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Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet‐induced hyperhomocysteinemia (HHcy). However, little is known about Hcy‐mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H2S) has potent antioxidant, anti‐inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H2S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy‐treated osteoblast cultures in vitro. MC3T3‐E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H2S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H2S level and expression of the cystathionine β‐synthase/Cystathionine γ‐lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy‐induced mitochondrial toxicity (production of total ROS and mito‐ROS, ratio of mitochondrial fission (DRP‐1)/fusion (Mfn‐2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy‐induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy‐induced mitochondrial dysfunction in cultured osteoblasts in vitro. [ABSTRACT FROM AUTHOR]

Abstract Periosteum plays an indispensable role in bone repair and reconstruction. To recapitulate the remarkable regenerative capacity of periosteum, a biomimetic tissue-engineered periosteum (TEP) was constructed via layer-by-layer bottom-up strategy utilizing polycaprolactone (PCL), collagen, and nano-hydroxyapatite composite nanofiber sheets seeded with bone marrow stromal cells (BMSCs). When combined with a structural bone allograft to repair a 4 mm segmental bone defect created in the mouse femur, TEP restored donor-site periosteal bone formation, reversing the poor biomechanics of bone allograft healing at 6 weeks post-implantation. Further histologic analyses showed that TEP recapitulated the entire periosteal bone repair process, as evidenced by donor-dependent formation of bone and cartilage, induction of distinct CD31high type H endothelium, reconstitution of bone marrow and remodeling of bone allografts. Compared to nanofiber sheets without BMSC seeding, TEP eliminated the fibrotic tissue capsule elicited by nanofiber sheets, leading to a marked improvement of osseointegration at the compromised periosteal site. Taken together, our study demonstrated a novel layer-by-layer engineering platform for construction of a versatile biomimetic periosteum, enabling further assembly of a multi-component and multifunctional periosteum replacement for bone defect repair and reconstruction. [ABSTRACT FROM AUTHOR]

Prenylation of bioactive natural compounds has been postulated to be able to enhance the utilization rate and affinity of the compounds with cell membranes, thus promote their bioactivities. Coumestrol, isolated from Medicago sativa, has been known as a phytoestrogen which has bone health benefits. In our previous work, psoralidin, a prenylated coumestrol, was proved to have a higher ability than coumestrol to promote bone formation and to attenuate resorption in vitro . However, it remains to be investigated whether psoralidin will have stronger bone health benefits than coumestrol. In the current study, psoralidin was isolated from Psoralea corylifolia L. and the osteotropic activities of coumestrol and psoralidin were compared in ovariectomized (OVX) rats. Both coumestrol and psoralidin were found to suppress OVX-induced bone loss in vivo , as shown by improved total bone mineral content (t-BMC) or density (t-BMD) and mineral apposition rate, bone biomechanical properties, microstructure and trabecular bone formation, enhanced osteogenic differentiation but suppressed adipogenic differentiation of bone marrow stromal cells (BMSCs), and activation of PI3K/Akt axis and downstream factors such as GSK3β/β-catenin and Nrf-2/HO-1. However, psoralidin was shown to have higher activities than coumestrol in the above measurements/indices. Our findings demonstrate that psoralidin, as a novel anti-osteoporosis candidate, could suppress bone loss in OVX rats and have better osteoprotective effects than coumestrol, which may be related to the presence of the isopentenyl group in psoralidin. [ABSTRACT FROM AUTHOR]

Bone homeostasis requires a balance between the bone formation of osteoblasts and bone resorption of osteoclasts to maintain ideal bone mass and bone quality. An imbalance in bone remodeling processes results in bone metabolic disorders such as osteoporosis. Hydrogen sulfide (H 2 S), a gasotransmitter, has attracted the focus of many researchers due to its multiple physiological functions. It has been implicated in anti-inflammatory, vasodilatory, angiogenic, cytoprotective, anti-oxidative and anti-apoptotic mechanisms. H 2 S has also been shown to exert osteoprotective activity through its anti-inflammatory and anti-oxidative effects. However, the underlying molecular mechanisms by which H 2 S mitigates bone diseases are not completely understood. Experimental evidence suggests that H 2 S may regulate signaling pathways by directly influencing a gene in the cascade or interacting with some other gasotransmitter (carbon monoxide or nitric oxide) or both. MicroRNAs (miRNAs) are short non-coding RNAs which regulate gene expression by targeting, binding and suppressing mRNAs; thus controlling cell fate. Certainly, bone remodeling is also regulated by miRNAs expression and has been reported in many studies. MicroRNAs also regulate H 2 S biosynthesis. The inter-regulation of microRNAs and H 2 S opens a new possibility for exploring the H 2 S-microRNA crosstalk in bone diseases. However, the relationship between miRNAs, bone development, and H 2 S is still not well explained. This review focuses on miRNAs and their roles in regulating bone remodeling and possible mechanisms behind H 2 S mediated bone loss inhibition, H 2 S-miRNAs crosstalk in relation to the pathophysiology of bone remodeling, and future perspectives for miRNA-H 2 S as a therapeutic agent for bone diseases. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicines (TCM) have been proven to prevent osteoporosis, but their clinical applications are not widely recognized due to their complicated ingredients. Psoralidin, a prenylated coumestan, has been reported to prevent bone loss of ovariectomized rats, but detailed mechanisms are still not clear. In current study, we found that both psoralidin and coumestrol promoted osteoblast proliferation and differentiation, as evidenced by improvements in cell proliferation and alkaline phosphatase activity; increased formation of ALP colonies and calcified nodules; enhanced secretion of collagen-I, BMP-2, osteocalcin and osteopontin; and stimulation of the expression of IGF-1, β-catenin, Runx-2, Osterix, and OPG, as well as the mRNA ratio of OPG/RANKL, while significantly decreasing the expression of RANKL. In addition, both psoralidin and coumestrol inhibited osteoclast formation and osteoclastic bone resorption, as demonstrated by the lower tartrate-resistant acid phosphatase activity and smaller area, with fewer resorption pits formed. Interestingly, psoralidin showed much stronger effects than coumestrol at enhancing osteoblast proliferation/differentiation or inhibiting osteoclast differentiation and bone resorption. Moreover, we found that both psoralidin and coumestrol suppressed COX-2 and ROS production in rat osteoblastic calvarias cells, and psoralidin showed stronger effects than coumestrol. Furthermore, we detected that by blocking estrogen receptors with ICI 182.780 (an estrogen receptor antagonist), the osteoprotective effects of psoralidin and coumestrol were also blocked. Our findings demonstrated that psoralidin and coumestrol exert their bone-protective effects by enhancing bone formation of osteoblasts and inhibiting bone resorption of osteoclasts. These roles might be mediated by their antioxidant activity and transduced through estrogen receptor signaling. [ABSTRACT FROM AUTHOR]

Abstract: Nitric oxide (NO) is an important intracellular and intercellular messenger, critically affecting bone metabolism. The purpose of this research is to investigate whether the effect of sinusoidal electromagnetic field (SEMF) on the differentiation and maturation of osteoblasts is mediated by the NO–cGMP–PKG signal pathway. We examined the impact of SEMF on nitric oxide synthase (NOS) activity, and found that l-NAME, nitric oxide synthase’s inhibitor, prevents SEMF-mediated increase in NOS activity and NO levels. We showed that an inhibitor of soluble guanylyl cyclase (ODQ) blocks the increase in cGMP levels triggered by exposure to SEMF. The inhibitor PDE5, which hydrolyzes 3′,5′-cyclic-GMP to 5′-GMP, prevents the SEMF’s stimulation of PKG activity. We also blocked the NO–cGMP–PKG pathway to determine whether the maturation and mineralization of osteoblasts, stimulated by SEMF, would be inhibited. This was evaluated by measuring alkaline phosphatase (ALP) activity, osterix gene expression and mineralized bone modulus. After treatment with SEMF, the NOS activity increases in comparison with the control group (P< 0.01), reaching the highest level after 0.5h. Osterix gene expression, ALP activity and mineralized bone nodules in the SEMF experimental group also increase significantly. However, these effects are partially blocked in the l-NAME treated cultures. Surprisingly, all the osteogenic markers in the SEMF+ l-NAME group were slightly higher than in the control culture, but lower than in the cells exposed to SEMF only. We conclude that the NO–cGMP–PKG signal pathway is activated by SEMF treatment, the stimulatory effect of SEMF on the differentiation and mineralization of osteoblasts is attenuated when the pathway is blocked. [Copyright &y& Elsevier]

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN− vessels exhibiting higher flowrate and higher oxygen tension (pO 2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN− vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering. [Display omitted] [ABSTRACT FROM AUTHOR]

Circulating nitrate is actively absorbed by salivary glands and secreted into the oral cavity, where it is reduced to nitrite by oral nitrate-reducing bacteria. This process has previously been considered harmful because nitrate and nitrite can promote the formation of potentially carcinogenic N-nitrosamines. However, recent studies have shown that nitrate may have other physiological functions, and it can serve as a precursor for the systemic production of nitric oxide (NO) and perform NO-like functions, such as promoting vasodilation, regulating metabolic diseases, alleviating senescence, and protecting the digestive system. Inside the oral cavity, NO is likely to inhibit sensitive species as part of the nonspecific oral immune system. Exogenous administration of nitrate can maintain a balance in the pH of saliva. Oral nitrate-reducing bacteria can control the progression of caries by metabolizing lactic acid and reducing its accumulation, which is beneficial to the homeostasis of the oral microecology. In the current manuscript, we reviewed nitrate-reducing bacteria and their nitrate-metabolizing functions during the development of caries. Furthermore, we listed the effects of probiotics and dietary modification, which may be a promising method to prevent the occurrence of caries. We believe that this review provides novel ideas for the prevention of caries and treatment in clinical settings. [ABSTRACT FROM AUTHOR]

Autogenous bone transplantation is a prevalent clinical method for addressing bone defects. However, the limited availability of donor bone and the morbidity associated with bone harvesting have propelled the search for suitable bone substitutes. Bio-inspired scaffolds, particularly those fabricated using electron beam melting (EBM) deposition technology, have emerged as a significant advancement in this field. These 3D-printed titanium alloy scaffolds are celebrated for their outstanding biocompatibility and favorable elastic modulus. Thermosensitive chitosan hydrogel, which transitions from liquid to solid at body temperature, serves as a popular carrier in bone tissue engineering. Icariin (ICA), known for its efficacy in promoting osteoblast differentiation from bone marrow mesenchymal stem cells (BMSCs), plays a crucial role in this context. We developed a system combining a 3D-printed titanium alloy with a thermosensitive chitosan hydrogel, capable of local bone regeneration and integration through ICA delivery. Our in vitro findings reveal that this system can gradually release ICA, demonstrating excellent biocompatibility while fostering BMSC proliferation and osteogenic differentiation. Immunohistochemistry and Micro-CT analyses further confirm the effectiveness of the system in accelerating in vivo bone regeneration and enhancing osseointegration. This composite system lays a significant theoretical foundation for advancing local bone regeneration and integration. [Display omitted] [ABSTRACT FROM AUTHOR]

Induction of some specific cell signaling molecules in peptide scaffold play a critical role in developing of functional substitutes for damaged tissue. In this study, a new peptide ELK8 (Ac-LELELKLK-CONH2) was designed by replacing Ala in EAK16 (Ac-AEAEAKAKAEAEAKAK-CONH2) with more hydrophobic residue Leu in order to enhance the strength of peptide. The functionalized peptides were obtained through directly coupling three functional motifs to C-terminal of ELK8, such as osteogenic growth peptide, osteopontin cell adhesion motif, and 2-unit RGD binding sequence. Then, the new self-assembling peptide scaffolds were fabricated by mixing three functionalized peptides severally with pure ELK8 at the ratio of 1:1 (v:v) and the nanostructures of self-assembling peptides were investigated with AFM and DLS. The osteogenic efficacy of designed peptides on mouse pre-osteoblast MC3T3-E1 were examined after cells were incubated in scaffolds for 14 d. The results showed that these functionalized peptides, ALK mix particularly, promoted the attachment, proliferation, and osteogenic differentiation of MC3T3-E1 cells by increasing in cell numbers, ALP activities and osteocalcin concentrations. [ABSTRACT FROM AUTHOR]

Alcohol is one of the most socially accepted addictive drugs in modern society. Its abuse affects virtually all organ systems with the central nervous system (CNS) being particularly vulnerable to excessive alcohol exposure. Alcohol exposure also causes profound damage to both the adult and developing brain. Excessive alcohol consumption induces numerous pathophysiological stress responses, one of which is the endoplasmic reticulum (ER) stress response. Potential mechanisms that trigger the alcohol induced ER stress response are either directly or indirectly related to alcohol metabolism, which include toxic levels of acetaldehyde and homocysteine, oxidative stress and abnormal epigenetic modifications. Growing evidence suggests that H 2 S is the most recently recognized gasotransmitter with tremendous physiological protective functions against oxidative stress induced neurotoxicity. In this review we address the alcohol induced oxidative stress mediated ER stress and the role of H 2 S in its mitigation in the context of alcohol neurotoxicity. Interruption of ER stress triggers is anticipated to have therapeutic benefits for alcohol mediated diseases and disorders. [ABSTRACT FROM AUTHOR]

Context: Radix Dipsaci is a kidney tonifying herbal medicine with a long history of safe use for treatment of bone fractures and joint diseases in China. Previous studies have shown that Radix Dipsaci extract (RDE) could prevent bone loss in ovariectomized rats. Objective: This study investigates the effect of RDE against bone loss induced by simulated microgravity. Materials and methods: A hindlimb unloading rat model was established to determine the effect of RDE on bone mineral density and bone microarchitecture. Twenty-four male Sprague-Dawley rats were divided into four groups ( n = 6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLU-ALN, 2.0 mg/kg/d), and hindlimb unloading treated with RDE (HLU-RDE, 500 mg/kg/d). RDE or ALN was administrated orally for 4 weeks. Results: Treatment with RDE had a positive effect on mechanical strength, BMD, BMC, bone turnover markers, and the changes in urinary calcium and phosphorus excretion. MicroCT analysis showed that RDE significantly prevented the reduction of the bone volume fraction, connectivity density, trabecular number, thickness, tissue mineral density, and tissue mineral content as well as improved the trabecular separation and structure model index. Discussion and conclusion: RDE was demonstrated to prevent the loss of bone mass induced by HLU treatment, which suggests the potential application of RDE in the treatment of microgravity-induced bone loss. [ABSTRACT FROM AUTHOR]

Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508-536 nm and fluorescence peaks at 524-550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates. [ABSTRACT FROM AUTHOR]

Osteoporosis is a common disease with wide prevalence, especially in the elderly population. Osteoporosis induced fractures not only decrease the patient's life quality, but also cause heavy financial burden to the society. Although current medications for osteoporosis are effective, numerous adverse effects have been observed accompanying their clinical applications. Effective prevention and therapy strategies with high safety are critical, which benefit both individual patients and the whole society. Traditional Chinese medicines have been used for thousands of years to treat bone related diseases in China and a number of modern preparations have been developed that are currently commercially available. In addition, several medicinal herbs demonstrated therapeutic effects against osteoporosis in animal models. This paper reviewed the anti-osteoporotic effects of traditional Chinese formulas, medicinal herbs and bioactive constituents based on clinical trials and in vivo animal studies. Due to the lack of rigorous studies to compare the effectiveness with conventional interventions, traditional formulas are recommended as alternative medications or supplements to treat osteoporosis at the current stage. Although there are abundant natural resources with anti-osteoporotic effects, either in the form of medicinal herbs or bioactive components, much work need to be accomplished before they are developed into potential drugs. [ABSTRACT FROM AUTHOR]

Du-Zhong has a long history of being used in traditional Chinese formulas to treat bone related diseases. The objective of the present study is to systematically investigate the effects of Du-Zhong cortex extract (DZCE) on disuse-induced osteoporosis. Rats were randomly divided into four groups, and three groups were treated with hind limb suspension (HLS). Control and HLS group received deionized distilled water, while the other two groups received alendronate (2.0 mg/kg/day) and DZCE (300 mg/kg/day) respectively by intragastric gavage for six weeks (two weeks prior to and during the four weeks of HLS). Dual-energy X-ray absorptiometry, assay of biochemical markers, and three-point bending test were employed to determine the effect of various treatments on bone mass, turnover, and strength. The trabecular bone microarchitecture was assessed by microCT analysis. DZCE could effectively prevent the bone loss induced by HLS, which was indicated by decreased levels of bone turnover markers as well as the changes in urinary calcium and phosphorus. The DZCE treatment also enhanced the biomechanical strength of bone and prevented the deterioration of trabecular bone microarchitecture. DZCE administration was able to prevent disuse-induced osteoporosis by regulating the bone metabolism, suggesting that DZCE could be used as an alternative therapy for the prevention of disuse-induced osteoporosis. [ABSTRACT FROM AUTHOR]