2), endoplasmic reticulum (Prx4), and peroxisomes (Prx5). We hypothesized that changes in estrogen levels may alter the expression and localization of Prxs in the bone and bone marrow. In this study, we analyzed the cellular localization and expression patterns of Prxs in the bone and bone marrow of rats as well as the effects of estrogens on their expression levels. We compared sham, ovariectomized, as well as ovariectomized/estrogen-replaced female rats of the same age. Western blot and immunohistochemistry revealed that all peroxiredoxins were abundantly expressed in different cell types of the bone and bone marrow such as osteocytes, chondrocytes, osteoclasts, bone lining cells, osteoblasts, and hematopoietic precursor cells. After ovariectomy, redox protein levels were significantly reduced in most cell types of the bone and bone marrow compartment. In addition to this, estrogen deficient animals showed increased levels of Prx1 in bone marrow supernatant suggesting a major redox dysregulation in this compartment. Estrogen replacement restored the levels of most redox proteins in the bone and bone marrow compartment and reduced the amount of extracellular Prx1. Based on these results, we conclude that estrogen deficiency negatively affects the ability of bone and bone marrow cells to maintain redox proteins at physiological levels. These results also reveal the widespread effects that are mediated by estrogen affecting different protein systems and tissues in the body. This work was funded by a Seed Discovery Grant (grant #3670920201102 to JG) from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electrospun nanofibers: building blocks for the repair of bone tissue.

Bone, one of the hardest structures of the body, is the basic constituent of the skeletal system, which gives the shape to the body, provides mechanical support for muscles and soft tissues, and provides movement. Even if there is no damage, bone remodeling is a permanent process to preserve and renew the structural, biochemical, and biomechanical integrity of bone tissue. Apart from the remodeling, bone healing is the highly complicated process of repairing deficiencies of bone tissue by the harmonious operation of osteoblasts, osteocytes, osteoclasts, and bone lining cells. Various materials can be used to both trigger the bone healing process and to provide mechanical support to damaged bone. Nanofiber scaffolds are at the forefront of these types of systems because of their extremely large surface area-to-volume ratio, small pore size, and high porosity. Nanofibers are known to be highly functional systems with the ability to mimic the structure and function of the natural bone matrix, facilitating osteogenesis for cell proliferation and bone regeneration. Electrospinning is an easy and fast method to produce non-woven structures consisting of continuous ultrafine fibers with diameters ranging from micrometers down to nanometers. The simplicity and cost-effectiveness of the electrospinning technique, its ability to use a wide variety of synthetic, natural, and mixed polymers, and the formation of highly porous and continuous fibers are the remarkable features of this method. The importance of nanofiber-based scaffolds in bone tissue regeneration is increasing because of suitable pore size, high porosity, osteoinduction, induction of bone growth with osteoconduction, adaptability to the target area, biodegradation, and appropriate mechanical properties, which are among the main parameters that are important in the design of polymeric bone grafts. The aim of this review is to cast light on the increasing use of nanofiber-based scaffolds in bone tissue regeneration and give an insight about bone regeneration, production techniques of the electrospun nanofibers, and varying formulation parameters in order to reach different drug delivery goals. This review also provides an extensive market research of electrospun nanofibers and an overview on scientific research and patents in the field.

Gadolinium-Based Contrast Agents and Free Gadolinium Inhibit Differentiation and Activity of Bone Cell Lineages.

Administration of gadolinium-based contrast agents (GBCA) in magnetic resonance imaging results in the long-term retention of gadolinium (Gd) in tissues and organs, including the bone, and may affect their function and metabolism. This study aims to investigate the effects of Gd and GBCA on the proliferation/survival, differentiation, and function of bone cell lineages. Primary murine osteoblasts (OB) and osteoclast progenitor cells (OPC) isolated from C57BL/6J mice were used to test the effects of Gd 3+ (12.5-100 μM) and GBCA (100-2000 μM). Cultures were supplemented with the nonionic linear Gd-DTPA-BMA (gadodiamide), ionic linear Gd-DTPA (gadopentetic acid), and macrocyclic Gd-DOTA (gadoteric acid). Cell viability and differentiation were analyzed on days 4-6 of the culture. To assess the resorptive activity of osteoclasts, the cells were grown in OPC cultures and were seeded onto layers of amorphous calcium phosphate with incorporated Gd. Gd 3+ did not affect OB viability, but differentiation was reduced dose-dependently up to 72.4% ± 6.2%-73.0% ± 13.2% (average ± SD) at 100 μM Gd 3+ on days 4-6 of culture as compared with unexposed controls ( P < 0.001). Exposure to GBCA had minor effects on OB viability with a dose-dependent reduction up to 23.3% ± 10.2% for Gd-DTPA-BMA at 2000 μM on day 5 ( P < 0.001). In contrast, all 3 GBCA caused a dose-dependent reduction of differentiation up to 88.3% ± 5.2% for Gd-DTPA-BMA, 49.8% ± 16.0% for Gd-DTPA, and 23.1% ± 8.7% for Gd-DOTA at 2000 μM on day 5 ( P < 0.001). In cultures of OPC, cell viability was not affected by Gd 3+ , whereas differentiation was decreased by 45.3% ± 9.8%-48.5% ± 15.8% at 100 μM Gd 3+ on days 4-6 ( P < 0.05). Exposure of OPC to GBCA resulted in a dose-dependent increase in cell viability of up to 34.1% ± 11.4% at 2000 μM on day 5 of culture ( P < 0.001). However, differentiation of OPC cultures was reduced on day 5 by 24.2% ± 9.4% for Gd-DTPA-BMA, 47.1% ± 14.0% for Gd-DTPA, and 38.2% ± 10.0% for Gd-DOTA ( P < 0.001). The dissolution of amorphous calcium phosphate by mature osteoclasts was reduced by 36.3% ± 5.3% upon incorporation of 4.3% Gd/Ca wt/wt ( P < 0.001). Gadolinium and GBCA inhibit differentiation and activity of bone cell lineages in vitro. Thus, Gd retention in bone tissue could potentially impair the physiological regulation of bone turnover on a cellular level, leading to pathological changes in bone metabolism.

Osteoprogenitor recruitment and differentiation during intracortical bone remodeling of adolescent humans.

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.

Active Osteoblasts or Quiescent Bone Lining Cells? Preosteoblasts Fate Orchestrated by Curvature and Stiffness of an In Vitro 2.5D Biomimetic Culture System.

Biomimetic cell culture systems are required to provide more physiologically relevant microenvironments for bone cells. Here, a simple 2.5D culture platform is proposed, combining adjustable stiffness and surface features that mimic bone topography by using sandpaper grits as master molds with two stiffness formulations of polydimethylsiloxane (PDMS). The subsequent replicas perfectly conform the grits and reproduce the corresponding negative relief with cavities separated by convex edges. Biomimicry is also provided by an extracellular matrix (ECM)-like thin film coating, using the layer-by-layer (LbL) method. The topographical features, alternating concave, and convex structures drive preosteoblasts organization and morphology. Strikingly, curvature orchestrates the commitment of preosteoblasts, with i) maturation to active osteoblasts able to produce a dense collagenous matrix that ultimately mineralizes in the cavities, and ii) edges hosting quiescent cells that synthetize a very thin immature collagen layer with no mineralization. In summary, the present in vitro culture system model offers a cell-instructive 2.5D microenvironment that controls preosteoblasts fate, leading to two coexisting subpopulations: mature osteoblasts and bone lining cells (BLC). This promising culture system opens new avenues to advanced tissue-engineered modeling and can be applied to precellularized bone biomaterials.

Dynamic bioinspired coculture model for probing ER+ breast cancer dormancy in the bone marrow niche.

Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor-positive (ER+) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER+ BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor-α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.

Abstract B023: Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells

Abstract Multiple myeloma (MM) is an incurable plasma cell cancer. Therapeutic advances have greatly improved patient survival; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive relapse. Tumor dormancy shares features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the treatment of MM, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that depends on interactions with the mesenchymal bone niche. GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of senescence after the initial stress response. Cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and stained for nuclei, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly growth arrested over 2 weeks compared to vehicle-treated cells. Co-culture with BMSCs significantly enhanced melphalan-treated 5TGM1 adherence compared to culture without BMSCs during media replacement. Of note, melphalan-treated 5TGM1 did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 also exhibited significantly greater adherence with BMSCs than vehicle-treated cells. Melphalan-treated 5TGM1 were significantly larger than vehicle-treated cells and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, which are all markers of cellular senescence. Our findings suggest that interactions with BMSCs promote the survival of melphalan-treated MM tumor cells. The enhanced adherence of these cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse. Citation Format: Angelo J. Guilatco, Gabriel Alvares Borges, Tamar Tchkonia, James L. Kirkland, Taxiarchis Kourelis, Matthew T. Drake, Megan Weivoda. Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B023.

Epigenetic Repression of RUNX2 and OSX Promoters Controls the Nonmineralized State of the Periodontal Ligament.

The nonmineralized state of the mammalian periodontal ligament is one of the hallmarks of vertebrate evolution as it provides resilient and nontraumatic tooth anchorage for effective predation. Here we sought to determine how the chromatin state of key mineralization gene promoters contributes to the nonmineralized periodontal ligament in the midst of fully mineralized alveolar bone and cementum anchor tissues. In developing mouse periodontal tissues, RUNX2 was localized to alveolar bone-lining cells, while OSX was localized throughout the periodontal ligament's soft tissue. Matching RT-PCR amplification data and western blot comparisons demonstrated that the expression of RUNX2 and OSX bone mineralization transcription factors was at least 2.5-fold elevated in alveolar bone osteoblasts versus periodontal ligament fibroblasts. ChIP enrichment data along the RUNX2 and OSX promoters revealed increased H3K4me3 marks in alveolar bone osteoblasts, while H3K9me3 and H3K27me3 marks were elevated in periodontal ligament fibroblasts. In support of an epigenetic mechanism responsible for the inhibition of mineralization gene expression in periodontal progenitors, histone methylation inhibitors DZNep and Chaetocin reactivated RUNX2 and OSX expression in periodontal progenitors and increased alkaline phosphatase and Alizarin Red, while the in vivo application of DZNep in rat maxillae resulted in aberrant mineralization in the periodontal ligament and a narrowing of the nonmineralized periodontal space. Together, these studies demonstrate that the nonmineralized state of the mammalian periodontal ligament is controlled by an epigenetic regulation of the RUNX2 and OSX key mineralization gene promoters.

Bone cells and their role in physiological remodeling.

This work compiles the characteristics of bone cells involved in the physiological bone remodeling. A narrative review of the literature was performed. Remodeling is a different process from modeling. Remodeling allows old or damaged bone tissue to be renewed, ensuring the maintenance of bone fracture resistance, as well as maintaining calcium and phosphorus homeostasis. We present the role of osteoclasts, a multinucleated cell with hematopoietic origin responsible for resorbing bone. The formation of osteoclasts depends on the cytokines macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) and can be blocked by osteoprotegerin. Furthermore, this review highlights the features of osteoblasts, polarized cubic cells of mesenchymal origin that deposit bone and also covers osteocytes and bone lining cells. This review presents the five fundamental phases of bone remodeling and addresses aspects of its regulation through hormones and growth factors. Knowledge of the current concepts of physiological bone remodeling is necessary for the study of the different pathologies that affect the bone tissue and thus helps in the search for new therapies.

Enhanced Survival and Adherence of Melphalan-Induced Senescent-like Dormant Multiple Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

Introduction: Multiple myeloma (MM) is an incurable plasma cell cancer and the second most common hematological malignancy in the United States. Therapeutic advances have greatly improved patient survival over the last decade; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive disease relapse. Tumor dormancy shares many features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the standard treatment of multiple myeloma such as melphalan, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains both growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that is dependent on interactions with the mesenchymal bone niche. Methods: GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of a senescent phenotype after the initial stress response. During this period, cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and assessed for nuclear foci, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Results: Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly (p<0.0001) growth arrested over the 2-week monitoring period (mean cumulative doublings: 1.26 ± 0.179) compared to vehicle-treated cells (5.84 ± 0.122). Co-culture with BMSCs significantly (p<0.0001) enhanced melphalan-treated 5TGM1 cell adherence (mean percent of total cells washed-off: 21.7% ± 4.2%) compared to culture without BMSCs (92.2% ± 4.3%) during media replacement. Of note, melphalan-treated 5TGM1 cells did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 cells also exhibited significantly greater (p<0.001) adherence with BMSCs (mean percent of total cells washed-off: 21.7% ± 4.2%) than vehicle-treated cells (48.9% ± 5.1%). Melphalan-treated 5TGM1 cells were significantly (p<0.0001) larger (median area in square microns: 63.96 [7.66-954.05]) than vehicle-treated cells (44.43 [7.66-434.32]) and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, all of which are markers of cellular senescence. Conclusions: Our findings suggest that interactions with BMSCs promote the survival of chemotherapy-treated MM tumor cells. The enhanced adherence of these melphalan-treated MM cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Impact of the host response and osteoblast lineage cells on periodontal disease

Periodontitis involves the loss of connective tissue attachment and alveolar bone. Single cell RNA-seq experiments have provided new insight into how resident cells and infiltrating immune cells function in response to bacterial challenge in periodontal tissues. Periodontal disease is induced by a combined innate and adaptive immune response to bacterial dysbiosis that is initiated by resident cells including epithelial cells and fibroblasts, which recruit immune cells. Chemokines and cytokines stimulate recruitment of osteoclast precursors and osteoclastogenesis in response to TNF, IL-1β, IL-6, IL-17, RANKL and other factors. Inflammation also suppresses coupled bone formation to limit repair of osteolytic lesions. Bone lining cells, osteocytes and periodontal ligament cells play a key role in both processes. The periodontal ligament contains cells that exhibit similarities to tendon cells, osteoblast-lineage cells and mesenchymal stem cells. Bone lining cells consisting of mesenchymal stem cells, osteoprogenitors and osteoblasts are influenced by osteocytes and stimulate formation of osteoclast precursors through MCSF and RANKL, which directly induce osteoclastogenesis. Following bone resorption, factors are released from resorbed bone matrix and by osteoclasts and osteal macrophages that recruit osteoblast precursors to the resorbed bone surface. Osteoblast differentiation and coupled bone formation are regulated by multiple signaling pathways including Wnt, Notch, FGF, IGF-1, BMP, and Hedgehog pathways. Diabetes, cigarette smoking and aging enhance the pathologic processes to increase bone resorption and inhibit coupled bone formation to accelerate bone loss. Other bone pathologies such as rheumatoid arthritis, post-menopausal osteoporosis and bone unloading/disuse also affect osteoblast lineage cells and participate in formation of osteolytic lesions by promoting bone resorption and inhibiting coupled bone formation. Thus, periodontitis involves the activation of an inflammatory response that involves a large number of cells to stimulate bone resorption and limit osseous repair processes.

Reactivation of Bone Lining Cells are Attenuated Over Repeated Anti-sclerostin Antibody Administration.

Reactivation of bone lining cells (BLCs) is a crucial mechanism governing the anabolic action of anti-sclerostin antibody (Scl-Ab) via modeling-based bone formation; however, it remains unclear whether this reactivation can be attenuated after persistent administration of Scl-Ab. Here, we aimed to investigate the reproducibility of persistent Scl-Ab administration for the reactivation of BLCs, and to elucidate the relationship between the activity of BLCs and serum levels of N-terminal procollagen type I (P1NP) during chronic Scl-Ab administration. We conducted an osteoblast lineage tracing study. Briefly, Dmp1-CreERt2(+):Rosa26R mice were injected with 1mg of 4-hydroxy-tamoxifen weekly from postnatal weeks four to eight. Mice were treated twice with either vehicle or Scl-Ab (25mg/kg) at weeks 12, 16, and 20, and were euthanized at weeks 8, 12, 13, 16, 17, 20, and 21 (4-6 mice in each group). After euthanization, the number and thickness of X-gal (+) cells on the periosteum of the femoral bones and the serum levels of P1NP were quantified at each time point. Scl-Ab induced a significant increase in the thickness of X-gal (+) cells on periosteal bone surfaces at postnatal weeks 13 (after 1st dose), 17 (after 2nd dose), and 21 (after 3rd dose) compared to that in vehicle-treated mice (all P < 0.001). In the Scl-Ab group, significant increases in the thickness of labeled cells were observed between weeks 16 and 17 and weeks 20 and 21 (both P < 0.001). The percentage increase in X-gal (+) cell thickness was 108.9% from week 12 to week 13, 54.6% from week 16 to week 17, and 49.2% from week 20 to week 21 in the Scl-Ab group. Although Scl-Ab treatment increased the serum levels of P1NP at postnatal weeks 13 and 17 compared with those at week 12 (P = 0.017 and P = 0.038, respectively), the same was not observed at week 21 (P = 0.296). A significant increase in P1NP levels was observed between weeks 16 and 17 and weeks 20 and 21 in the Scl-Ab group (P = 0.005 and P = 0.007, respectively). The percentage increase in P1NP levels was 141.7% from weeks 12 to 13, 114.8% from weeks 16 to 17, and 99.4% from weeks 20 to 21. Serum P1NP levels were positively correlated with X-gal (+) cell thickness (R2 = 0.732, P < 0.001). Reactivation of BLCs is modestly attenuated, but reproducible, during persistent Scl-Ab administration. Serum P1NP levels appear to be an indicator of the impact of Scl-Ab on the conversion of BLCs into mature osteoblasts on periosteal bone surfaces, thus contributing to modeling-based bone formation.

Reprogramming bone progenitor identity and potency through control of collagen density and oxygen tension

SummaryThe biophysical microenvironment of the cell is being increasingly used to control cell signaling and to direct cell function. Herein, engineered 3D tuneable biomimetic scaffolds are used to control the cell microenvironment of Adipose-derived Mesenchymal Stromal Cells (AMSC), which exhibit a collagen density-specific profile for early and late stage bone cell lineage status. Cell potency was enhanced when AMSCs were cultured within low collagen density environments in hypoxic conditions. A transitional culture containing varied collagen densities in hypoxic conditions directed differential cell fate responses. The early skeletal progenitor identity (PDPN+CD146−CD73+CD164+) was rescued in the cells which migrated into low collagen density gels, with cells continuously exposed to the high collagen density gels displaying a transitioned bone-cartilage-stromal phenotype (PDPN+CD146+CD73−CD164-). This study uncovers the significant contributions of the physical and physiological cell environment and highlights a chemically independent methodology for reprogramming and isolating skeletal progenitor cells from an adipose-derived cell population.

Bone Lining Cells Could Be Sources of Bone Marrow Adipocytes.

BackgroundRecently, lineage-tracing studies demonstrated that parathyroid hormone and anti-sclerostin antibody (Scl-Ab) can convert bone lining cells (BLCs) into active osteoblasts. However, BLCs might also be differentiated into other lineages. Here we investigated whether BLCs could differentiate into bone marrow adipocytes (BMAds) and whether Scl-Ab could suppress this process.MethodsDmp1-CreERt2:mTmG mice were injected with 0.5 mg of 4-hydroxytamoxifen once weekly from postnatal week 4 to week 8. The mice were treated with either vehicle or rosiglitazone for 8 weeks (weeks 12–20). Moreover, they were administered either vehicle or Scl-Ab (50 mg/kg) twice weekly for 4 weeks (weeks 16–20, N = 4–6/group). We chased the GFP+ cells from the endosteal surface to the bone marrow (BM) of the femur. Using immunohistochemical staining, the numbers of perilipin+ or GFP+/perilipin double+ cells in the BM were quantified. In addition, serum N-terminal propeptide of type I procollagen (P1NP) levels were measured at each time point, and bone mass was analyzed at 20 weeks using micro-computed tomography.ResultsScl-Ab administration significantly reversed the decreases in bone parameters induced by rosiglitazone. Plump GFP+ cells, presumably active osteoblasts, and extremely flat GFP+ cells, presumably BLCs, were present on the endosteal surface of the femur at 8 and 12 weeks, respectively, in line with prior findings. When we chased the GFP+ cells, rosiglitazone significantly increased the number of GFP/perilipin double+ BMAds compared to the effects of the vehicle (P < 0.001), and overlapping Scl-Ab administration decreased the number of GFP/perilipin double + BMAd compared to rosiglitazone alone (P < 0.001). In addition, we found that osteoblast lineage cells such as BLCs might express PPARγ on immunohistochemical staining. When rosiglitazone was administered to Rip-Cre:mTmG mice, GFP+ cells were not present on the endosteal surface or in the BM of the femur; however, they were present in the pancreas.ConclusionBLCs could be sources of BMAds, and rosiglitazone could stimulate the differentiation of osteoblast lineage cells into BMAds. Suppression of the differentiation of osteoblast lineage cells into BMAds might contribute to anabolic effects resulting from the pharmacologic inhibition of sclerostin.

Diabetes impairs fracture healing through disruption of cilia formation in osteoblasts.

Diabetes-associated fracture risk and impaired fracture healing represents a serious health threat. It is well known that type 1 diabetes mellitus (T1DM) impairs fracture healing due to its effect on osteoblasts and their progenitor cells. Previous studies have showed that primary cilia and intraflagellar transport protein 80 (IFT80) are critical for bone formation. However, whether TIDM impairs fracture healing due to influencing ciliary gene expression and cilia formation is unknown. Here, we investigated the effect of T1DM on primary cilia in a streptozotocin induced diabetes mouse model and examined the impact of cilia on fracture healing in osteoblasts by deletion of IFT80 in osteoblast linage using osterix (OSX)-cre (OSXcretTAIFT80f/f). The results showed that diabetes inhibited ciliary gene expression and primary cilia formation to an extent that was similar to normoglycemic mice with IFT80 deletion. Moreover, diabetic mice and normoglycemic mice with cilia loss in osteoblasts (OSXcretTAIFT80f/f) both exhibited delayed fracture healing with significantly reduced bone density and mechanical strength as well as with reduced expression of osteoblast markers, decreased angiogenesis and proliferation of bone lining cells at the fracture sites. In vitro studies showed that advanced glycation end products (AGEs) downregulated IFT80 expression in osteoblast progenitors. Moreover, AGEs and IFT80 deletion significantly reduced cilia number and length which inhibited differentiation of primary osteoblast precursors. Thus, this study for the first time report that primary cilia are essential for bone regeneration during fracture healing and loss of cilia caused by diabetes in osteoblasts resulted in defective diabetic fracture healing.

Corrigendum: Correction of Acknowledgments. Transformation of Mature Osteoblasts into Bone Lining Cells and RNA Sequencing-Based Transcriptome Profiling of Mouse Bone during Mechanical Unloading

Corrigendum: Correction of Acknowledgments. Transformation of Mature Osteoblasts into Bone Lining Cells and RNA Sequencing-Based Transcriptome Profiling of Mouse Bone during Mechanical Unloading

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.