Exosomes from M2c macrophages alleviate intervertebral disc degeneration by promoting synthesis of the extracellular matrix via MiR-124/CILP/TGF-ß.

Immuno-inflammation is highly associated with anabolic and catabolic dysregulation of the extracellular matrix (ECM) in the nucleus pulposus (NP), which dramatically propels intervertebral disc degeneration (IVDD). With the characteristics of tissue remodeling and regeneration, M2c macrophages have attracted great attention in research on immune modulation that rebuilds degenerated tissues. Therefore, we first demonstrated the facilitating effects of M2c macrophages on ECM anabolism of the NP in vitro. We subsequently found that exosomes from M2c macrophages (M2c-Exoss) mediated their metabolic rebalancing effects on the ECM. To determine whether M2c-Exoss served as positive agents protecting the ECM in IVDD, we constructed an M2c-Exos-loaded hyaluronic acid hydrogel (M2c-Exos@HA hydrogel) and implanted it into the degenerated caudal disc of rats. The results of MRI and histological staining indicated that the M2c-Exos@HA hydrogel alleviated IVDD in vivo in the long term. To elucidate the underlying molecular mechanism, we performed 4D label-free proteomics to screen dysregulated proteins in NPs treated with M2c-Exoss. Cartilage intermediate layer protein (CILP) was the key protein responsible for the rebalancing effects of M2c-Exoss on ECM metabolism in the NP. With prediction and verification using luciferase assays and rescue experiments, miR-124-3p was identified as the upstream regulator in M2c-Exoss that regulated CILP and consequently enhanced the activity of the TGF-ß/smad3 pathway. In conclusion, we demonstrated ameliorating effects of M2c-Exoss on the imbalance of ECM metabolism in IVDD via the miR-124/CILP/TGF-ß regulatory axis, which provides a promising theoretical basis for the application of M2c macrophages and their exosomes in the treatment of IVDD.

Black-Phosphorus-Nanosheet-Reinforced Coating of Implants for Sequential Biofilm Ablation and Bone Fracture Healing Acceleration.

Incurable implant-related infection may cause catastrophic consequences due to the existence of a biofilm that resists the infiltration of host immune cells and antibiotics. Innovative approaches inspired by nanomedicine, e.g., engineering innovative multifunctional bionic coating systems on the surface of implants, are becoming increasingly attractive. Herein, 2D black phosphorus nanosheets (BPs) were loaded onto a hydroxyapatite (HA)-coated metal implant to construct a BPs@HA composite coating. With its photothermal conversion effect and in situ biomineralization, the BPs@HA coating shows excellent performances in ablating the bacterial biofilm and accelerating fracture healing, which were verified through both in vitro and in vivo studies. Moreover, differentially expressed genes of bone formation and bone mesenchymal stem cells (BMSCs) regulated by the BPs@HA coating were identified using absolute quantitative transcriptome sequencing followed by the screening of gene differential expressions. A functional enrichment analysis reveals that the expression of core markers related to BMSC differentiation and bone formation could be effectively regulated by BPs through a metabolism-related pathway. This work not only illustrates the great potential in clinical application of the BPs@HA composite coating to eliminate bacteria and accelerate bone fracture healing but also contributes to an understanding of the underlying molecular mechanism of osteogenesis physiological function regulation based on an analysis of absolute quantitative transcriptome sequencing.

The Role of Conformational Dynamics and Allostery in the Control of Distinct Efficacies of Agonists to the Glucocorticoid Receptor.

Glucocorticoid receptor (GR) regulates various cellular functions. Given its broad influence on metabolic activities, it has been the target of drug discovery for decades. However, how drugs induce conformational changes in GR has remained elusive. Herein, we used five GR agonists (dex, AZ938, pred, cor, and dibC) with different efficacies to investigate which aspect of the ligand induced the differences in efficacy. We performed molecular dynamics simulations on the five systems (dex-, AZ938-, pred-, cor-, and dibC-bound systems) and observed a distinct discrepancy in the conformation of the cofactor TIF2. Moreover, we discovered ligand-induced differences regarding the level of conformational changes posed by the binding of cofactor TIF2 and identified a pair of essential residues D590 and T39. We further found a positive correlation between the efficacies of ligands and the interaction of the two binding pockets' domains, where D590 and T739 were involved, implying their significance in the participation of allosteric communication. Using community network analysis, two essential communities containing D590 and T739 were identified with their connectivity correlating to the efficacy of ligands. The potential communication pathways between these two residues were revealed. These results revealed the underlying mechanism of allosteric communication between the ligand-binding and cofactor-binding pockets and identified a pair of important residues in the allosteric communication pathway, which can serve as a guide for future drug discovery.

Local delivery and controlled release of miR-34a loaded in hydroxyapatite/mesoporous organosilica nanoparticles composite-coated implant wire to accelerate bone fracture healing.

Immediate mechanical stability is a prerequisite for fracture healing. In addition to bringing immediate mechanical stability in fracture site, implants with bioactive coating can release active substance to accelerate bone-fracture healing. However, limited drug-loading capacity of established coatings weakens their biological functions, which urges the engineering of more effective coating biomaterials for accelerating fracture healing. Herein, mesoporous organosilica nanoparticles (MONs), as miR-34a delivers, are loaded onto hydroxyapatite (HA)-coated Kirschner wire to engineer a HA/MONs@miR-34a composite coating. The composite coating can effectively deliver miR-34a into osteoclasts, generate gene dose-dependent inhibiting effect on differentiation and resorptive activity of osteoclasts by regulating multiple downstream gene expression at the early stage of fracture healing, which additionally exhibits decent bone regeneration potentials as evidenced in rat tibial fracture model. In particular, differentially expressed genes regulated by miR-34a are identified using RNA-seq followed by bioinformatics analysis. Functional enrichment analysis reveals that genes with altered expression mainly distribute in mainly distribute in DNA replication and cell cycle, which are associated with the development of osteoclasts. This work not only demonstrates the high clinical translation potential of HA/MONs@miR-34a to accelerate fracture healing, but also reveals the underlying molecular mechanism of regulating physiological functions of osteoclasts based on analysis of singlecell RNA sequencing.

Optimization, characterization and evaluation of ZnO/polyvinylidene fluoride nanocomposites for orthopedic applications: improved antibacterial ability and promoted osteoblast growth.

Herein, electrospun zinc oxide nanoparticle/poly (vinylidene fluoride) (ZnONP/PVDF) composite fiber membranes were designed, fabricated, and tested for improved orthopedic applications. A single factor screening study was conducted to determine the optimal ZnONP/PVDF formulation based on osteoblast (bone forming cells) proliferation and antibacterial properties. Further, ZnONP/PVDF materials were characterized for their morphology, crystallinity, roughness, piezoelectric properties, and chemistry to understand such cell results. The optimal concentration of high molecular weight PVDF (18%, w/v) and a low concentration of ZnONPs (1 mg/ml) were identified for electrospinning at room temperature in order to inhibit bacterial colonization (without resorting to antibiotic use) and promote osteoblast proliferation. Compared to no ZnO/PVDF scaffold without Piezo-excited group,the study showed that on the 1 mg/ml ZnO/PVDF scaffolds with piezo-excitation, the density of SA and E.coli decreased by 68% and 56%.The density of osteoblasts doubled within three days(compared to the control). In summary, ZnONP/PVDF composite fiber membranes were formulated by electrospinning showing an exceptional ability to eliminate bacteria colonization while at the same time promote osteoblast functions and, thus, they should be further studied for a wide range of orthopedic applications.

An easy long-acting BMP7 release system based on biopolymer nanoparticles for inducing osteogenic differentiation of adipose mesenchymal stem cells.

In contrast to the early acting bone morphogenetic protein 2, bone morphogenetic protein 7 (BMP7) plays a decisive role mainly in the late stages of bone formation. To overcome deactivation and degradation of expensive BMP7, we designed a novel long-acting BMP7 release system based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) nanoparticles to enable the induction of osteogenic differentiation in human adipose mesenchymal stem cells (ADSCs). In order to improve the encapsulation efficiency of BMP7 and avoid damage by organic solvents, BMP7 was modified and protected using the biosurfactant soybean lecithin. In an in vitro test, BMP7-soybean lecithin-P34HB nanoparticles (BMP7-SPNPs) showed a short initial burst of BMP7 release during the first 24h, followed by a steady increase to a cumulative 80% release in 20days. Compared with the rapid release of control P34HB nanoparticles without soybean phospholipids loaded with BMP7 without soybean lecithin, BMP7-SPNPs significantly reduced the initial burst of BMP7 release and stabilized the content of BMP7 to allow long-term osteogenic differentiation during the late phase of bone development. Human ADSCs treated with BMP7-SPNPs showed higher alkaline phosphatase activity and higher expression levels of genetic markers of osteogenic differentiation compared with the control group. Thus, the results indicate that BMP7-SPNPs can be used as a rapid and long-acting BMP7 delivery system for osteogenic differentiation.

Dual targeting curcumin loaded alendronate-hyaluronan- octadecanoic acid micelles for improving osteosarcoma therapy.

INTRODUCTION: Curcumin (CUR) is a general ingredient of traditional Chinese medicine, which has potential antitumor effects. However, its use clinically has been limited due to its low aqueous solubility and bioavailability. In order to improve the therapeutic effect of CUR on osteosarcoma (i.e., bone cancer), a multifunctional micelle was developed here by combining active bone accumulating ability with tumor CD44 targeting capacity. METHODS: The CUR loaded micelles were self-assembled by using alendronate-hyaluronic acid-octadecanoic acid (ALN-HA-C18) as an amphiphilic material. The obtained micelles were characterized for size and drug loading. In addition, the in vitro release behavior of CUR was investigated under PBS (pH 5.7) medium containing 1% Tween 80 at 37℃. Furthermore, an hydroxyapatite (the major inorganic component of bone) affinity experiment was studied. In vitro antitumor activity was evaluated. Finally, the anti-tumor efficiency was studied. RESULTS: The size and drug loading of the CUR loaded ALN-HA-C18 micelles were about 118 ± 3.6 nm and 6 ± 1.2%, respectively. CUR was released from the ALN-HA-C18 micelles in a sustained manner after 12 h. The hydroxyapatite affinity experiment indicated that CUR loaded ALN-HA-C18 micelles exhibited a high affinity to bone. CUR loaded ALN-HA-C18 micelles exhibited much higher cytotoxic activity against MG-63 cells compared to free CUR. Finally, CUR loaded ALN-HA-C18 micelles effectively delayed anti-tumor growth properties in osteosarcoma bearing mice as compared with free CUR. CONCLUSION: The present study suggested that ALN-HA-C18 is a novel promising micelle for osteosarcoma targeting and delivery of the hydrophobic anticancer drug CUR.

Preliminary studies on the anti-osteoporosis activity of Baohuoside I.

The aim of the present study was to investigate the anti-osteoporotic activity of Baohuoside I, an active component of Herba Epimedii. Effects of Baohuoside I on the differentiation of BMSCs and the formation of adipocytes were evaluated using alkaline phosphatase staining and methylene blue staining method, respectively. Osteoporosis model was established in ovariectomized rats prior to the measurement of the serum SOD and MDA levels as well as the expression of inflammatory cytokines protein in the rats' tissues after treatment with Baohuoside I using ELISA assay kits. The estrogen-like effect of Baohuoside I was also measured on HeLa cells. The positive rates of ALP staining in Baohuoside I groups were significantly higher (p < 0.01) compared with the normal group, with no obvious adipocyte formation observed in the groups that received Baohuoside I treatments. The levels of inflammatory markers (IL-1ß, TNF-α, IL-6 and IL-8) in the treated groups were significantly lower (p < 0.05) than in the model group. Likewise, the treated groups exhibited a significantly higher (p < 0.05) serum levels of MDA compared with the model group, while SOD levels were markedly lower (p < 0.05) in a dose-dependent fashion. Baohuoside I showed no estrogen-like effect on HeLa cells upon treatment with the drug. Collectively, these results indicated that the anti-osteoporotic activity of Baohuoside I could be related to the induction of BMSCs differentiation into osteoblasts coupled with the inhibition of adipocyte formation, regulation of immune functions, and antioxidant activity.

Effect of needle diameter, type and volume of contrast agent on intervertebral disc degeneration in rats with discography.

PURPOSE: Discography can increase disc degeneration, but the influence of different discography variables on the degeneration of discs has not been reported. The aim of this study was to investigate the effects of discography variables of needle diameter, type of contrast agent and volume of contrast agent on disc degeneration. METHODS: Three separate experiments examined needle diameter, and type and volume of contrast agent. Coccygeal discs (Co7-10) adult male rats were used. X-rays were used to detect the disc height degeneration index at 1, 2 and 4 weeks after the procedure. MRI was used to study the changes in the disc structure and the signal intensity of IVD 2 and 4 weeks after the procedure. Disc water content and histology were measured at 4 weeks after the procedure. RESULTS: A 21-g needle significantly increased disc degeneration when compared with the 30-g needle as detected by X-ray, MRI, disc water content and histology (P < 0.05). Two microlitres of iodine significantly decreased the disc MRI signal and water content at 4 weeks compared with the same volume of normal saline (P < 0.05). Three microlitres of iodine significantly increased disc degeneration when compared with 2 µl iodine, as detected by X-ray, MRI, disc water content and histology at 4 weeks (P < 0.05). CONCLUSION: To reduce disc degeneration after discography, it may be best to choose a smaller needle size, minimize the use of contrast agent and use non-ionic contrast agents with osmotic pressure similar to the intervertebral disc. These slides can be retrieved under Electronic Supplementary Material.

Early-stage autophagy protects nucleus pulposus cells from glucose deprivation-induced degeneration via the p-eIF2α/ATF4 pathway.

Autophagy is a double-edged sword in cellular survival, but its effects on nucleus pulposus (NP) cells are yet to be clarified. This study explored the role and molecular mechanisms of autophagy in the survival of NP cells under nutrient deprivation. Glucose limitation induced time-dependent morphological changes, proteoglycan degradation, and apoptosis in NP cells. Glucose deprivation triggered the activation of early-stage autophagy, evident as increases in LC3-II and ATG12-ATG5 expression and the number of GFP-LC3 puncta. Importantly, at early time points, the autophagy inhibitor 3-MA significantly enhanced the apoptosis of NP cells, suggesting that early-stage autophagy protects cells against glucose deprivation. Interestingly, the p-eIF2α/ATF4 unfolded protein response pathway was activated in NP cells deprived of glucose, and a deficiency in ATF4 attenuated the activation of early-stage autophagy and increased apoptosis. ATF4 silencing also inhibited the late-stage accumulation of reactive oxygen species (ROS) and apoptosis. Together, our results demonstrate an interplay between endoplasmic reticulum stress, ROS production, and the early-stage autophagy induced by glucose deprivation in NP cells. These findings may provide a therapeutic strategy for the treatment of degenerative intervertebral disc disease.

[Osteogenetic capacity of cross-linked adjustable anti-tuberculosis drug sustained-release artificial composite].

OBJECTIVE: To explore the osteogenetic capacity of cross-linked adjustable anti-tuberculosis drug sustained-release artificial composite (TPB/SA-RFP/PLA). METHODS: The model of femur bone defect was established in rabbits.TPB/SA-RFP/PLA complex was implanted into defect parts in the experimental group while TPB/SA/PLA in the blank control group. At Weeks 4, 8 and 12, gross specimens received radiographic, histological and immunohistochemical examinations to determine the osteogenetic performance of TPB/SA-RFP/PLA. RESULTS: As compared with the control group, TPB/SA-RFP/PLA complex had excellent osteogenic capacities while the TPB/SA/PLA group had no obvious osteogenic difference. Lane-sandhu histological and radiographic ratings demonstrated significant difference between TPB/SA-RFP/PLA (8.3 ± 0.3) and blank groups (2.2 ± 0.4) (P < 0.05). And TPB/SA/PLA showed no significant intragroup significance (P > 0.05). Two groups immunohistochemical Alkaline phosphatase was strongly positive in two test groups and weakly positive in the control group. CONCLUSION: TPB/ SA-RFP/PLA has excellent profiles of bone conductivity and regeneration.And the incorporation of rifampin does not affect its osteogenetic capacity.