Resorption Pits Research Articles

The anatomy, phylogenetic relationships, and autecology of the carnivorous lizard “Saniwa” feisti Stritzke, 1983 from the Eocene of Messel, Germany

The evolution and interrelationships of carnivorous squamates (mosasaurs, snakes, monitor lizards, Gila Monsters) are a contentious part of reptile systematics and go to the heart of conflict between morphological and molecular data in inferring evolutionary history. One of the best-preserved fossils in this motley grouping is “Saniwa” feisti Stritzke, 1983, represented by complete skeletons from the early-middle Eocene of Messel, Germany. We re-describe it on the basis of superficial examination, stereoradiography, and high-resolution X-ray computed tomography of new and published specimens. The scalation of the lizard is unique, consisting of small, keeled scales on the head (including a row of enlarged medial supraorbitals) and large, rhomboidal, keeled scales (invested by osteoderms) that covered the rest of the body. Two paired longitudinal rows of enlarged scales ran down the neck. The head was laterally compressed and box-shaped due to the presence of a strong canthal-temporal ridge; the limbs and tail were very long. Notable osteological features include: a toothed, strap-like vomer; septomaxilla with a long posterior process; palpebral with a long posterolateral process; a lacrimal boss and a single lacrimal foramen; a well-developed cultriform process of the parabasisphenoid; two hypoglossal (XII) foramina in addition to the vagus; a lack of resorption pits for replacement teeth; and possibly the presence of more than one wave of developing replacement teeth per locus. There are no osteological modifications suggestive of an intramandibular hinge, but postmortem displacement of the angular-prearticular-surangular complex in multiple specimens suggests that there might have been some degree of mobility in the lower jaw based on soft-tissue modifications. Using phylogenetic analyses on a data-set comprising 473 morphological characters and 46 DNA loci, we infer that a monophyletic Palaeovaranidae Georgalis, 2017, including Eosaniwa Haubold, 1977, lies on the stem of Varanidae Merrem, 1820, basal to various Cretaceous Mongolian taxa. We transfer feisti to the new genus Paranecrosaurus n. gen. Analysis of gut contents reveals only the second known specimen of the cryptozoic lizard Cryptolacerta hassiaca Müller, Hipsley, Head, Kardjilov, Hilger, Wuttke & Reisz, 2011, confirming a diet that was at least partly carnivorous; the preservation of the teeth of C. hassiaca suggests that the gastric physiology of Paranecrosaurus feisti (Stritzke, 1983) n. comb. had high acidity but low enzyme activity. Based on the foregoing and linear discriminant function analysis, we reconstruct P. feisti n. comb., as a powerful, widely roaming, faunivorous-carnivorous stem monitor lizard with a sensitive snout. If the molecular phylogeny of anguimorphs is correct, then many of the features shared by Helodermatidae Gray, 1837 and Varanidae must have arisen convergently, partly associated with diet. In that case, a reconciliation of morphological and molecular data would require the discovery of equally primitive fossils on the helodermatid stem.

Glaucocalyxin A suppresses osteoclastogenesis induced by RANKL and osteoporosis induced by ovariectomy by inhibiting the NF-κB and Akt pathways

Ethnopharmacological relevanceGlaucocalyxin A (GLA), the most abundant active component of the aboveground sections of Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, possesses various pharmacological activities, such as antioxidant, antithrombosis, anticoagulation, antibacterial, antitumor, anti-inflammatory activities. According to previous studies, inflammation is closely associated with osteoclast differentiation and activity. Although GLA has demonstrated effective anti-inflammatory properties, its effects on osteoclast differentiation remain unclear. Aim of the studyTo examine the possible inhibitory effects of GLA and its molecular mechanisms in osteogenesis induced by RANKL as well as ovariectomy (OVX)-induced osteoporosis (OP) in mice. Materials and methodsTartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and a bone resorption pit assay were applied for identifying the effects of GLA on the differentiation of osteoclasts and the function of bone resorption. The mRNA expression of the genes related to osteoclast differentiation was measured by quantitative PCR. Protein expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-fos and phosphorylation of inhibitor of nuclear factor kappa B (IκBα), protein kinase B (AKT), c–Jun N–terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 in RANKL-induced osteoclasts was determined using western blotting. The effect of GLA on OP was studied using a mouse model of OVX. ResultsAt nontoxic concentrations ≤0.5 μM in vitro, GLA suppressed the formation of osteoclasts induced by RANKL with the decreased number and area size of TRAP-positive multinuclear osteoclasts, and the resorption of bone function by reducing F-actin ring number and bone resorption pit areas. It also reduced the expression of the genes specific for osteoclasts, which included genes encoding NFATc1, cathepsin K, c-fos, TRAP, vacuolar-type ATPase d2, and dendritic cell-specific transmembrane protein. Moreover, GLA repressed NF-κB and Akt pathway activation induced by RANKL. Micro-CT analysis of femur samples indicated decreased bone loss and greater trabecular bone density after GLA treatment, which showed that GLA played a protective role by inhibiting bone loss in OVX-induced OP mice in vivo. ConclusionsOur study is the first to show that GLA has significant therapeutic potential in OP, which is the disease of osteoclast increase caused by estrogen deficiency.

Interleukin-1 induces receptor activator of nuclear factor-κB ligand-independent osteoclast differentiation in RAW264.7 cells.

Interleukin-1 (IL-1) is a pro-inflammatory cytokine which induces bone destruction in various diseases, such as osteoporosis and rheumatoid arthritis. RAW264.7 cells are frequently used in studies as osteoclast precursors, however it remains unclear whether IL-1 can induce osteoclast differentiation from RAW264.7 cells without the stimulation of receptor activator of nuclear factor-κB ligand (RANKL). Hence, the present study aimed to investigate the effects of IL-1 on the formation of osteoclasts from RAW264.7 cells. The cell viability was determined via the Cell Counting Kit-8 (CCK-8) assay. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR, respectively. Tartrate-resistant acid phosphatase (TRAP) staining and the resorption pit assay were performed to determine the formation and activity of osteoclasts. A significantly increased quantity of osteoclasts were found in the IL-1 group compared with the control group, and also in the RANKL+IL-1 group compared with the RANKL group. In addition IL-1 significantly increased both the protein and mRNA expression of specific genes associated with osteoclastogenesis, including nuclear factor of activated T cells cytoplasmic 1, matrix metalloprotein-9, cathepsin K and TRAP. The findings of the present study suggested that IL-1 can induce osteoclast differentiation and upregulate the quantity of osteoclasts differentiated from RAW264.7 cells. These results may lay a foundation for further study of diseases involving inflammation-associated bone loss. The combined blockade of IL-1 and RANKL may be effective for the prevention of inflammatory bone loss.

6'-O-Galloylpaeoniflorin Attenuates Osteoclasto-genesis and Relieves Ovariectomy-Induced Osteoporosis by Inhibiting Reactive Oxygen Species and MAPKs/c-Fos/NFATc1 Signaling Pathway.

Emerging evidence suggests bright prospects of some natural antioxidants in the treatment of osteoporosis. 6′-O-Galloylpaeoniflorin (GPF), an antioxidant isolated from peony roots (one of very widely used Oriental medicines, with various anti-inflammatory, antitumor, and antioxidant activities), shows a series of potential clinical applications. However, its effects on osteoporosis remain poorly investigated. The current study aimed to explore whether GPF can attenuate osteoclastogenesis and relieve ovariectomy-induced osteoporosis via attenuating reactive oxygen species (ROS), and investigate the possible mechanism. After the culture of primary murine bone marrow-derived macrophages/monocytes were induced by the use of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL) and then treated with GPF. Cell proliferation and viability were assessed by Cell Counting Kit-8 (CCK-8) assay. Thereafter, the role of GPF in the production of osteoclasts and the osteogenic resorption of mature osteoclasts were evaluated by tartrate-resistant acid phosphatase (TRAP) staining, podosome belt formation, and resorption pit assay. Western blotting and qRT-PCR examination were performed to evaluate proteins’ generation and osteoclast-specific gene levels, respectively. The ROS generation in cells was measured in vitro by 2′,7′-Dichlorodi-hydrofluorescein diacetate (DCFH-DA). Ovariectomy-induced osteoporosis mouse administered with GPF or vehicle was performed to explore the in vivo potential of GPF, then a micro-CT scan was performed in combination with histological examination for further analysis. GPF suppressed the formation of osteoclasts and podosome belts, as well as bone resorption when induced by RANKL through affecting intracellular ROS activity, MAPKs signaling pathway, and subsequent NFATc1 translocation and expression, as well as osteoclast-specific gene expression in vitro. In vivo study suggested that exposure to GPF prevented osteoporosis-related bone loss in the ovariectomized mice. These findings indicate that GPF attenuates osteoclastogenesis and relieves ovariectomy-induced osteoporosis by inhibiting ROS and MAPKs/c-Fos/NFATc1 signaling pathway. This suggested that GPF may be potentially used to treat bone diseases like periodontitis, rheumatoid arthritis, and osteoporosis associated with osteoclasts.

Inhibition of osteoclast activities by SCPC bioceramic promotes osteoblast-mediated graft resorption and osteogenic differentiation.

Maximizing vital bone in a grafted site is dependent on a number of factors. These include resorption or turnover of the graft material, stimulation of bone formation pathway without a need for biological molecules added to the site and inhibition of cellular activities that compromise the mineralization of new bone matrix. In the present study, the dissolution profile of silica-calcium phosphate composite (SCPC) in physiological solution was measured and the data were fed to (ANN-NARX) prediction model to predict the time required for complete dissolution. The inductively coupled plasma-optical emission spectrometer ionic composition analysis of the culture medium incubated for 3 days with SCPC showed 57% decrease in Ca concentration and a significant increase in the concentration of Si (13.5 ± 1.8μg/ml), P (249.4 ± 22 μg/ml), and Na (9.3 ± 0.52 μg/ml). In conjunction with the release of Si, P, and Na ions, the bone resorptive activity of osteoclasts was inhibited as indicated by the significant decrease in multinucleated tartrate resistant acidic phosphate stained cells and the volume of resorption pits on bone slices. In contrast, addition of SCPC to hBMSC cultured in conventional medium promoted higher Runt-related transcription factor 2 (p < .05), osteocalcin (p < .01), and bone sialo protein (p < .01) than that expressed by control cells grown in the absence of SCPC. The predicted dissolution time of 200 mg of porous SCPC particles in 10ml phosphate buffered saline is 6.9months. An important byproduct of the dissolution is inhibition of osteoclastic activity and promotion of osteoblastic differentiation and hence bone formation.

Liquid-phase ASEM imaging of cellular and structural details in cartilage\xa0and bone formed during endochondral ossification: Keap1-deficient osteomalacia

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1−/− femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro.

Glucocorticoids are widely used to treat various diseases including rheumatoid arthritis (RA); however, one of the most frequent and severe adverse effects is glucocorticoid-induced osteoporosis (GIOP). Iguratimod (IGU) is a novel conventional synthetic disease-modifying anti-rheumatic drug developed in Japan. The aim of this study is to investigate the effects of IGU on glucocorticoid-induced disorder of bone metabolism in vitro. In osteoclastogenesis of mouse bone marrow-derived cells, tartrate-resistant acid phosphatase staining, resorption pit assay, western blotting, real-time polymerase chain reaction (PCR), and mRNA sequencing were performed. In osteoblastogenesis of MC3T3-E1 cells, alkaline phosphatase (ALP) staining and activity, alizarin red staining, and mRNA sequencing were performed, and real-time PCR and western blotting were conducted in MC3T3-E1 cells and murine osteocyte-like cell line MLO-Y4 cells. IGU significantly suppressed a dexamethasone-induced increase in osteoclasts, differentiation, and bone resorption activity by inhibition of the receptor activator of the nuclear factor kappa-B (RANK)/tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)/nuclear factor kappa-B (NFκB)-p52 pathway. In MC3T3-E1 cells, IGU significantly upregulated dexamethasone-induced downregulation of ALP activity, bone mineralization, and osteoblast-related gene andprotein expression. In MLO-Y4 cells, IGU significantly upregulated dexamethasone-induced downregulation of the gene expression of ALP and osteocalcin, and also downregulated receptor activator of NFκB ligand (RANKL)/osteoprotegerin gene expression ratio without dexamethasone. These results suggest that IGU may improve glucocorticoid-induced disorder of bone metabolism and may exhibit positive effects against GIOP associated with RA.

How to Get Them off?-Assessment of Innovative Techniques for Generation and Detachment of Mature Osteoclasts for Biomaterial Resorption Studies.

The fusion process of mononuclear monocytes into multinuclear osteoclasts in vitro is an essential process for the study of osteoclastic resorption of biomaterials. Thereby biomaterials offer many influencing factors such as sample shape, material composition, and surface topography, which can have a decisive influence on the fusion and thus the entire investigation. For the specific investigation of resorption, it can therefore be advantageous to skip the fusion on samples and use mature, predifferentiated osteoclasts directly. However, most conventional detachment methods (cell scraper, accutase), lead to a poor survival rate of osteoclasts or to a loss of function of the cells after their reseeding. In the present study different conventional and novel methods of detachment in combination with different culture surfaces were investigated to obtain optimal osteoclast differentiation, yield, and vitality rates without loss of function. The innovative method—using thermoresponsive surfaces for cultivation and detachment—was found to be best suited. This is in particular due to its ability to maintain osteoclast activity, as proven by TRAP 5b-, CTSK-activity and resorption pits on dentin discs and decellularized osteoblast-derived matrix plates. In conclusion, it is shown, that osteoclasts can be predifferentiated on cell culture dishes and transferred to a reference biomaterial under preservation of osteoclastic resorption activity, providing biomaterial researchers with a novel tool for material characterization.

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL.

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

The effect of surgical suture material on osteoclast generation and implant-loosening.

Background: Implant loosening - either infectious or aseptic- is a still a major complication in the field of orthopaedic surgery. In both cases, a pro-inflammatory peri-prosthetic environment is generated by the immune system - either triggered by bacteria or by implant wear particles - which leads to osteoclast differentiation and osteolysis. Since infectious cases in particular often require multiple revision surgeries, we wondered whether commonly used surgical suture material may also activate the immune system and thus contribute to loss of bone substance by generation of osteoclasts.Methods: Tissue samples from patients suffering from infectious implant loosening were collected intraoperatively and presence of osteoclasts was evaluated by histopathology and immunohistochemistry. Further on, human monocytes were isolated from peripheral blood and stimulated with surgical suture material. Cell supernatant samples were collected and ELISA analysis for the pro-inflammatory cytokine IL-8 was performed. These experiments were additionally carried out on ivory slices to demonstrate functionality of osteoclasts. Whole blood samples were incubated with surgical suture material and up-regulation of activation-associated cell surface markers CD11b and CD66b on neutrophils was evaluated by flow cytofluorometry analysis.Results: We were able to demonstrate that multinucleated giant cells form in direct vicinity to surgical suture material. These cells stained positive for cathepsin K, which is a typical protease found in osteoclasts. By in vitro analysis, we were able to show that monocytes differentiated into osteoclasts when stimulated with surgical suture material. Resorption pits on ivory slices provided proof that the osteoclasts were functional. Release of IL-8 into cell supernatant was increased after stimulation with suture material and was further enhanced if minor amounts of bacterial lipoteichoic acid (LTA) were added. Neutrophils were also activated by surgical suture material and up-regulation of CD11b and CD66b could be seen.Conclusion: We were able to demonstrate that surgical suture material induces a pro-inflammatory response of immune cells which leads to osteoclast differentiation, in particular in combination with bacterial infection. In conclusion, surgical suture material -aside from bacteria and implant wear particles- is a contributing factor in implant loosening.

The effects of vasoactive intestinal peptide on RANKL-induced osteoclast formation.

BackgroundCongenital pseudarthrosis of the tibia is a rare disease characterized by an imbalance in bone remodeling. Vasoactive intestinal peptide (VIP) has been proven to modulate bone resorption and the formation of osteoclasts. This study aimed to explore the effects of VIP on the homeostasis of bone metabolism in diverse in vitro systems.MethodsBone marrow-derived macrophages (BMMs) were differentiated into tartrate-resistant acid phosphatase-positive cells through incubation with receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In vitro resorption pit detection was carried out to assess the effects of VIP on osteoclastic activity. Rat osteosarcoma cell line ROS 17/2.8 was cultured alone or co-cultured with rat BMMs in the presence or absence of VIP at various concentrations. The expression levels of RANKL, RANK, OPG, NF-κB, IL-6, ERK, CAII, and GAPDH were determined by qRT-PCR and WB assay.ResultsVIP was observed to repress osteoclast differentiation without affecting the number of osteoclast precursor cells. Furthermore, the modulation of the RANKL/osteoprotegerin (OPG), nuclear factor-κB (NF-κB), and extracellular signal-regulated kinase (ERK) signaling pathways were involved in the inhibitive influence of VIP upon bone erosion. Additionally, VIP affected the expression levels of osteoclastic factors including RANKL, OPG, and interleukin-6 in osteoblast cells. Furthermore, the expression levels of RANKL and RANK were increased, while OPG expression was reduced after treatment with VIP in the co-culture of ROS 17/2.8 and rat BMMs. ERK and NF-κB signal pathways were demonstrated to be involved in the effect of VIP in the co-culture system.ConclusionsVIP plays a critical role in bone remodeling and might serve as a potential target in the development of treatments for congenital pseudarthrosis of the tibia.

Suppressive effects of an apoptotic mimicry prepared from jumbo-flying squid-skin phospholipids on the osteoclastogenesis in receptor activator of nuclear factor kappa B ligand/macrophage colony-stimulating factor-induced RAW 264.7 cells.

Liposomes containing docosahexaenoic acid (DHA) and phosphatidylserine were claimed to inhibit osteoclast formation and bone resorption in the inflammatory status. Herein, we proposed that an apoptotic mimicry (SQ liposome) prepared from squid-skin phospholipids can explore the suppressive osteoclastogenesis. The intermolecular fatty-acid composition in the phospholipid of squid-skin extract was analyzed by GC-FID. The SQ liposome structure was characterized by size distribution and zeta potential (ζ). RAW 264.7 cell is used to study the effect of SQ liposomes on osteoclast differentiation. Secretion of prostaglandin E2 (PGE2) and transforming growth factor-β (TGF-β) from RAW 264.7 cells were assayed. Antiosteoclastogenesis effects were performed via the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) counting, bone resorption pit assay, and TRAP activity analysis. The specific gene expressions related to antiosteoclastogenesis were also detected. An apoptotic mimicry through the use of a single-layer liposome (SQ liposome) with phosphatidylserine exposure contains DHA (28.7%) and eicosapentaenoic acid (EPA, 11.8%). Co-treatment with receptor activator of nuclear factor kappa B ligand (RANKL)/macrophage colony-stimulating factor induced RAW 264.7-cell differentiation into mature osteoclasts, thus enhancing PGE2 and TGF-β secretion. However, cotreatment with 1 mg/mL of SQ liposome restored (p < 0.05) the cell viabilities under the RANKL stress. Increased PGE2 levels was downregulated (p < 0.05) in cotreatments with 0.11 and 0.33 mg/mL of SQ liposome, but on the TGF-β levels were not (p > 0.05) influenced in SQ liposome cotreatments. Cotreatments with 0.33-1 mg/mL of SQ liposome suppressed (p < 0.05) the osteoclast maturation (such as decreased MNCs and bone pit formation), inhibited TRAP activities, and downregulated the osteoclastogenesis-related gene expressions. In summary, current data support that a possible prevention of our prepared SQ liposomes which are rich in DHA and EPA on bone loss is through the suppression of osteoclastogenesis. Moreover, based on the results from this study an in vivo study warrants a further investigation.

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7-related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl- /H+ -exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Crosstalk between adipocytes and M2 macrophages compensates for osteopenic phenotype in the Lrp5-deficient mice.

A loss-of-function mutation in the Lrp5 gene in mice leads to a low bone mass disorder due to the inhibition of the canonical Wnt signaling pathway; however, the role of bone marrow microenvironment in mice with this mutation remains unclear. In this study, we evaluated proliferation and osteogenic potential of mouse osteoblasts using the MTT assay and Alizarin red staining. The levels of alkaline phosphatase, tartrate-resistant acid phosphatase, and adiponectin in culture supernatants were measured using the enzyme-linked immunosorbent assay. Osteoclast bone resorbing activity was evaluated by toluidine staining and the number and area of bone resorption pits were determined. We observed increased osteogenesis in osteoblasts co-cultured with the BM-derived myeloid cells compared to the osteoblasts cultured alone. Mice with global Lrp5 deletion had a relatively higher bone density compared to the mice carrying osteoblast/osteocyte-specific Lrp5 deletion. An increased frequency of M2 macrophages and reduced expression of inflammatory cytokines were detected in the myeloid cells derived from the bone marrow of mice with global Lrp5 deletion. Higher adipogenic potential and elevated levels of adiponectin in the global Lrp5 deletion mice contributed to the preferential M2 macrophage polarization. Here, we identified a novel systemic regulatory mechanism of bone formation and degradation in mice with global Lrp5 deletion. This mechanism depends on a crosstalk between the adipocytes and M2 macrophages in the bone marrow and is responsible for partly rescuing osteopenia developed as a result of decreased Wnt signaling.

Natural Antibiotic Oregano in Hydroxyapatite-Coated Titanium Reduces Osteoclastic Bone Resorption for Orthopedic and Dental Applications.

Traditional infection prevention and treatment methods include synthetic antibiotics, which can cause severe adverse side effects. Carvacrol and thymol are biologically active monoterpenoid extractants from oregano leaves with antibiotic capabilities; however, little is known regarding their effects on bone tissue engineering. The objective of this work is to understand their effects on osteogenesis, specifically with osteoblast and osteoclast cells, from surface-modified Ti6Al4V with plasma sprayed hydroxyapatite (HA) coatings. This system is an alternative to cemented implants to aid in bone healing. Results reveal that full carvacrol release from the HA matrix is successful in aqueous environments and modulation of release kinetics can also be made using polycaprolactone (PCL) and polyethylene glycol (PEG) polymers. From HA-pressed disc samples in physiological pH, full carvacrol release is achieved in 10 days using PCL/PEG, about 95% release in 50 days using no polymer, and 60% in 50 days when using a PCL coating. Without polymer, full carvacrol release is achieved after 3 days from HA coatings in both physiological pH and acidic pH, mimicking the post-surgery environment. The release is assessed as a diffusion-based mechanism in phosphate-buffered saline but degradation-based mechanism in acetate buffer solution. Carvacrol and thymol show bacterial inhibition of Staphylococcus epidermidis and no cytotoxic effects on osteoblast proliferation in vitro. Carvacrol and thymol also induce a significant 7% reduction in osteoclast tartrate-resistant acid phosphatase (TRAP) activity, caused by poorly attached cellular morphologies, leading to an approximately 65% reduction in osteoclast resorption pit formation. Our goal is to demonstrate a natural medicinal system that can support bone healing while providing infection prevention and reducing costly revision surgeries for orthopedic and dental applications.

Measuring mineralised tissue formation and resorption in a human 3D osteoblast-osteoclast co-culture model.

In vitro tissue engineered bone constructs have been developed, but models which mimic both formation and resorption in parallel are still lacking. To be used as a model for the bone remodeling process, the formation and resorption of mineralised tissue volume over time needs to be visualised, localised and quantified. The goal of this study was to develop a human 3D osteoblast-osteoclast co-culture in which 1) osteoblasts deposit mineralised matrix, 2) monocytes differentiate into resorbing osteoclasts, and 3) the formation and resorption of mineralised matrix could be quantified over time using micro-computed tomography (μCT). Mesenchymal stromal cells were seeded on silk fibroin scaffolds and differentiated towards osteoblasts to create mineralised constructs. Thereafter, monocytes were added and differentiated towards osteoclasts. The presence of osteoblasts and osteoclasts was confirmed using immunohistochemistry. Osteoclastic activity was confirmed by measuring the increased release of osteoclast marker tartrate resistant acid phosphatase (TRAP), suggesting that osteoclasts were actively resorbing mineralised tissue. Resorption pits were visualised using scanning electron microscopy. Mineralised matrix formation and resorption were quantified using μCT and subsequent scans were registered to visualise remodelling. Both formation and resorption occurred in parallel in the co-culture. The resorbed tissue volume exceeded the formed tissue volume after day 12. In conclusion, the current model was able to visualise, localise and quantify mineralised matrix formation and resorption. Such a model could be used to facilitate fundamental research on bone remodeling, facilitate drug testing and may have clinical implications in personalised medicine by allowing the use of patient cells.

Anti-Myeloma Drug Melflufen Inhibits RANKL-Stimulated Osteoclastogenesis By Suppressing Proliferation of CD14+ Precursor Cells

Background Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate (PDC) that targets aminopeptidases and rapidly releases alkylating agents into tumor cells. Osteolytic lesions are a hallmark of multiple myeloma and occur in almost all MM patients, causing severe bone pain and pathological fractures. Previous reports indicate that melphalan enhances osteoclastogenesis. On other other hand, melflufen is able to suppress proliferation of malignant myeloid cells including osteoclast precursors, monocytes. In the present study we therefore evaluated the direct effects of melflufen and melphalan on human osteoclastogenesis using cultured peripheral blood CD14+ monocytes. Methods CD14+ osteoclast precursors were isolated from peripheral blood. Osteoclastogenesis was stimulated by adding RANKL and M-CSF to CD14+ osteoclast precursors and assessed by tartrate-resistant acid phosphatase (TRAP) staining using para-nitrophenyl phosphate (PNPP). Osteoclastic activity was assessed by two assays. First, TRAP activity was measured by quantification of resorption pits on osteo assay synthetic surface made of crystalline calcium phosphate coating mimicking living bone material. Second, collagen degradation was quantified by release of C-terminal telopeptide of type I collagen (CTx) from mineralized bone slices. The proliferation of mature osteoclasts and their precursors in the presence of melflufen and melphalan was assessed using cytotoxicity assays. RNA isolated from osteoclast precursors and mature osteoclasts was subject to gene expression analysis by microarray. Results Melflufen inhibited RANKL-induced osteoclast formation at 1 µM concentration, while 25 µM melphalan was needed to initiate osteoclastogenesis inhibition. However, only extremely high concentrations of melflufen (10 µM) and melphalan (100 µM) affected mature osteoclasts and their TRAP activity. Cytotoxicity assays have demonstrated that melflufen, but not melphalan, can inhibit proliferation of monocytes, osteoclast progenitor cells, at clinically relevant concentrations. When added during osteoclastogenesis, melflufen has significantly inhibited TRAP phosphatase activity on crystalline calcium phosphate-coated surface at 1 µM, whereas 25 µM of melphalan was required to achieve the same effect. As for collagen degradation, the addition of 1 µM melflufen completely suppressed the CTx fragment release. In a parallel assay, 10 µM of melphalan was necessary to produce the same effect. Noteworthy, we did not observe any positive effect of melphalan on osteoclastogenesis or proliferation of osteoclasts. Gene expression analysis showed that mature osteoclasts express higher levels of bone lytic genes ACP5/TRAP, CTSK, MMP2, MMP7, and MMP9. In addition, CD276 and PDCD1LG2 mRNAs encoding B7 family immune checkpoint molecules B7-H3 and PD-L2 are also strongly up-regulated upon osteoclastogenesis. Discussion Myeloma patients often suffer from severe bone pain associated with osteolytic bone lesions. These lesions are the result of excessive osteoclastogenesis induced by multiple myeloma in the bone marrow microenvironment. Moreover, this process creates an immunosuppressive microenvironment since osteoclasts express immune checkpoint molecules known to negatively control activity of T lymphocytes (PD-L2, B7-H3) and NK cells (B7-H3). In this study, we have shown that a novel anti-myeloma PDC melflufen efficiently inhibits osteoclastogenesis and associated collagen degradation and bone resorption when used at clinically relevant concentrations. Melphalan could also induce a reduction of bone resorption and collagen release, however high and clinically irrelevant concentrations were necessary to obtain these effects. Given these results, it is plausible that melflufen-treated MM patients might achieve a reduction in bone pain, and this should be assessed in more detail in the follow-up studies. Also, elimination of immunosuppressive osteoclasts by melflufen may provide a synergistic effect between melflufen and immunotherapy while treating MM patients. This hypothesis is currently being tested in clinical trials. Disclosures Byrgazov: Oncopeptides AB: Current Employment. Gullbo:Theradex: Current Employment; Oncopeptides AB: Consultancy, Current Employment, Current equity holder in publicly-traded company. Slipicevic:Oncopeptides AB: Current Employment. Lehmann:Oncopeptides AB: Current Employment, Current equity holder in publicly-traded company. Larsson:Oncopeptides AB: Current equity holder in publicly-traded company. Fryknäs:Oncopeptides AB: Research Funding.

Glycyrrhizin improves bone metabolism in ovariectomized mice via inactivating NF-κB signaling

Objectives Postmenopausal osteoporosis (PMO) is a prevalent metabolic bone disease with high morbidity and serious complications. Here, we studied the effect of glycyrrhizin on bone metabolism using the ovariectomized (OVX) mouse model. Methods Osteoclast-related gene expression and osteoclastic function were evaluated in RAW264.7 cells and bone marrow-derived monocytes (BMMs) by real-time polymerase chain reaction and bone resorption assay. For animal studies, female C57BL/6J mice were randomly divided into sham operated, OVX and OVX with glycyrrhizin groups. Bone mass and trabecular microarchitecture were analyzed by micro-computed tomography, dual X-ray absorptiometry, and histomorphometric analysis. Receptor activator of nuclear factor-κB (NF-κB) ligand-induced osteoclastogenesis and the NF-κB signaling pathway were studied by tartrate-resistant acid phosphatase staining and western blotting, respectively. Results Glycyrrhizin inhibits RANKL-induced expression of Nfatc-1, c-Fos, Trap, Ds-stamp, and Ctsk in RAW264.7 cells. Also, fewer bone resorption pits form when BMMs are incubated in the presence of glycyrrhizin. Glycyrrhizin ameliorates bone loss and improves trabecular bone parameters in OVX mice. BMMs isolated from OVX mice show higher ability of RANKL-induced osteoclastogenesis, which is tremendously reversed by glycyrrhizin. There is significantly higher phosphorylation of IκB-α at Ser32 and NF-κB p65 at Ser536, as well as increased protein levels of c-FOS and NFATc-1 in BMMs of OVX mice, which are all greatly suppressed by glycyrrhizin. Conclusions Our findings imply that glycyrrhizin is a potential efficient adjuvant therapeutic for PMO.

Growth factors produced by bone marrow stromal cells on nanoroughened titanium-aluminum-vanadium surfaces program distal MSCs into osteoblasts via BMP2 signaling.

Statement of Clinical Significance: There remains the need to develop materials and surfaces that can increase the rate of implant osseointegration. Though osteoanabolic agents, like bone morphogenetic protein (BMP), can provide signaling for osteogenesis, the appropriate design of implants can also produce an innate cellular response that may reduce or eliminate the need to use additional agents to stimulate bone formation. Studies show that titanium implant surfaces that mimic the physical properties of osteoclast resorption pits regulate cellular responses of bone marrow stromal cells (MSCs) by altering cell morphology, transcriptomes, and local factor production to increase their differentiation into osteoblasts without osteogenic media supplements required for differentiation of MSCs on tissue culture polystyrene (TCPS). The goal of this study was to determine how cells in contact with biomimetic implant surfaces regulate the microenvironment around these surfaces in vitro. Two different approaches were used. First, unidirectional signaling was assessed by treating human MSCs grown on TCPS with conditioned media from MSC cultures grown on Ti6Al4V biomimetic surfaces. In the second set of studies, bidirectional signaling was assessed by coculturing MSCs grown on mesh inserts that were placed into culture wells in which MSCs were grown on the biomimetic Ti6Al4V substrates. The results show that biomimetic Ti6Al4V surface properties induce MSCs to produce factors within 7 days of culture that stimulate MSCs not in contact with the surface to exhibit an osteoblast phenotype via endogenous BMP2 acting in a paracrine signaling manner.

240. A subtractive biomimetic titanium surface promotes bony ongrowth in an ovine lumbar interbody fusion model

BACKGROUND CONTEXT Osseointegration, direct functional contact between bone and an implant surface, is a key process in achieving a rigid fusion and ultimately a successful clinical outcome following interbody fusion surgery. Surface properties of an implant (chemistry, topography, roughness) directly affect the healing response at the bone-implant interface. Surface modifications may enhance osseointegration on implants compared to those that have machined surfaces. A number of techniques exist that can provide macro- and microscale modifications to implant surfaces. Laser-etching is a subtractive technique that allows for controlled, intentionally designed features to be imparted on a surface. An implant surface that mimics the natural osteogenic environment with pores inspired by osteoclast resorption pits may trigger osteoblast cellular signaling, leading to positive downstream effects like implant osseointegration. PURPOSE The purpose of this study was to compare osseointegration of lumbar interbody fusion devices made from polyetheretherketone (PEEK), smooth titanium-alloy (sTAV), and laser-etched biomimetic TAV (rTAV) in a preclinical ovine model. STUDY DESIGN/SETTING In vivo ovine animal study. PATIENT SAMPLE N/A OUTCOME MEASURES Microcomputed tomography (microCT) and histomorphometric analyses were performed to quantify bone volume (BV) within the spacers (ingrowth) and total bone growth onto the surface of the spacer (ongrowth), respectively. BV was measured as the sum of the volume of bone in the central and both lateral graft windows. Bone ongrowth was assessed as the bone apposition ratio (BAR), the percent of the total length of the implant with direct bony contact on the interbody surface (linear apposition). Only superior and inferior surfaces of the implants were considered in BAR analysis, as the laser-etching treatment was only applied to these sides. METHODS Eighteen mature female sheep underwent two-level (L2–L3 and L4–L5) lateral interbody fusion using either PEEK, sTAV, or rTAV spacers (n=6 levels per group per time point). Animals were sacrificed at 6 and 12 weeks postoperatively. BV and BAR were evaluated at the two postoperative time points. RESULTS The rTAV implants demonstrated a trend towards higher BV than PEEK and sTAV implants at the 6-week postoperative time points (179.5±70.7 mm3, 116.9±43.0 mm3, and 108.7±15.2 mm3, respectively) (0.05≤p 0.05). At 6 weeks, the rTAV implants had significantly greater BAR than sTAV (24.9%±17.6% and 3.4±2.6%, respectively) (p CONCLUSIONS Bony ongrowth during interbody fusion is necessary for optimal implant fixation. Interbodyspacers with a biomimetic, laser-etched surface treatment demonstrated more bony ongrowth when compared to sTAV at the initial 6-week time point and continued to demonstrate robust bony ongrowth at 12 weeks. Although a small sample size limited this study's statistical power, these results demonstrate that implants with biomimetic surface modifications may promote robust bony ongrowth and ingrowth. FDA DEVICE/DRUG STATUS SINTROS (Globus Medical) (Approved for this indication)