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Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis, osteopenia, and other bone-related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD). Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to assess the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by using high-resolution quantitative micro–computed tomography and the three-dimensional reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. SCD female mice in particular are prone to trabecular bone loss, whereas cortical bone degradation occurs in both sexes. We also describe the impact of genetic knockdown of cathepsin K– and E-64–mediated cathepsin inhibition on SBD.

The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.

Background: Breast cancer (BC) produces bone resorptive cytokines and growth factors which accelerate the development of osteoclasts (OCs), leading to osteolytic bone metastases. In the Long-term Odanacatib Fracture Trial (LOFT), the skeletal-metastasized breast cancer subjects who received odanacatib (ODN) had a delayed tumour progression and skeletal tumour burden as a result of anti-resorptive effects through inhibition of cathepsin K (CTSK). In this study, we explored the effect of ODN, a CTSK inhibitor, on the paracrine pro-osteoclast activity of breast cancer cells. Methods: Immunohistochemistry study was performed to demonstrate CTSK and PTHrP expression in the samples of primary breast carcinoma. Expression of CTSK mRNA and protein was confirmed by the reverse transcription PCR and western blotting analysis in two human breast cancer cell lines MDA-MB-231 and MCF-7 BC cell lines. Cells were incubated with sub-lethal amounts of ODN, and their conditioned supernatants were assessed for their capacity to differentiate PBMCs of healthy donors into osteoclast and its interference on bone resorbing activities. We also measured the mRNA levels of major pro-osteoclast (pro-OC) factors in ODN-treated breast cancer cells and their secreted levels by semi-quantitative reverse transcription PCR and protein expression by immunoblotting. Results: Different staining intensity was observed in samples containing PTHrP and CTSK in various histological grades of breast carcinoma. Significant positive relationship was found between CTSK expression and histological grade of BC and presence or absence of distant metastasis.The present study results also indicate that ODN has no effects on OCs number , however; ODN decreases the mRNA expression of secreted pro-OC factors such as PTHrP, CXCR-4, and TNFα. Immuno blot indicates that ODN treatment decreased the protein expression of CTSK, IL-6, and IL-1β, and thus lowered protein levels paralleled the defective phosphorylation of NF-κB. Moreover, there was a significant reduction in the level of growth factors such as IGF-1, PDGF and TGFβ expression at transcriptional level after ODN treatment as compared to control. Conclusion: ODN has shown to prevent osteolytic metastasis by interacting with the NF-κB pathway; inhibiting bone resorptive cytokines, and growth factors. This effect can also be take into account the delayed development of metastatic bone disease found in the long-term odanacatib fracture trial (LOFT) study.

Background There is a knowledge gap about how equine medial femoral condyle (MFC) subchondral radiolucencies (SR) arise and evolve. Osteoclasts are believed to have a role, but have not been studied in situ. Objectives To measure and compare osteoclast density and the percentage of chondroclasts in healthy and MFC SR specimens from juvenile Thoroughbreds. Study design Cadaveric study. Methods Medial femoral condyles from a tissue bank of equine stifles were studied. Inclusion criteria were MFCs (≤8 months old) with a computed tomography SR lesion and histological focal failure of endochondral ossification (L group). Contralateral, lesion-free, MFCs were a control group (CC). Osteochondral slabs were cut through the lesion (L), a healthy site immediately caudal to the lesion, (internal control; IC) and the contralateral, site - matched controls (CC). Histological sections were immunostained with Cathepsin K for osteoclast counting. Osteoclasts in contact with the growth cartilage (chondroclasts) were also counted. The sections were segmented into regions of interest (ROI) at different depths in the subchondral bone: ROI1 (0-1mm), ROI2 (1-3mm) and ROI3 (3-6mm). Osteoclasts were counted and the bone area measured in each ROI to calculate their density. Chondroclasts were counted in ROI1 . Results Sections were studied from L and IC (n=6) and CC sites (n=5). Osteoclast density was significantly higher in ROI1 when compared with ROI3 in all groups. Although higher osteoclast density was measured in ROI1 in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI1 was lower in the L sections when compared with controls but no significant differences was detected. Main limitations Limited sample size. Conclusions Osteoclasts are important actors in MFC subchondral bone development, digesting both growth cartilage (chondroclasts) and bone, but the pathophysiology of early MFC SRs cannot be explained solely by an increased osteoclast presence in the subchondral bone.

Osteomyelitis associated with periprosthetic joint infection (PJI) signals a chronic infection and the need for revision surgery. An osteomyelitic bone exhibits distinct morphological features, including evidence for osteolysis and an accelerated bone remodelling into poorly organised, poor-quality bone. In addition to immune cells, various bone cell-types have been implicated in the pathology. The present study sought to determine the types of bone-cell activities in human PJI bones. Acetabular biopsies from peri-implant bone from patients undergoing revision total hip replacement (THR) for chronic PJI (with several identified pathogens) as well as control bone from the same patients and from patients undergoing primary THR were analysed. Histological analysis confirmed that PJI bone presented increased osteoclastic activity compared to control bone. Analysis of osteocyte parameters showed no differences in osteocyte lacunar area between the acetabular bone taken from PJI patients or primary THR controls. Analysis of bone matrix composition using Masson’s trichrome staining and second-harmonic generation microscopy revealed widespread lack of mature collagen, commonly surrounding osteocytes, in PJI bone. Increased expression of known collagenases, such as matrix metallopeptidase (MMP) 13, MMP1 and cathepsin K (CTSK), was measured in infected bone compared to non-infected bone. Human bone and cultured osteocyte-like cells experimentally exposed to Staphylococcus aureus exhibited strongly upregulated expression of MMP1, MMP3 and MMP13 compared to non-exposed controls. In conclusion, the study identified previously unrecognised bone-matrix changes in PJI caused by multiple organisms deriving from osteocytes. Histological examination of bone collagen composition may provide a useful adjunct diagnostic measure of PJI.

Objectives The objective of this study was to determine the effects of antimicrobial photodynamic therapy (aPDT) with indocyanine green (ICG) and toluidine blue (TB) on protease activity (matrix-bound cathepsin K and matrix metalloproteinase (MMP) and dentin bond strength. Methods Caries-free human third molars were assigned to five groups: 1—control group, 2—application of ICG with activation using an 810 nm diode (aPDT), 3—application of ICG, 4—application of TB with activation using a 660 nm diode (aPDT), and 5—application of TB. For the enzymatic investigation, dentin beams were incubated for either 3 days or 3 weeks. Aliquots of the incubation media were analyzed by ELISA for CTX (C-terminal cross-linked telopeptide of type I Collagen) and ICTP (cross-linked carboxy-terminal telopeptide of type I collagen). For microtensile bond strength testing (μTBS), composite resins were layered onto the tooth surface; the samples were then subjected to μTBS. Kruskall–Wallis and Mann–Whitney U tests were applied for statistical analysis of CTX and ICTP, one way-ANOVA and Tukey’s test were applied for statistical analysis of μTBS. Results Pretreating the dentin matrices with aPDT decreased the endogenous protease activity. ICG with laser activation resulted in the highest μTBS. Therefore, aPDT should be considered as a treatment method because it can reduce MMP-mediated dentin degradation and increase the μTBS. Significance Inhibiting endogenous protease activity improves the stability of the dentin–adhesive bond and the durability of the bond strength.

Igor Aleksander Bednarski,1 Magdalena Ciążyńska,2 Jacek Kabziński,3 Ireneusz Majsterek,3 Dorota Sobolewska-Sztychny,1 Joanna Narbutt,1 Aleksandra Lesiak1 1Department of Dermatology, Pediatric Dermatology and Dermatological Oncology, Medical University of Lodz, Lodz, 91-347, Poland; 2Nicolaus Copernicus Multidisciplinary Centre for Oncology and Traumatology, Lodz, 93-513, Poland; 3Department of Chemistry and Clinical Biochemistry, Medical University of Lodz, Lodz, 90-136, PolandCorrespondence: Igor Aleksander BednarskiDepartment of Dermatology, Pediatric Dermatology and Dermatological Oncology, Medical University of Lodz, Kniaziewicza 1/5, Lodz, 91-347, PolandEmail igorbednarskiv@gmail.comIntroduction: Photoaging is a premature skin aging developing secondarily to the excessive exposure to ultraviolet radiation. Due to its complexity, an exact mechanism of photoaging has not been found yet; however, recent research has shown two new emerging players in this process – cathepsin K and progerin.Aim: To evaluate how different wavelengths of ultraviolet radiation (UVA, narrowband UVB and broadband UVB) influence cathepsin K and progerin protein and mRNA expression in dermal cultured fibroblasts.Materials and Methods: Primary human dermal fibroblasts (Detroit 551, ATCC CCL-110) were cultured and irradiated with UVA, narrowband UVB (UVBnb) and broadband UVB (UVBwb). Fibroblasts were irradiated with 2 protocols: single high-dose exposure to UVR with protein/mRNA extraction immediately after exposure, 24 h after exposure and 48 h after exposure, and repeated (0 h, 24 h and 48 h) low-dose exposure to UVR with protein/mRNA extraction 48 h after first exposure.Results: Single high doses of UVA, UVBwb and UVBnb resulted in decreased expression of cathepsin K and progerin protein/mRNA in all subsequent time points. Repeated exposure to low doses of UVA results in significant increase of progerin mRNA and significant decrease of progerin protein after 48 h, but repeated exposure to UVBwb and UVBnb resulted in decreased progerin mRNA and protein expression. Repeated exposure to UVA, UVBwb and UVBnb resulted in decreased cathepsin K protein and mRNA expression.Conclusion: The results suggest that there could be another progerin/cathepsin K regulatory pathway, which has not been described yet. Being contradictory with previous research, the influence of ultraviolet radiation on progerin and cathepsin K needs to be further elucidated.Keywords: progerin, cathepsin K, photoaging

Pycnodysostosis is a rare autosomal recessive condition caused by the mutation of CTSK gene. CTSK regulates the activity of Cathepsin K which is responsible for osteoclast-mediated bone resorption. This mutation causes the bones to become dense, sclerotic, brittle, and thus, prone to fracture. Affected individuals have normal cognitive development and life expectancy, however, the quality of life depends on the early diagnosis of the condition. The patient presents with striking clinical (short stature, brachydactyly) and radiological (frontal and parieto-occipital bossing, open sutures, and fontanelles, acro-osteolysis of terminal phalanges) features making the diagnosis clinico-radiographic. In atypical or mild cases with overlapping features, gene mapping is advocated. A plethora of dental anomalies and characteristic craniofacial dysmorphia puts the dentist in a position to diagnose such a case.

Introduction. Unfavourable factors of the working environment affect bone tissue, increasing the risk of developing osteoporosis in workers of harmful industries, which is an urgent social and hygienic problem due to the high prevalence and severity of medical and social consequences. The aim of the study was to investigate the impact of industrial vibration and physical overload on the markers of bone metabolism in industrial workers. Materials and methods. One hundred fifty-two employees of mining and mechanical engineering enterprises in contact with industrial vibration and physical overload were examined. The assessment of bone tissue metabolism in the workers was carried out by the method of solid-phase enzyme immunoassay to determine biochemical markers of bone metabolism: bone alkaline phosphatase, osteocalcin, C-terminal serum telopeptide, cathepsin K, as well as indicators of calcium-phosphorus metabolism. X-ray densitometry was used to assess bone mineral density. Results. There was a significant decrease in the markers of osteogenesis: the concentration of CSF (14.26 ± 4.13 and 18.69 ± 5.28, respectively, p

Bone is the common extra-hepatic site for cancer metastasis. Hepatic cancer is associated with a higher incidence of pathological fracture. However, this important regulatory mechanism remains unexplored. Thus, exosome-mediated cell-cell communication between hepatocellular cancer and bone might be key to osteolytic bone destruction. Huh-7 exosomes were characterized for size and exosome marker expressions (CD63, Alix). Exosome mediated osteoclast differentiation in the RAW 264.7 cells was monitored from day 1 to 6 and multinucleated osteoclast formation and bone resorption activity were analyzed. The osteoclastogenic factor expressions in the exosomes and osteoclast differentiation markers such as tumor necrosis factor receptor 6 (TRAF6), nuclear factor κB (NF-κB), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and cathepsin K (CTSK) were analyzed using western blot. Exosomes released by liver cancer cells (Huh-7) promoted osteoclast differentiation in RAW 264.7 cells. Analysis of osteoclastogenic factors in the exosomes showed that exosomes were specifically enriched with tumor necrosis factor α (TNF-α). Huh-7 exosomes promoted osteoclast differentiation by significantly increasing the number of TRAP-positive multi nucleated osteoclasts and resorption pits. Importantly, exosomes upregulated osteoclast markers TRAF6, NF-κB, and CTSK expressions. Further, neutralizing exosomal TNF-α reverted exosome-mediated osteoclast differentiation in RAW 264.7 cells. Collectively, our findings show that cellular communication of exosomal TNF-α from hepatocellular cancer cells (Huh-7) regulates osteoclast differentiation through NF-κB/CTSK/TRAP expressions. Thus, exosomal TNF-α might act as an important therapeutic target to prevent hepatocellular cancer mediated pathological bone disease.

Osteoporosis is the most common cause of fractures in middle-aged and elderly people. Fracture repair can be difficult due to the decreased bone volume in osteoporosis patients and implants are often required. In this study, a slow-release system for microstructured titanium (Micro-Ti) was designed to promote osteogenesis and inhibit osteoclastogenesis. Firstly, Micro-Ti was prepared on titanium surfaces by dual acid etching. Micro-Ti was covered with naringin (NA), chitosan (CHI) and gelatin (GEL) multilayers through layer by layer technique, which is denoted as LBL (NA) coated-Ti. Osteoblasts (ME3T3-E1) and macrophages (RAW 264.7) were cultured on untreated and treated titanium surfaces in vitro. Osteoblasts grown on LBL (NA) coated-Ti showed higher alkaline phosphatase (ALP) and mineralization, consistent with qRT-PCR analysis of osteoblast genes including runt-related transcription factor 2 (Runx2), ALP, collagen I (Col I), osteocalcin (OCN), osteopontin (OPN), and osteoprotegerin (OPG). In contrast, acid tartarate-resistant phosphatase activity and the expression of osteoclastic differentiation related genes comprising of cathepsin K (CTSK), nuclear factor of activated T cells (NFAT), tartrate resistant acid phosphatase (TRAP) and V-ATPase (VATP) in osteoclasts were significantly reduced on LBL (NA) coated-Ti surfaces compared with other groups. These results indicate that microstructured titanium functionalized by naringin inserted multilayers enhanced the differentiation of osteoblasts and inhibited osteoclast formation. The proposed approach in this research provides a novel way to modify titanium-based implants for fracture repair in osteoporosis patients.

OBJECTIVE: Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). Our previous study showed that the serum levels of C3/C4 in MM patients were significantly positively correlated with the severity of bone disease. However, the mechanism of C3a/C4a in osteoclasts MM patients remains unclear. METHODS: The formation and function of osteoclasts were analyzed after adding C3a/C4a in vitro. RNA-seq analysis was used to screen the potential pathways affecting osteoclasts, and the results were verified by Western blot, qRT-PCR, and pathway inhibitors. RESULTS: The osteoclast area per view induced by 1 μg/mL (mean ± SD: 50.828 ± 12.984%) and 10 μg/mL (53.663 ± 12.685%) of C3a was significantly increased compared to the control group (0 μg/mL) (34.635 ± 8.916%) (P < 0.001 and P < 0.001, respectively). The relative mRNA expressions of genes, OSCAR/TRAP/RANKL/cathepsin K, induced by 1 μg/mL (median: 5.041, 3.726, 1.638, and 4.752, respectively) and 10 μg/mL (median: 5.140, 3.702, 2.250, and 5.172, respectively) of C3a was significantly increased compared to the control group (median: 3.137, 2.004, 0.573, and 2.257, respectively) (1 μg/mL P = 0.001, P = 0.003, P < 0.001, and P = 0.008, respectively; 10 μg/mL: P < 0.001, P = 0.019, P < 0.001, and P = 0.002, respectively). The absorption areas of the osteoclast resorption pits per view induced by 1 μg/mL (mean ± SD: 51.464 ± 11.983%) and 10 μg/mL (50.219 ± 12.067%) of C3a was also significantly increased (33.845 ± 8.331%) (P < 0.001 and P < 0.001, respectively) compared to the control. There was no difference between the C4a and control groups. RNA-seq analysis showed that C3a promoted the proliferation of osteoclasts using the phosphoinositide 3-kinase (PI3K) signaling pathway. The relative expressions of PIK3CA/phosphoinositide dependent kinase-1 (PDK1)/serum and glucocorticoid inducible protein kinases (SGK3) genes and PI3K/PDK1/p-SGK3 protein in the C3a group were significantly higher than in the control group. The activation role of C3a in osteoclasts of MM patients was reduced by the SGK inhibitor (EMD638683). CONCLUSIONS: C3a activated osteoclasts by regulating the PI3K/PDK1/SGK3 pathways in MM patients, which was reduced using a SGK inhibitor. Overall, our results identified potential therapeutic targets and strategies for MBD patients.

Aims To investigate the effect of dencichine on osteoclastogenesis in vivo and in vitro. Methods RANKL-induced osteoclastogenesis were treated with different concentrations of dencichine. Pit forming assays were applied to evaluate the degree of bone resorption. Osteoclastogenic markers were detected by real-time quantitative PCR (RT-qPCR) and Western blot. Micro CT was conducted to investigate the effects of dencichine on osteoclastogenesis in ovariectomized (OVX) mice. Results Dencichine suppressed osteoclastogenesis through the inhibition of phosphorylation of p65, p50 (NF-κB pathway), p38, ERK and JNK (MAPKs pathway) in vitro. Furthermore, dencichine inhibited the function of osteoclasts in a dose-dependent manner. In addition, the expression levels of the nuclear factor of activated T cells 1 (NFATc1) and osteoclastogenesis markers were decreased by dencichine, including MMP-9, Cathepsin K (CTSK), Tartrate-Resistant Acid Phosphatase (TRAP), C-FOS, dendritic cell specific transmembrane protein (DC-STAMP). In vivo data proved that dencichine alleviated ovariectomy-induced bone loss and osteoclastogenesis in mice. Conclusion Our results demonstrate that dencichine alleviates OVX-induced bone loss in mice and inhibits RANKL-mediated osteoclastogenesis via inhibition of NF-κB and MAPK pathways in vitro, suggesting that dencichine might serve as a promising candidate for treatment of bone loss diseases, including PMOP and rheumatoid arthritis.

Knee osteoarthritis (KOA) is a chronic joint disorder involving the articular cartilage and tissues around the synovial joint. The key objective of this study was to determine the effect of miR-186-5p administration on the expression of pathogenic signalling in the chondrocytes using a surgical destabilization of the medial meniscus (DMM) model of KOA, and to testify the mechanism of P2X7-mediated regulation of RUNX2/ADAMTS5 axis by miR-186 in the KOA rats. After eight weeks of intra-articular injection of the miR-186-5p and negative control lentivirus samples, the knee cartilage tissues were subjected to histopathological analysis Safranin-O/Fast green staining. Further, the articular chondrocytes were separated and analysed for various proteins including P2X7, cathepsin-K, RUNX2 and ADAMTS5 using Western blotting method. We observed that the protein expressions of P2X7, cathepsin-K/RUNX2/ADAMTS5, and also MMP-13 were upmodulated in the KOA rats, while intra-articular miR-186-5p lentivirus administration prevented these aberrations. Hence, the study concludes that miR-186 orchestrates P2X7 expression and the P2X7-mediated cathepsin-K/RUNX2/ADAMTS5 axis and regulates the pathogenesis of KOA. In light of this evidence, we propose that molecular therapeutic interventions targeting miR-186 activation might attenuate osteoarthritic cartilage degeneration.

The CD200–CD200R immunoregulatory signaling axis plays an etiologic role in the survival and spread of numerous cancers, primarily through suppression of antitumor immune surveillance. Our previous work outlined a prometastatic role for the CD200–CD200R axis in cutaneous squamous cell carcinoma (cSCC) that is independent of direct T-cell suppression but modulates the function of infiltrating myeloid cells. To identify effectors of the CD200–CD200R axis important for cSCC metastasis, we conducted RNA sequencing profiling of infiltrating CD11B+Cd200R+ cells isolated from CD200+ versus CD200-null cSCCs and identified the cysteine protease cathepsin K (Ctsk) to be highly upregulated in CD200+ cSCCs. CD11B+Cd200R+ cells expressed phenotypic markers associated with myeloid-derived suppressor cell–like cells and tumor-associated macrophages and were the primary source of Ctsk expression in cSCC. A Cd200R+ myeloid cell–cSCC coculture system showed that induction of Ctsk was dependent on engagement of the CD200–CD200R axis, indicating that Ctsk is a target gene of this pathway in the cSCC tumor microenvironment. Inhibition of Ctsk, but not matrix metalloproteinases, significantly blocked cSCC cell migration in vitro. Finally, targeted CD200 disruption in tumor cells and Ctsk pharmacologic inhibition significantly reduced cSCC metastasis in vivo. Collectively, these findings support the conclusion that CD200 stimulates cSCC invasion and metastasis via induction of Ctsk in CD200R+ infiltrating myeloid cells. Significance: These findings highlight the relationship between CD200–CD200R and cathepsin K in cutaneous squamous cell carcinoma metastasis and suggest that either of these components may serve as a viable therapeutic target in this disease.

Cathepsin K (CatK), encoded by CtsK gene in human, is involved in bone remodeling through ossification. The objective of the work conducted here was to express catalytic domains of CtsK gene in bacterial expression system as an initial step, facilitating recombinant production of human CatK for downstream applications in pharmacology. Four healthy human blood samples were collected. Genomic DNA was extracted using FlexiGene® whole blood DNA extraction kit. Upon quantification of DNA through NanodropTM spectrophotometer, sufficient quantity and quality was observed. CtsK gene was amplified by Polymerase Chain Reaction (PCR) using two pairs of primers tagged with restriction endonuclease sites of Sal1 and HindIII facilitating molecular cloning and visualized by Agarose Gel Electrophoresis (AGE). Two different bands of size 545bp and 265bp were observed. The bands were dissected and gel purified using GenaxxonTM gel purification kit and sequentially double digested by restriction enzymes; SalI and HindIII. Vector PBS was also subjected to sequential double digestion using same enzymes and visualized via AGE. Double digested insert of size 265bp and vector were ligated using T4 DNA Ligase (all enzymes from PromegaTM). On another trail, ligation of the PCR product with band size 265bp to pGEM-TTM easy vector system (from PromegaTM) was also done and transformed to Top10 Escherichia coli competent cells for expression separately. Cells were grown in LB media in presence of XGAL, IPTG and Ampicillin and transformed cells were screened. In the restriction enzyme digestion and ligation setup, since the insert and vector were both double digested, it is confirmed that white colonies obtained were Escherichia coli cells were transformed with the desired recombinant vector and is therefore confirmatory. In the case of pGEM-TTM ligation, a colony PCR was done using the white colonies obtained and product size was confirmed via AGE. In conclusion, the objective of study was successfully achieved, by expressing a catalytic domain of CtsK. Developments and improvements could be made for expression of entire CatK gene and downstream production of the Cathepsin K protein for effective therapeutic purpose.

Background: A cross-linking agent commonly used for cancer chemotherapy is a platinum compound such as cisplatin. However, with the acquisition of cellular drug resistance and adverse side effects, the potency of cisplatin is, therefore, often tempered. To overcome these issues, the present study has established the use of cathepsin k (CTSK) inhibitor as a potent chemosensitizer. Methods: The cytotoxic effect of cisplatin and odanacatib (ODN) on two different breast cancer patient- derived cell lines, MDA-MB-231 and MCF-7, was assessed by MTT-based colorimetric assay. The drug interaction coefficient CDI was used to evaluate the synergistically inhibitory impact of the drug combination and immunoblot was used to examine the expression of certain proteins responsible for cell survival and the mechanism of apoptosis. Results: In this study, we found that IC50 of ODN in combination with cisplatin (half of IC25) induced a synergistic cytotoxic effect in different breast cancer cells. Diminished expression of Bcl-2 and increased expression of Bax aroused the cytochrome release, that triggered caspase-9 and -3 activation in the combinatorial group. ODN with a lower dose of cisplatin significantly inhibited the protein expression of novel chemoresistant factors such as STAT3, NFκB and IL-6. Conclusion: This study highlights the potential effects of the combination of ODN with a reduced dose of cisplatin on improving the growth inhibition and apoptosis-inducing effect on breast cancer cells via combined inhibition of NF-κB-induced IL-6 and STAT3 activation. The study result suggests that the further development of this novel inhibitor in combination with a low dose of standard cisplatin-based chemotherapy may contribute to an alternative treatment option for certain cancers.

Bacterial products can stimulate inflammatory reaction and activate immune cells to enhance the production of inflammatory cytokines, and finally promote osteoclasts recruitment and activity, leading to bone destruction. Unfortunately, effective preventive and treatment measures for inflammatory osteolysis are limited and usually confuse the orthopedist. Astragalus polysaccharide (APS), the main extractive of Astragali Radix, has been widely used for treating inflammatory diseases. In the current study, in vitro and in vivo experimental results demonstrated that APS notably inhibited osteoclast formation and differentiation dose‐dependently. Moreover, we found that APS down‐regulated RANKL‐related osteoclastogenesis and levels of osteoclast marker genes, such as NFATC1, TRAP, c‐FOS and cathepsin K. Further underlying mechanism investigation revealed that APS attenuated activity of MAPK signalling pathways (eg ERK, JNK and p38) and ROS production induced by RANKL. Additionally, APS was also found to suppress LPS‐related inflammatory osteolysis by decreasing inflammatory factors' production in vivo. Overall, our findings demonstrate that APS effectively down‐regulates inflammatory osteolysis due to osteoclast differentiation and has the potential to become an effective treatment of the disorders associated with osteoclast.

The only difference between fractured and non-fractured postmenopausal women with PHPT of same sex, age, and BMI was a significantly mean higher serum k-periostin level. K-periostin value was associated with fracture at any site (odds ratio 1.044, 95% CI 1.005–1.091, p = 0.03). To assess serum k-periostin fragment levels in patients with primary hyperparathyroidism (PHPT), fractured and non-fractured matched for sex, age, and body mass index. Twenty-five Caucasian fractured postmenopausal women with PHPT (group Fx) and 25 PHPT non-fractured (group NFx) were enrolled. Each patient underwent DXA scan at lumbar, hip, and forearm, spine X-ray, and biochemical evaluation of calcium metabolism. For k-periostin analyses, we utilized a specific ELISA test that detects CatK-generated fragment levels in the bloodstream. We found no difference in mean BMD and bone turnover marker values between Fx and NFx groups. Prevalence of osteoporosis was not significantly different in Fx vs NFx (72% vs 60%, p = 0.55). Among Fx, 16% reported multiple fractures, 28% morphometric vertebral fractures, 4% femoral fractures, 28% non-vertebral non-femoral fractures, and 8% wrist fractures. The only detectable difference between Fx and NFx group was a significantly mean higher k-periostin serum level (46.2 ± 21.4 vs 34.7 ± 13.5 ng/ml, p = 0.02). K-periostin was associated with fracture at any site (odds ratio 1.044, 95% CI 1.005–1.091, p = 0.03). No difference in mean k-periostin values was found between patients with vertebral fracture vs those with non-vertebral fracture, and between those with multiple fractures vs those with single fracture. Serum k-periostin is significantly associated with fracture in PHPT. If confirmed by further studies, k-periostin could be considered a new marker of bone fragility in PHPT, independently of BMD.

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti‐inflammation and anti‐oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose‐dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast‐specific genes, such as tartrate‐resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V‐ATPase d2) and dendritic cell–specific transmembrane protein (DC‐STAMP), were down‐regulated by galangin treatment. Furthermore, the results of the pit formation assay and F‐actin ring staining revealed impaired osteoclastic bone resorption in the galangin‐treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C‐Jun and C‐Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)‐induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF‐κB signalling pathways.

Osteoporosis is defined as the thinning of bone resulting from the imbalance between bone resorption and bone formation, the two cellular mechanisms that regulate the remodeling of skeletal tissue. This loss of bone ultimately results in an increased risk of fracture. One therapeutic approach to treating patients with osteoporosis is to inhibit the process of bone resorption. This has been clinically manifested by the development of a number of large and small molecules that include calcitonin, integrin inhibitors, cathepsin K inhibitors, OPG/RANKL inhibitors, bisphosponates, and SERMS. This chapter describes recent advances in the anti-resorptive field with a focus on clinically validated targets and the molecules that have been developed to regulate these pathways. Keywords: Osteoporosis; Osteoclast; Osteoblast; Calcitonin; Cathepsin K Inhibitors; Osteoprotegerin; RANKL; Bisphosponates; Selective Estrogen Receptor Modulators; SERMs

Pycnodysostosis is a rare autosomal recessive disease caused by a mutation in the cathepsin K enzyme gene, a protease that is expressed primarily in osteoclasts and is responsible for bone matrix degradation. The presentation is usually accompanied by short stature, osteoesclerosis, craniofacial dysmorphia and bone fragility. Some papers provide surgical options for fractures of long bones in this type of patients, but none are presenten in European Caucasian patients. The case presented is of a Spanish Caucasian European male with bilateral femoral fracture treated by endomedular nailing.

Abstract:: Osteoporosis is a pathological loss of bone mass due to an imbalance in bone remodeling where osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation resulting in skeletal fragility and fractures. Anti-resorptive agents, such as bisphosphonates and SERMs, and anabolic drugs that stimulate bone formation, including PTH analogues and sclerostin inhibitors, are current treatments for osteoporosis. Despite their efficacy, severe side effects and loss of potency may limit the long term usage of a single drug. Sequential and combinational use of current drugs, such as switching from an anabolic to an anti-resorptive agent, may provide an alternative approach. Moreover, there are novel drugs being developed against emerging new targets such as Cathepsin K and 17β-HSD2 that may have less side effects. This review will summarize the molecular mechanisms of osteoporosis, current drugs for osteoporosis treatment, and new drug development strategies.

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non‐immune cells help initiating activation of myeloid cells for differentiation, whereas hyper‐activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp–derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone‐related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co‐cultured in a contact‐free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP‐9 assessed by quantitative RT‐PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT‐PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up‐regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose‐dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M‐CSF phosphorylate AKT and activate PI3K‐AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG‐mediated inhibition of the downstream activation of PI3K‐AKT signalling pathway was similar to the DPSC co‐culture–mediated inhibition of OC differentiation. This study provides novel evidence of DPSC‐mediated inhibition of osteoclastogenesis mechanisms.

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.

Introduction Leg ulcers are a frequently observed medical problem affecting 3–5% of the general population over 65 years of age. The most common factor responsible for the development of leg ulcers is chronic venous insufficiency (CVI). It is believed that during the formation of an ulcer there are two processes occurring simultaneously, extracellular matrix (ECM) degradation and angiogenesis in which several proteins including matrix metalloproteinases, angiogenic and regulatory factors are engaged. Aim To determine the serum concentration of matrix metalloproteinase-1 (MMP-1), -9 (MMP-9), tissue inhibitors of metalloproteinases-1 (TIMP-1), angiogenin and vascular endothelial growth factor (VEGF) in patients suffering from venous leg ulcers and in the healthy control group. Material and methods The study group consisted of 71 Caucasians (39 patients, 32 controls). To evaluate the serum concentration of MMP-1, MMP-9, TIMP-1, VEGF and angiogenin, the ELISA technique was used. Results Mean MMP-1 and MMP-9 concentrations in the study group were 14.16 ±2.98 and 12.45 ±3.85 ng/ml, respectively, and in controls 6.08 ±2.51 ng/ml and 6.77 ±2.41 ng/ml, respectively and both differences were statistically significant (p < 0.001). There was no significant difference between the study and the control group in TIMP-1 concentration. Mean VEGF and ANG concentrations in the study group were 589.3 ±346.2 pg/ml and 1802.0 ±415.7 pg/ml, respectively, and in controls 220.3 ±110.4 pg/ml and 1229.0 ±337.7 pg/ml, respectively and both differences were statistically significant (p < 0.001). Conclusions Lack of significant differences in the concentration of TIMP-1 between the control and the study group confirms that proteolysis is a hallmark of CVI, but increased concentration of VEGF and angiogenin in the study group compared to the control group shows that angiogenesis occurs simultaneously with ECM remodelling.

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.

Bone is one of the dynamic tissues in the human body that undergoes continuous remodelling through subsequent actions of bone cells, osteoclasts, and osteoblasts. Several signal transduction pathways are involved in the transition of mesenchymal stem cells into osteoblasts. These primarily include Runx2, ATF4, Wnt signaling and sympathetic signalling. The differentiation of osteoclasts is controlled by M-CSF, RANKL, and costimulatory signalling. It is well known that bone remodelling is regulated through receptor activator of nuclear factor-kappa B ligand followed by the binding to RANK, which eventually induces the differentiation of osteoclasts. The resorbing osteoclasts secrete TRAP, cathepsin K, MMP-9 and gelatinase to digest the proteinaceous matrix of type I collagen and form a saucer-shaped lacuna along with resorption tunnels in the trabecular bone. Osteoblasts secrete a soluble decoy receptor, osteoprotegerin that prevents the binding of RANK/RANKL and thus moderating osteoclastogenesis. Moreover, bone homeostasis is also regulated by several growth factors, cytokines, calciotropic hormones, parathyroid hormone and sex steroids. The current review presents a correlation of the probable molecular targets underlying the regulation of bone mass and the role of essential metabolic elements in bone remodelling. Targeting these signaling pathways may help design newer therapies for treating bone diseases.

BACKGROUND Periodontitis and orthodontic treatment can lead to inflammatory root resorption (IRR) through an unclear mechanism. Chemerin, a novel chemoattractant protein, is closely associated with inflammation, affects osteoblast and osteoclast differentiation, and may play a role in IRR. We aimed to explore possible roles of the chemerin/ChemR23 interaction in cementoblast function and IRR and reveal a new IRR therapeutic target. METHODS Cementoblast function-related gene and protein expression in the immortalized murine cementoblast cell line OCCM-30 after treatment with chemerin and siChemR23 was examined by qRT-PCR and Western blotting. The roles of the MAPK and PI3K-Akt signaling pathways were studied using specific inhibitors. Cementoblast cytokine production under different treatment conditions was measured by enzyme-linked immunosorbent assay and qRT-PCR. Additionally, we modeled IRR in wild-type and chemerin-overexpressing mice and injected transgenic mice with anti-ChemR23 antibody to block ChemR23. We then calculated the root resorption volume and examined periodontal tissue cathepsin K, Runx2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression. RESULT Chemerin suppressed cementoblast differentiation and mineralization and exerted a proinflammatory effect on cementoblasts. These effects were partially reversed by siChemR23 and reversed to different extents by p38, Erk1/2 and PI3K-Akt pathway inhibition, suggesting p38, Erk1/2 and PI3K-Akt pathways as signaling pathways downstream of chemerin/ChemR23. In vivo, chemerin overexpression worsened IRR. Moreover, chemerin expression was positively correlated with TNF-α, IL-6, and cathepsin K expression and negatively correlated with Runx2 expression. ChemR23 downregulation reversed these effects. CONCLUSION Chemerin/ChemR23 induced TNF-α and IL-6 expression dependent on Erk1/2, p38 MAPK, and PI3K-Akt signaling pathway activation, thereby regulating cementoblast function and affecting IRR.

Melatonin, secreted in a typical diurnal rhythm pattern, has been reported to prevent osteoporosis; however, its role in osteoclastogenesis remains unclear. In the present study, the ability of melatonin to inhibit receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclastogenesis and the associated mechanism were investigated. Raw264.7 cells were cultured with RANKL (100 ng/ml) and macrophage colony‑stimulating factor (M‑CSF; 30 ng/ml) for 7 days, and tartrate‑resistant acid phosphatase (TRAP) staining was used to detect osteoclastogenesis following treatment with melatonin. In addition, the effect of melatonin on cathepsin K and microRNA (miR)‑882 expression was investigated via western blotting and reverse transcription‑quantitative PCR. Melatonin significantly inhibited RANKL‑induced osteoclastogenesis in Raw264.7 cells. From bioinformatics analysis, it was inferred that nuclear receptor subfamily 1 group D member 1 (NR1D1/Rev‑erbα) may be a target of miR‑882. In vitro, melatonin upregulated Rev‑erbα expression and downregulated miR‑882 expression in the osteoclastogenesis model. Rev‑erbα overexpression boosted the anti‑osteoclastogenesis effects of melatonin, whereas miR‑882 partially diminished these effects. The present results indicated that the miR‑882/Rev‑erbα axis may serve a vital role in inhibiting osteoclastogenesis following RANKL and M‑CSF treatment, indicating that Rev‑erbα agonism or miR‑882 inhibition may represent mechanisms through which melatonin prevents osteoporosis.

Approximately 90% of patients with advanced breast cancer develop bone metastases; an event that results in severe decrease of quality of life and a drastic deterioration in prognosis. Therefore, to increase the survival of breast cancer patients, the development of new therapeutic strategies to impair metastatic process and skeletal complications is critical. Previous studies on the role of cathepsin K (CTSK) in metastatic spreading led to several strategies for inhibition of this molecule such as MIV-711 (Medivir), balicatib and odanacatib (ODN) which were on trial in the past. The present study intended to assess the anti-metastatic efficacy of ODN in breast cancer cells. Human breast cancer cell lines MDA-MB-231 were treated with different concentrations of ODN and performed invasion, adhesion and migration assays and, RT-PCR and western blot to evaluate the effect of ODN on the metastatic potential of breast cancer cells. ODN markedly decreased wound healing cell migration, invasion and adhesion at a dose dependent manner. ODN inhibits cell invasion by decreasing the matrix metalloproteinase (MMP-9) with the upregulation of TIMP-1 expression. ODN effectively inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal Kinase (JNK), and blocked the expression of β-integrins and FAK proteins. ODN also significantly inhibited PI3K downstream targets Rac1, Cdc42, paxillin and Src which are critical for cell adhesion, migration and cytoskeletal reorganization. ODN exerts anti-metastatic action through inhibition of signaling pathway for MMP-9, PI3K and MAPK. This indicates potential therapeutic effects of ODN in the treatment of metastatic breast cancer.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Osteoporosis disturbs the balance of bone metabolism, and excessive bone resorption causes a decrease in bone density, thus increasing the risk of fracture. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea. EGCG has a variety of pharmacological activities. Recently, it was reported that EGCG inhibits osteoclast differentiation, but the details of the mechanism underlying the EGCG-mediated suppression of osteoclastogenesis are unknown. In this study, we investigated the effects of EGCG on several signaling pathways in osteoclastogenesis. EGCG suppressed the expression of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), the master regulator of osteoclastogenesis. EGCG decreased the expression of cathepsin K, c-Src, and ATP6V0d2 and suppressed bone resorption. We also found that EGCG upregulated heme oxygenase-1 (HO-1) and suppressed the extracellular release of high-mobility group box 1 (HMGB1). In addition, EGCG decreased the expression of the receptor for advanced glycation end products (RAGE), which is the receptor of HMGB1, in osteoclastogenesis. In summary, our study showed that EGCG could inhibit osteoclast differentiation through the downregulation of NFATc1 and the suppression of the HO-1-HMGB1-RAGE pathway. EGCG might have the potential to be a lead compound that suppresses bone resorption in the treatment of osteoporosis.

Background Neutralising antibodies to sclerostin (Scl-Ab) have shown significant potential to induce bone formation and decrease bone resorption, increase strength and substantially reduce fracture risk in animal studies and clinical trials. Mechanical loading negatively regulates sclerostin expression, and sclerostin has been shown to induce RANKL synthesis in osteocytes. However, how Scl-Ab governs osteocyte regulation of osteoclast differentiation and function is not fully understood. We have recently discovered that osteoblasts and osteocytes alter osteoclastogenic signalling (RANKL/OPG) during estrogen-deficiency, and that osteoblast-induced osteoclastogenesis and resorption are exacerbated. However, it is not known whether estrogen deficient osteocytes exacerbate osteoclastogenesis. The aims of this study were to (1) establish whether osteocytes induce osteoclastogenesis and bone resorption during estrogen deficiency in vitro (2) investigate whether the sclerostin antibody can revert osteocyte-mediated osteoclastogenesis and resorption by attenuating RANKL/OPG expression. Results Using conditioned media and co-culture experiments we found increased osteocyte-induced osteoclastogenesis and bone resorption in estrogen deficient conditions. This is the first study to report that administration of Scl-Ab has the ability to revert osteocyte-mediated osteoclastogenesis and resorption by decreasing RANKL/OPG ratio expression and increasing WISP1 expression in estrogen deficient osteocytes. Conclusions This study provides an enhanced understanding of the biological changes underpinning decreases in bone resorption following Scl-Ab treatment observed in vivo by revealing that Scl-Ab can reduce pro-osteoclastogenic cell signalling between osteocytes and osteoclasts.

Background: In treating peri-implantitis, dentists pay attention to drugs that do not contain chemically aggressive agents. They rather use those having natural mechanisms for normalization of the functional activity of cells and tissues in addition to local antiseptic and antibacterial agents. The aim of the study was to determine the clinical effectiveness of nicotinamide adenine nucleotide hydride dental gel in the treatment of peri-implantitis. Materials and methods: Depending on the therapeutic tactics patients (n = 40), we divided patients into two groups. In Group 1 (n=20), we used a standard treatment of peri-implantitis including professional hygiene measures, antiseptic treatment and the use of antibacterial agents. In Group 2 (n = 20), the complex treatment included photodynamic effects of 1% photosensitizer gel instead of the antibacterial therapy. Results: Due to the use of the photodynamic therapy in addition to standard treatment of group 2 patients, the concentration of a tartrate-resistant acid phosphatase in the peri-implantation exudate reached the level of healthy people in the gingival fluid. In addition, the concentrations of cathepsin K and interleukin-6 were as close as possible to the control level. Conclusion: The use of NADH for peri-implantitis helps to eliminate inflammatory phenomena and stimulates the regeneration of the oral mucosa.

A study involving 45 patients with ulcerative colitis found that the activity of the Cathepsin K bone resorption marker measured by an enzyme-linked immunosorbent assay in gingival fluid could be used as a non-invasive control over the development of steroid osteoporosis caused by systemic use of glucocorticoids. When the concentration of Cathepsin K in the gingival fluid exceeded 3.7 pmol/L, the risk of developing osteoporosis in patients with ulcerative colitis taking glucocorticosteroids on a regular basis increased by 7.9 times with a diagnostic accuracy of 82.6% (p=0.002). As a result of the study, an effective method for non-invasive assessment of osteoresorbable side effects of glucocorticosteroids in autoimmune diseases was proposed.

The intracellular accumulation of α-synuclein (α-syn) amyloid fibrils is a hallmark of Parkinson’s disease. Because lysosomes are responsible for degrading aggregated species, enhancing lysosomal function could alleviate the overburden of α-syn. Previously, we showed that cysteine cathepsins (Cts) is the main class of lysosomal proteases that degrade α-syn, and in particular, CtsL was found to be capable of digesting α-syn fibrils. Here, we report that CtsK is a more potent protease for degrading α-syn amyloids. Using peptide mapping by liquid chromatography with mass spectrometry, critical cleavage sites involved in destabilizing fibril structure are identified. CtsK is only able to devour the internal regions after the removal of both N- and C-termini, indicating their protective role of the amyloid core from proteolytic attack. Our results suggest that if overexpressed in lysosomes, CtsK has the potential to ameliorate α-syn pathology.

Cardiovascular (CV) disease remains the leading cause of mortality among end-stage kidney disease (ESKD) patients. Cathepsin-K (CatK), a small cysteine protease involved in bone and extracellular matrix remodeling, has recently emerged as a key-factor in the pathogenesis of various conditions predisposing to CV disease, including atherosclerosis, obesity, diabetes, and vascular calcification. In this pilot prospective study, we aimed at evaluating the clinical significance and the predictive power of CatK in a small cohort of hemodialysis (HD) patients.Cathepsin-K was measured in 54 prevalent HD patients and in 30 controls together with routine parameters. Patients were then followed up to 26 months and the time of cardiovascular death (endpoint of the study prospective phase) recorded.CatK levels were increased in the HD cohort as compared with controls (p 0.001). In HD patients, CatK was also independently correlated to PTH (β = 0.368; p = 0.001), alkaline phosphatase (β = 0.383; p 0.001), C-reactive protein (β = 0.260; p = 0.01), and white cell count (β = - 0.219; p = 0.02). After baseline assessment, patients were followed for CV death (mean follow-up 24.8 ± 3.1 months). Kaplan-Meier analysis showed a worsen survival (log-rank p = 0.04) in HD patients with CatK levels 440 pg/mL (best ROC-derived cut-off with 69.6% sensitivity and 79.8% specificity) with a crude HR (Mantel-Haenszel) of CV death of 3.46 (95% CI 1.89-13.44).In prevalent HD patients, altered CatK levels may reflect mineral dysmetabolism and inflammation, and predict CV death in the mid-term. These preliminary findings prompt the rationale for further investigations on larger cohorts to validate CatK as a biomarker for improving CV risk stratification in ESKD.

In the case of complete luxation with dental trauma, delayed replantation leads to a higher probability of root resorption. We evaluated the efficacy of N-acetylcysteine (NAC) as a root canal medication for trauma-associated root resorption. A rat dental trauma model was used. In the test group, mesial root pulpectomy was carried out after tooth extraction. NAC was applied as a root canal medication followed by replantation. We euthanized the rats after two weeks, excised the maxilla, and prepared thin sections, followed by H-E and TRAP staining. Furthermore, RAW264.7 cells were treated with RANKL and NAC. The effects of NAC on osteoclast differentiation were investigated by determining the numbers of osteoclasts and nuclei and quantifying osteoclast-related mRNA expression levels by real-time PCR. The level of root resorption and number of odontoclasts tended to be lower in the NAC groups than in the control group. In terms of osteoclast differentiation, the total number of osteoclasts was lower in both the 5 and 10 mmol/L NAC groups than in the control group. Real-time PCR revealed that the expression levels of RANK, NFATc1, DC-STAMP, CD47, and cathepsin K were lower in the 10 mmol/L NAC group. NAC tended to inhibit root resorption and reduce the number of odontoclasts. NAC also inhibited the expression of osteoclast-related genes. Overall, NAC reduced the number of odontoclasts, inhibited bone resorption, and influenced the inhibition of osteoclast differentiation, fusion, and functions. Therefore, we suggest that NAC may be beneficial in the treatment of root resorption. (C) 2020 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.

The endothelin system has an important role in bone modelling during orthodontic tooth movement (OTM); however, little is known about the involvement of endothelin B receptors (ETB) in this process. The aim of this study was to evaluate the role of ETB in bone modelling during OTM using ETB knockout rats (ETB-KO). Thirty-two male rats were divided into 4 groups (n = 8 per group): the ETB-KO appliance group, ETB-KO control group, wild type (ETB-WT) appliance group, and ETB-WT control group. The appliance consisted of a super-elastic closed-coil spring placed between the first and second left maxillary molar and the incisors. Tooth movement was measured on days 0 and 35, and maxillary alveolar bone volume, osteoblast, and osteoclast volume were determined histomorphometrically on day 35 of OTM. Next, we determined the serum endothelin 1 (ET-1) level and gene expression levels of the osteoclast activity marker cathepsin K and osteoblast activity markers osteocalcin and dentin matrix acidic phosphoprotein 1 (DMP1) on day 35. The ETB-KO appliance group showed significantly lower osteoblast activity, diminished alveolar bone volume and less OTM than the ETB-WT appliance group. Our results showed that ETB is involved in bone modelling in the late stage of OTM.