lncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived from Adipose-Derived Stem Cells for the Treatment of Osteoporosis

Background. Osteoporosis is a worldwide medical and socioeconomic burden characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study was aimed at investigating the role of ADSCs-Exos and a novel long noncoding RNA KCNQ1OT1 played in osteoporosis as well as the underlying mechanism. Methods. Primary osteoblasts were treated with different doses of tumor necrosis factor-α (TNF-α) (0, 1, 2.5, 5, and 10 ng/ml) and then cocultured with ADSCs-Exos or exosome-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3, and Bax was determined by Western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results. TNF-α dose-dependently increased miR-141-5p expression, inhibited viability, and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or cocultured with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of KCNQ1OT1 by luciferase reporter assay. Conclusions. ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment.

Angiogenesis by BMP-2-PDLLA-Biohybrids in Co-Culture with Osteoblasts and Endothelia

Adsorbate biohybrids for BMP-2 delivery based on electrospun PDLLA-nanofiber fleeces loaded with 0.6 μg/cm2 rhBMP-2COL (E. coli) strongly induced microvessel- like structures when incubated for 14 days in cocultures of human outgrowth endothelial cells (OEC) together with human primary osteoblasts (pOB). Higher loaded rhBMP-2COL biohybrids (4.6 μg/cm2) were low- to noninductive. Adsorbate biohybrids of rhBMP-2CHO (CHO cells) loaded with 1.0-6.0 μg/cm2 induced only a classic cobblestone- like growth pattern without any microvessels. Concentration- response experiments (7 days) indicate a binding affinity of rhBMP-2COL for microvessel induction in the picomolar range with nanomolar concentrations being non-inductive

Lactoferrin Promotes the Autophagy Activity During Osteoblast Formation via BCL2-Beclin1 Signaling

Lactoferrin, as the main component of milk, can maintain osteoblast formation, which is conducive to the prevention and treatment of osteoporosis. Lactoferrin also serves as an autophagy regulator, especially in osteoblasts. This study aimed to explore the significance of autophagy in osteoblast formation regulated by Lactoferrin and the internal mechanism. Our results showed that Lactoferrin enhanced the autophagy activity of primary osteoblasts. Importantly, Lactoferrin inhibited BCL2 expression and the co-immunoprecipitation of BCL2 and Beclin1 in osteoblasts. Moreover, Lactoferrin-promoted autophagy was reversed by BCL2 overexpression or Beclin1 inhibition with spautin-1 in osteoblasts. In conclusion, Lactoferrin can inhibit BCL2 expression in osteoblasts, further enhancing Beclin1-dependent autophagy activation.

LncRNA-KCNQ1OT1: a potential target in exosomes derived from adipose-derived stem cells for the treatment of osteoporosis

Background. Osteoporosis is a worldwide medical and socioeconomic threat characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study aimed to investigate the role of ADSCs-Exos and a noval long none coding RNA KCNQ1OT1 (lnc-KCNQ1OT1) played in osteoporosis as well as the mechanism. Methods. Primary osteoblasts were treated with different doses of TNF-α (0, 1, 2.5, 5, 10 ng/ml) and then co-cultured with ADSCs-Exos or exosomes-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3 and Bax was determined by Western blot. Cell viability and apoptosis were assessed by cell counting kit 8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results. Tumor necrosis factor-α (TNF-α) dose dependently increased miR-141-5p expression, inhibited viability and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or co-culture with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of lnc-KCNQ1OT1 by luciferase reporter assay. Conclusions. ADSCs-Exos attenuated cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos had a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos could be promising agents in osteoporosis treatment.

Counter-Acting Candida albicans-Staphylococcus aureus Mixed Biofilm on Titanium Implants Using Microbial Biosurfactants

This study aimed to grow a fungal-bacterial mixed biofilm on medical-grade titanium and assess the ability of the biosurfactant R89 (R89BS) coating to inhibit biofilm formation. Coated titanium discs (TDs) were obtained by physical absorption of R89BS. Candida albicans-Staphylococcus aureus biofilm on TDs was grown in Yeast Nitrogen Base, supplemented with dextrose and fetal bovine serum, renewing growth medium every 24 h and incubating at 37 °C under agitation. The anti-biofilm activity was evaluated by quantifying total biomass, microbial metabolic activity and microbial viability at 24, 48, and 72 h on coated and uncoated TDs. Scanning electron microscopy was used to evaluate biofilm architecture. R89BS cytotoxicity on human primary osteoblasts was assayed on solutions at concentrations from 0 to 200 μg/mL and using eluates from coated TDs. Mixed biofilm was significantly inhibited by R89BS coating, with similar effects on biofilm biomass, cell metabolic activity and cell viability. A biofilm inhibition >90% was observed at 24 h. A lower but significant inhibition was still present at 48 h of incubation. Viability tests on primary osteoblasts showed no cytotoxicity of coated TDs. R89BS coating was effective in reducing C. albicans-S. aureus mixed biofilm on titanium surfaces and is a promising strategy to prevent dental implants microbial colonization.

Transforming growth factor-β1-induced N-cadherin drives cell–cell communication through connexin43 in osteoblast lineage

Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

Luteolin stimulates proliferation and inhibits late differentiation of primary rat calvarial osteoblast induced by high-dose Dexamethasone via Sema3A /NRP1/Pleixin A1

Background: Although Semaphorin 3A (Sema3A)/ Neuropilin-1(NRP1)/Plexin A1 is one of the important targets in bone metabolism, few studies are done on this target in the glucocorticoids-induced osteoporosis. Luteolin is a chemical component of Honeysuckle and it has various bioactivities. The effect and study of Luteolin on the glucocorticoids-induced osteoporosis (primary osteoblasts model) remain unknown. Objective: The aim of this study was to investigate the action of Sema3A/ NRP1/Plexin A1 in Luteolin-induced osteoprotection against high-dose Dexamethasone. Method: The effect of luteolin on the proliferation, late differentiation, and apoptosis of primary osteoblasts model of glucocorticoids-induced osteoporosis in vitro as well as the expression of Sema3A/NRP1/Plexin A1 are investigated by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Cell Counting Kit-8, reverse transcriptionquantitative real-time polymerase chain reaction, western bolting and so on. Results: Suckling SD rats’ calvarial osteoblasts were isolated and identified. The glucocorticoids-induced primary osteoporosis cell model was established by 100 µM Dexamethasone in 48 h. (P<0.01) Luteolin promotes osteoblasts either in physiological condition (without Dexamethasone) or pathological condition (with Dexamethasone) at 1 µM concentration for 48h. (P<0.01). Luteolin partly reverses down-regulated expression of proliferation markers such as proliferating cell nuclear and CyclinD1. (P<0.01) Similarly, Luteolin partly reverses up-regulated expression of apoptosis markers such as Bax/B-cell lymphoma-2. (P<0.01) The expression of mRNA and protein of Sema3A/NRP1/Plexin A1 decreased in model one which significantly increased in Luteolin protecting one. (P<0.05) Interestingly, the late differentiation marker such as osteocalcin and collagenase sharply decreased in Luteolin protecting group compared with model one. (P<0.01) Conclusion: The thesis concludes that Luteolin promoted the proliferation of osteoblast and inhibited its apoptosis and late differentiation in this glucocorticoids-induced primary osteoporosis cell model. This function may be related to the expression of up-regulated Sema3A/NRP1/Plexin A1. Therefore, Luteolin may be a potential medicine for the glucocorticoids-induced osteoporosis