Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs.

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreERT2 , Col2a1-Cre; Col2a1-CreERT2 , Shh-Cre, Shh-CreERT2 , and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreERT2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreERT2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreERT2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.

METTL3 promotes experimental osteoarthritis development by regulating inflammatory response and apoptosis in chondrocyte.

OBJECTIVE: This study was to investigate the functional role of RNA methyltransferase METTL3, an enzyme catalyzes the formation of N6-methyladenosine (m6A) on the target mRNA, in the development of osteoarthritis (OA) and the underlying mechanism. METHODS: Cytokine IL-1ß was used to stimulate the chondroprogenitor cell line ATDC5 cells to mimic the inflammatory condition in vitro. The level of METTL3 mRNA and m6A as well as inflammatory cytokines were detected by qRT-PCR. Cell activity was detected by CCK-8. The rate of apoptotic cell was measured by flow cytometry. Western blot was used to detect the levels of NF-κB signaling molecules and collagen in cells. Methylation inhibitor cycloleucine and methyl donor betaine were used to treat collagenase-induced OA mice. RESULTS: In IL-1ß-treated ATDC5 cells, the METTL3 mRNA levels and the percentage of m6A methylated mRNA of total mRNA were increased in a dose-dependent manner. Silencing of METTL3 by shRNA reduced the percentage of IL-1ß-induced apoptosis, suppressed IL-1ß-induced increased inflammatory cytokines levels and activation of NF-κB signaling in chondrocytes. Moreover, silencing of METTL3 promotes degradation of extracellular matrix (ECM) by reducing the expression of MMP-13 and Coll X, elevating the expression of Aggrecan and Coll II. In a OA mouse model induced by collagenase, injection of methylation inhibitor cycloleucine or methyl donor betaine does not affects METTL3 mRNA expression, but significantly inhibits or promotes the total level of m6A as well as inflammatory condition and ECM degradation, respectively. CONCLUSION: METTL3 has a functional role in mediates osteoarthritis progression by regulating NF-κB signaling and ECM synthesis in chondrocytes that shed insight on developing preventive and curative strategies for OA by focusing on METTL3 and mRNA methylation.

SNHG16 promotes osteosarcoma progression and enhances cisplatin resistance by sponging miR-16 to upregulate ATG4B expression.

BACKGROUND: Human osteosarcoma is the most common primary cancer of the bone. Multiple mechanisms underlying cell growth, apoptosis, bone development, and drug resistance are important in the development of osteosarcoma therapy, which remains to be fully studied. METHODS: We collected thirty-paired tumor tissues and the adjacent normal ones from osteosarcoma patients. Two osteosarcoma cell lines (SAOS2, U2OS) were used for in vitro experiments. RT-qPCR and Western blot were used for gene expression detection. We applied starBase to predict the potential binding sites. Then, the luciferase reporter assay was used to confirm the potential direct interaction. Besides, we applied CCK-8, EdU assay, and flow cytometric assays to detect cell growth and apoptosis rate. We used wound healing and transwell assays to determine cell migration and invasion abilities. Additionally, we constructed a cisplatin-resistant osteosarcoma cell line to study the potential impact of the regulatory axis on drug resistance. RESULTS: Small nucleolar RNA host gene 16 (SNHG16) and autophagy-related 4B (ATG4B) were significantly upregulated in osteosarcoma tissues than the normal ones, and the higher expression level of SNHG16 predicted a poor prognosis in osteosarcoma patients. By contrast, the expression level of miR-16 was markedly lower in tumor tissues and was negatively correlated with SNHG16 (p < 0.001). SNHG16 was shown to promote cell growth, migration, and invasion, while miR-16 reversed this impact. Meanwhile, overexpression of ATG4B significantly promoted the development of osteosarcoma cells attenuated by SNHG16 knockdown or miR-16 mimics. Specifically, overexpression of ATG4B promoted cisplatin-induced autophagy and inhibited cell apoptosis rate, which enhanced the cisplatin resistance in osteosarcoma cell lines. CONCLUSIONS: Overall, our findings showed the importance of the regulatory axis of SNHG16/miR-16/ATG4B underlying osteosarcoma progression and chemoresistance to cisplatin. This research would benefit the therapy development in the treatment of osteosarcoma.

Osteocyte-intrinsic mTORC1 signaling restrains trabecular bone accrual in mice.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling plays important physiological roles in bone homeostasis by regulating multiple steps of osteoblast differentiation as well as its activity. However, its potential role in osteocytes has not been explored. In this study, we deleted Raptor, a specific and essential component of mTORC1, in osteocytes using Dmp1-Cre. Deletion of Raptor in osteocytes did not affect bone development and growth, but caused compartment-specific effects on bone mass. Osteocyte-specific deletion of Raptor had no obvious effect on cortical bone compartments, but led to increased trabecular bone mass. Mechanistically, Raptor deletion resulted in decreased bone resorption without altering bone formation activity. Thus, our study revealed an unexpected role of osteocyte-intrinsic mTORC1 signaling in limiting trabecular bone mass, suggesting that osteocyte-specific inhibition of mTORC1 may be used as a novel approach to treatment of osteoporosis.

Piperlongumine, a Piper longum-derived amide alkaloid, protects mice from ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis via suppression of p38 and JNK signaling.

Postmenopausal osteoporosis (PMOP) is a metabolic bone disease that results from overproduction and hyperactivation of osteoclasts caused by insufficient estrogen in women after menopause. Current therapeutic strategies are mainly focused on treating PMOP patients who have already developed severe bone loss or even osteoporotic fractures. Obviously, a better strategy is to prevent PMOP from occurring in the first place. However, such reagents are largely lacking. Piperlongumine (PLM), an amide alkaloid extracted from long pepper Piper longum, exhibits the anti-osteoclastogenic effect in normal bone marrow macrophages (BMMs) and the protective effect against osteolysis induced by titanium particles in mice. This study examined the preventive effect of PLM on PMOP and explored the potential mechanism of this effect using both ovariectomized mice and their primary cells. The result showed that PLM (5 and 10 mg kg-1) administered daily for 6 weeks ameliorated ovariectomy-induced bone loss and osteoclast formation in mice. Further cell experiments showed that PLM directly suppressed osteoclast formation, F-actin ring formation, and osteoclastic resorption pit formation in BMMs derived from osteoporotic mice, but did not obviously affect osteogenic differentiation of bone marrow stromal cells (BMSCs) from these mice. Western blot analysis revealed that PLM attenuated maximal activation of p38 and JNK pathways by RANKL stimulation without affecting acute activation of NF-κB, AKT, and ERK signaling. Furthermore, PLM inhibited expression of key osteoclastogenic transcription factors NFATc1/c-Fos and their target genes (Dcstamp, Atp6v0d2, Acp5, and Oscar). Taken together, our findings suggest that PLM inhibits osteoclast formation and function by suppressing RANKL-induced activation of the p38/JNK-cFos/NFATc1 signaling cascade, thereby preventing ovariectomy-induced osteoporosis in mice. Thus, PLM can potentially be used as an anti-resorption drug or dietary supplement for the prevention of PMOP.

3D-printed antibiotic-loaded bone cement spacers as adjunctive therapy for hip periprosthetic infection after arthroplasty: A clinical assessment.

OBJECTIVE: To explore the effect of three-dimensional (3D) printing to create personalized antibiotic-loaded bone cement (ALBC) spacers to assist in treatment of periprosthetic infection after total hip arthroplasty (THA). METHODS: The data of 40 patients with postoperative infection after THA were analysed retrospectively. The patients were divided into two groups: the 3D-printing group (age 47-78 years, n = 20) and the conventional group (age 57-78 years, n = 20). In stage I surgery, 3D-printed silicone moulds were used to create ALBC spacers for the 3D-printing group, while traditional manual methods were used to create spacers for the conventional group. After the infection was controlled, both groups underwent conventional hip revision surgery (stage II surgery). All patients were evaluated using the Harris Hip Score (HHS) (primary outcome) for hip function. RESULTS: All 40 patients had follow-up data from 3 months after stage I surgery and 12 months after stage II surgery. The intergroup difference in HHS was 11.25 points [97.5% confidence interval (CI) 7.92-14.58; P < 0.01] at 3 months after stage I surgery, and 9.15 points (97.5% CI 4.82-13.48; P < 0.01) at 12 months after stage II surgery. The overall difference between the two groups was 9.55 points (97.5% CI 5.83-13.27; P < 0.01), which was significant (P < 0.05). CONCLUSION: During the follow-up period, the hip function of the 3D-printing group was superior to that of the conventional group following the treatment of infections after THA.

Burden of diabetes attributable to dietary cadmium exposure in adolescents and adults in China.

At present, the health risk assessment of cadmium exposure has become a major focus of environmental health research. However, there is still a lack of systematic research on the burden of diabetes (DM) attributable to dietary cadmium exposure in adolescents and adults in China. Using the top-down method, the blood cadmium level (B-Cd) of Chinese adolescents and adults from 2001 to 2023 was combined with the relative risk (RR) of cadmium-induced diabetes to calculate the population attribution score (PAF). Subsequently, PAF was used to assess the disease burden (DB) of diabetes caused by cadmium exposure, expressed in disability adjusted life years (DALYs), and attribution analysis was carried out for cadmium exposure from different sources. The average blood cadmium concentration in Chinese adolescents and adults was 1.54 ± 1.13 µg/L, and the burden of DM attributable to cadmium exposure was 56.52 (44.81, 70.33) × 105 DALYs. The contribution rate of dietary cadmium exposure was 59.78%, and the burden of DM attributable to dietary cadmium exposure was 337.86 (267.85, 420.42) × 108 DALYs. In addition, the highest blood cadmium concentrations were found in Henan, Shanxi, and Jiangxi provinces, while the highest burden of DM attributable to cadmium exposure was found in Jiangsu, Henan, and Guangdong provinces. Cadmium exposure is a risk factor for DM, and we need to take comprehensive action to reduce the burden of DM attributable to dietary cadmium from health, economic, and social perspectives.

Genome-wide identification, expression analysis, and potential roles under low-temperature stress of bHLH gene family in Prunus sibirica.

The basic helix-loop-helix (bHLH) family is one of the most well-known transcription factor families in plants, and it regulates growth, development, and abiotic stress responses. However, systematic analyses of the bHLH gene family in Prunus sibirica have not been reported to date. In this study, 104 PsbHLHs were identified and classified into 23 subfamilies that were unevenly distributed on eight chromosomes. Nineteen pairs of segmental replication genes and ten pairs of tandem replication genes were identified, and all duplicated gene pairs were under purifying selection. PsbHLHs of the same subfamily usually share similar motif compositions and exon-intron structures. PsbHLHs contain multiple stress-responsive elements. PsbHLHs exhibit functional diversity by interacting and coordinating with other members. Twenty PsbHLHs showed varying degrees of expression. Eleven genes up-regulated and nine genes down-regulated in -4°C. The majority of PsbHLHs were highly expressed in the roots and pistils. Transient transfection experiments demonstrated that transgenic plants with overexpressed PsbHLH42 have better cold tolerance. In conclusion, the results of this study have significant implications for future research on the involvement of bHLH genes in the development and stress responses of Prunus sibirica.

Surface-Modified Nano-Hydroxyapatite Uniformly Dispersed on High-Porous GelMA Scaffold Surfaces for Enhanced Osteochondral Regeneration.

Purpose: This study aims to investigate the impact of enhancing subchondral bone repair on the efficacy of articular cartilage restoration, thereby achieving improved osteochondral regeneration outcomes. Methods: In this study, we modified the surface of nano-hydroxyapatite (n-HAp) through alkylation reactions to prepare n-HApMA. Characterization techniques, including X-ray diffraction, infrared spectroscopy scanning, thermogravimetric analysis, particle size analysis, and electron microscopy, were employed to analyze n-HApMA. Bioinks were prepared using n-HApMA, high porosity GelMA hydrogel, and adipose tissue derived stromal cells (ADSCs). The rheological properties of the bioinks during photocuring were investigated using a rheometer. Based on these bioinks, a biphasic scaffold was constructed. The viability of cells within the scaffold was observed using live-dead cell staining, while the internal morphology was examined using scanning electron microscopy. The stiffness of the scaffold was evaluated through compression testing. Scaffolds were implanted into the osteochondral defects of New Zealand rabbit knees, and microCT was utilized to observe the subchondral bone repair. Hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and Safranin O/Fast Green staining were performed to assess the regeneration of subchondral bone and cartilage. Furthermore, immunohistochemical staining was employed to detect the expression of osteogenic and chondrogenic-related molecules. Results: Scaffold characterization revealed that surface modification enables the uniform distribution of n-HApMA within the GelMA matrix. The incorporation of 5% n-HApMA notably enhanced the elastic modulus and stiffness of the 6% high-porosity GelMA in comparison to n-HAp. Moreover, in-vivo study showed that the homogeneous dispersion of n-HApMA on the GelMA matrix facilitated the osteogenic differentiation of adipose-derived stem cells (ADSCs) and promoted osteochondral tissue regeneration. Conclusion: These findings suggest potential applications of the n-HApMA/GelMA composite in the field of tissue engineering and regenerative medicine.

HDAC inhibitor quisinostat prevents estrogen deficiency-induced bone loss by suppressing bone resorption and promoting bone formation in mice.

Postmenopausal osteoporosis (PMOP) is a metabolic skeletal disorder characterized by reduced bone mass and impaired bone microarchitecture resulting in increased bone fragility and fracture risk. PMOP is primarily caused by excessive osteoclastogenesis induced by estrogen deficiency. Quisinostat (Qst) is a potent hydroxamate-based second-generation inhibitor of histone deacetylases (HDACs) that can inhibit osteoclast differentiation in vitro, and protect mice from titanium particle-induced osteolysis in vivo. However, whether Qst has therapeutic potential against PMOP remains unclear. In the present study, we evaluated the therapeutic efficacy of Qst on PMOP, using a murine model of ovariectomy (OVX)-induced osteoporosis. We examined the body weight, femur length, and histology of major organs, and showed that Qst did not cause obvious toxicity in mice. Micro-computed tomography and histological analyses revealed that Qst treatment prevented OVX-induced trabecular bone loss both in femurs and vertebrae. Moreover, ELISA showed that Qst decreased the serum levels of the osteoclastic bone resorption marker CTX-1, whereas increased the levels of the osteoblastic bone formation marker Osteocalcin in OVX mice. Consistent with the CTX-1 results, TRAP staining showed that Qst suppressed OVX-induced osteoclastogenesis. Mechanistically, we showed that Qst suppressed RANKL-induced osteoclast differentiation in part by inhibiting p65 nuclear translocation. Collectively, our results demonstrated that Qst can ameliorate estrogen deficiency-induced osteoporosis by inhibiting bone resorption and promoting bone formation in vivo. In summary, our study provided the first preclinical evidence to support Qst as a potential therapeutic agent for PMOP prevention and treatment.

Genetic diversity of Prunus armeniaca L. var. ansu Maxim. germplasm revealed by simple sequence repeat (SSR) markers.

The genetic diversity and genetic structure of P. armeniaca var. ansu were analyzed based on SSR markers. The aim was to provide scientific basis for conservation, efficient utilization, molecular marker assisted breeding and improved variety selection of P. armeniaca var. ansu germplasm resources. The results showed that the level of genetic diversity within the population was high. Among the 30 SSR markers, the mean number of observed alleles was 11.433, the mean number of effective alleles was 4.433, the mean of Shannon information index was 1.670, and the mean of polymorphic information content was 0.670. Among the eight provenances, Tuanjie Township, Xinyuan County, Xinjiang had the highest genetic diversity. The observed alleles, effective alleles, Shannon information index and Nei's gene diversity index among provenances were higher than those within provenances. Based on Bayesian mathematical modeling and UPGMA cluster analysis, 86 P. armeniaca var. ansu accessions were divided into three subpopulations and four groups, which reflected individual differences in provenances. Subpopulations classified by Bayesian mathematical modeling and groups classified by UPGMA cluster analysis were significantly correlated with geographical provenance (Sig<0.01) and the provenances significantly impacted classification of groups. The provenances played an important role in classification of groups. The genetic distance between Tuanjie Township of Xinyuan County and Alemale Township of Xinyuan County was the smallest, while the genetic relationship between them was the closest and the degree of genetic differentiation was small.

Triptolide ameliorates osteoarthritis by regulating nuclear factor kappa B-mediated inflammatory response.

OBJECTIVES: Osteoarthritis (OA) is a joint degenerative disease that commonly occurs in older people and affect the quality of life. Triptolide (TPL), a compound derived from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory properties. Here, we investigated the therapeutic effect of TPL on the experimental OA as well as the underlying molecular mechanisms. METHODS: OA models were established using monosodium iodoacetate (MIA) or surgery. The arthritis score and paw withdrawal threshold value of knees were used to evaluate the degree of arthritis. The level and expression of proinflammatory cytokines were evaluated by quantitative real-time PCR and ELISA kits. KEY FINDINGS: In surgery and MIA-induced OA rats, TPL alleviated arthritis symptoms and reduced inflammatory cytokine production in serum. In primary chondrocytes, TPL dose-dependently reversed lipopolysaccharide (LPS)-induced cell proliferation. Moreover, LPS-induced cell apoptosis and the expressions of proinflammatory cytokines interleukin-(IL-)6, IL-8, IL-1ß, IL-12, tumour necrosis factor-α (TNF-α) and interferon-gamma (INF-γ) were also attenuated by TPL. Mechanistically, the therapeutic effects of TPL on OA were effective by dampening nuclear factor kappa B (NF-κB) activity leading to reduced proinflammatory cytokines production and inflammatory response. CONCLUSIONS: TPL acts as an effective therapeutic drug for OA by mediating NF-κB signalling, thereby leading to the reduced proinflammatory cytokines production and inflammatory response.

Low-Intensity Pulsed Ultrasound Alleviates Osteoarthritis Condition Through Focal Adhesion Kinase-Mediated Chondrocyte Proliferation and Differentiation.

OBJECTIVE: Osteoarthritis (OA) is a prevalent chronic multifactorial degenerative disease characterized by joint tissue inflammation, osteophyte formation, subchondral bone sclerosis, and articular cartilage degradation. Low-intensity pulsed ultrasound (LIPUS), a noninvasive ultrasound technique, is widely used to attenuate diseases. The aim of this study was to investigate whether LIPUS can ameliorate OA, and to explore its underlying molecular mechanism. DESIGN: The OA model was established in a C57BL/6 mouse by the anterior cruciate ligament transaction method. OA was assessed using arthritis scoring and weightbearing parameters. Chondrocyte proliferation was detected by a CCK-8 assay. The levels of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α) in synovial fluid of the mice were measured by enzyme-linked immunosorbent assay. RESULTS: In OA mice, the arthritis score and weightbearing abilities were dramatically improved by LIPUS treatment. LIPUS also remarkably declined the levels of inflammatory cytokines IL-6, IL-8, and TNF-α in synovial fluid of OA mice. Moreover, LIPUS promoted chondrocyte proliferation and differentiation by activating focal adhesion kinase (FAK) signaling. Inhibition of FAK significantly blocked LIPUS-mediated cell proliferation and differentiation in vitro, as well as inflammation condition in OA mice. CONCLUSION: LIPUS alleviates OA through promoting chondrocytes proliferation and differentiation by activating FAK, which could act as an intervening target for OA treatment.

A modified tape transfer approach for rapidly preparing high-quality cryosections of undecalcified adult rodent bones.

BACKGROUND/OBJECTIVE: Histology-based analyses are important tools to dissect cellular and molecular mechanisms of skeletal homeostasis, diseases, and regeneration. The success of these efforts is highly dependent on rapidly obtaining high-quality sections of mineralized skeletal tissues suitable for various analyses. However, the current techniques for preparing such sections are still far from satisfactory. This study aimed to develop a new approach for preparing high-quality undecalcified bone sections applicable to various histological analyses. METHODS: Two important modifications were made to the conventional Cryojane Tape-Transfer System, including utilization of an optimized adhesive to prepare adhesive glass slides for improving the transfer efficiency, and a cheap conventional benchtop UV transilluminator for UV curing. Cryosections of undecalcified rodent bones were prepared using this modified tape transfer approach, and their tissue morphology and structural integrity were visually examined. A variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as 5-Ethynyl-2'-deoxyuridine (EdU) and TUNEL assays, were performed on these sections. RESULTS: We developed a modified version of tape transfer approach that can prepare cryosections of undecalcified rodent adult bones within 4 days at a low cost. Bone sections prepared by this approach exhibited good tissue morphology and structural integrity. Moreover, these sections were applicable to a variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as EdU and TUNEL assays. CONCLUSION: The tape transfer approach we developed provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections valuable for bone research. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our research provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections that can be potentially used for accurate diagnosis of various bone disorders and evaluation of the efficacy of different therapies in the treatment of these diseases.

USP7 Inhibition Alleviates H2O2-Induced Injury in Chondrocytes via Inhibiting NOX4/NLRP3 Pathway.

Osteoarthritis (OA), the most common form of arthritis, is a very common joint disease that often affects middle-aged to elderly people. However, current treatment options for OA are predominantly palliative. Thus, understanding its pathological process and exploring its potential therapeutic approaches are of great importance. Rat chondrocytes were isolated and exposed to hydrogen peroxide (H2O2) to mimic OA. The effects of H2O2 on ubiquitin-specific protease 7 (USP7) expression, reactive oxygen species (ROS) levels, proliferation, inflammatory cytokine release, and pyroptosis were measured. USP7 was knocked down (KD) or overexpressed to investigate the role of USP7 in OA. Co-immunoprecipitation (Co-IP) was used to study the interaction between USP7 and NAD(P)H oxidases (NOX)4 as well as NOX4 ubiquitination. NOX4 inhibitor was applied to study the involvement of NOX4 in USP7-mediated OA development. USP7 inhibitor was given to OA animals to further investigate the role of USP7 in OA in vivo. Moreover, H2O2 treatment significantly increased USP7 expression, enhanced ROS levels, and inhibited proliferation in rat chondrocytes. The overexpression of USP7 enhanced pyroptosis, ROS production, interleukin (IL)-1ß and IL-18 levels, and the expression level of NLRP3, GSDMD-N, active caspase-1, pro-caspase-1, matrix metalloproteinases (MMP) 1, and MMP13, which was abolished by ROS inhibition. The USP7 KD protected rat chondrocytes against H2O2-induced injury. Co-IP results showed that USP7 interacted with NOX4, and USP7 KD enhanced NOX4 ubiquitinylation. The inhibition of NOX4 blocked the pro-OA effect of USP7. Moreover, the USP7 inhibitor given to OA animals suppressed OA in vivo. USP7 inhibited NOX4 ubiquitination for degradation which leads to elevated ROS production. ROS subsequently activates NLPR3 inflammasome, leading to enhanced production of IL-1ß and IL-18, GSDMD-N-dependent pyroptosis, and extracellular matrix remodeling. Thus, UPS7 contributes to the progression of OA via NOX4/ROS/NLPR3 axis.

Retracted Article: Macrophage-derived exosomes mediate osteosarcoma cell behavior by activating AKT signaling.

Osteosarcoma is the most common type of bone tumor, which severely threatens the health of adolescents and young adults. Tumor-infiltrating macrophages have been shown to mediate cancer progression via extracellular vesicles. However, their potential mechanisms in osteosarcoma progression and in drug-resistance are still not yet known. The macrophage cell line THP1 was stimulated by phorbol myristate acetate (PMA) to secrete exosomes. The exosomes isolated from THP1 were characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and by a western blot. Cell proliferation was determined using CCK-8. A transwell assay and flow cytometry were conducted to detect cell migration and apoptosis, respectively. The expression levels of AKT and its phosphorylation status were determined using a western blot. PMA-treated activated THP1 cells secreted an abundance of exosomes with the characteristics of being less than 200 nm in diameter, and showing the robust expression of exosome markers CD63 and CD81. The THP1-derived exosomes promoted cell proliferation, migration and drug-resistance to the chemical drug docetaxel in both osteosarcoma cell lines MG63 and 143B. The inhibition of the generation of exosomes by the knockdown of ALIX clearly suppressed the cell proliferation, migration and drug-resistance. Mechanistically, the THP1-derived exosomes activated AKT signaling by inducing the increased expression of the phosphorylated AKT at serine 473 (p-AKT). The AKT inhibitor MK2206 significantly abolished exosome-mediated cell proliferation and drug-resistance in osteosarcoma cells. In summary, our data demonstrated that macrophage-derived exosomes promoted osteosarcoma progression and drug-resistance by activating AKT signaling that could be used as a potential molecular target for osteosarcoma treatment.

Genetic and pharmacological activation of Hedgehog signaling inhibits osteoclastogenesis and attenuates titanium particle-induced osteolysis partly through suppressing the JNK/c-Fos-NFATc1 cascade.

Rationale: Wear particle-induced periprosthetic osteolysis (PPO) is a common long-term complication of total joint arthroplasty, and represents the major cause of aseptic loosening and subsequent implant failure. Previous studies have identified the central role of osteoclast-mediated bone resorption in the pathogenesis of PPO. Thus, therapeutic approaches of inhibiting osteoclast formation and activity are considered to be of great potential to prevent and treat this osteolytic disease. Hedgehog (Hh) signaling has been shown to play an important role in promoting osteoblast differentiation and bone formation. While Hh signaling is also implicated in regulating osteoclastogenesis, whether it can directly inhibit osteoclast differentiation and bone resorption remains controversial. Moreover, its potential therapeutic effects on PPO have never been assessed. In this study, we explored the cell-autonomous role of Hh signaling in regulating osteoclastogenesis and its therapeutic potential in preventing wear particle-induced osteolysis. Methods: Hh signaling was activated in macrophages by genetically ablating Sufu in these cells using LysM-Cre or by treating them with purmorphamine (PM), a pharmacological activator of Smoothened (Smo). In vitro cell-autonomous effects of Hh pathway activation on RANKL-induced osteoclast differentiation and activity were evaluated by TRAP staining, phalloidin staining, qPCR analyses, and bone resorption assays. In vivo evaluation of its therapeutic efficacy against PPO was performed in a murine calvarial model of titanium particle-induced osteolysis by µCT and histological analyses. Mechanistic details were explored in RANKL-treated macrophages through Western blot analyses. Results: We found that Sufu deletion or PM treatment potently activated Hh signaling in macrophages, and strongly inhibited RANKL-induced TRAP+ osteoclast production, F-actin ring formation, osteoclast-specific gene expression, and osteoclast activity in vitro. Furthermore, we found that Sufu deletion or PM administration significantly attenuated titanium particle-induced osteoclast formation and bone loss in vivo. Our mechanistic study revealed that activation of Hh signaling suppressed RANKL-induced activation of JNK pathway and downregulated protein levels of two key osteoclastic transcriptional factors, c-Fos and its downstream target NFATc1. Conclusions: Both genetic and pharmacological activation of Hh signaling can cell-autonomously inhibit RANKL-induced osteoclast differentiation and activity in vitro and protect against titanium particle-induced osteolysis in vivo. Mechanistically, Hh signaling hinders osteoclastogenesis partly through suppressing the JNK/c-Fos-NFATc1 cascade. Thus, Hh signaling may serve as a promising therapeutic target for the prevention and treatment of PPO and other osteolytic diseases.

Comparison of Proximal Femoral Nail Antirotation and Dynamic Hip Screw Internal Fixation on Serum Markers in Elderly Patients with Intertrochanteric Fractures.

OBJECTIVE: To compare effect of proximal femoral nail antirotation (PFNA) and dynamic hip screw (DHS) internal fixation on serum inflammatory mediators (CRP, IL-1, IL-6 and TNF-α), myocardial injury markers (cTnT, CK-MB), and Myo-heart failure marker (BNP) in elderly patients with intertrochanteric fractures. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Orthopedics, The Second Affiliated Hospital of Xuzhou Medical University, China, from January 2016 to February 2018. METHODOLOGY: A total of 114 patients with intertrochanteric fractures were randomly divided into Group A and Group B, with 57 cases in each group. Group A was treated with PFNA and Group B with DHS internal fixation. Serum CRP, IL-1, IL-6, TNF-α, cTnT, CK-MB, Myo, BNP and surgical indication were compared. RESULTS: Operation time and weight-bearing time in Group A were shorter than Group B (both p<0.001). Intraoperative blood loss and postoperative drainage volume in Group A were lower than Group B (both p<0.001). On the 7ᵗʰ day after surgery, serum CRP, IL-1, IL-6, TNF-α, cTnT, CK-MB, Myo and BNP in Group A were lower than Group B (all p<0.001). CONCLUSION: Compared with DHS, PFNA effectively reduced serum inflammatory mediators with less damage to cardiac function and myocardium in elderly patients with intertrochanteric fractures.

Low dose of indomethacin and Hedgehog signaling inhibitor administration synergistically attenuates cartilage damage in osteoarthritis by controlling chondrocytes pyroptosis.

Blockade of Hedgehog signaling can prevent osteoarthritis (OA) syndromes. However, the amelioration of related inflammation condition is limited. The purpose of this study was to observe the effect of combined use of Hedgehog signaling inhibitor GANT-61 and common clinical anti-inflammatory drug indomethacin on cartilage injury and inflammation in experimental OA mice. We found that GANT-61 and indomethacin synergistically attenuate cartilage damage and serum levels of inflammatory cytokines TNF-α, IL-2 and IL-6 in OA mice. Moreover, in vitro treatment of GANT-61 and indomethacin synergistically reduced the mRNA expression of TNF-α, IL-2 and IL-6 in lipopolysaccharide (LPS)-stimulated C28/I2 chondrocytes. Mechasnistic studies showed that GANT-61 and indomethacin synergistically attenuate the expressions of cell pyroptosis-related genes caspase-1, IL-1ß and IL-18 at mRNA and protein level. To conclude, our study showed that GANT-61 and indomethacin had a synergistically ameliorating effect on osteoarthritis by mediating chondrocytes pyroptosis.