Poly-2-methyl-2-oxazoline-modified bioprosthetic heart valve leaflets have enhanced biocompatibility and resist structural degeneration.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ in vitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)-modified BP in vitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Carbon dioxide addition to coral reef waters suppresses net community calcification.

Coral reefs feed millions of people worldwide, provide coastal protection and generate billions of dollars annually in tourism revenue. The underlying architecture of a reef is a biogenic carbonate structure that accretes over many years of active biomineralization by calcifying organisms, including corals and algae. Ocean acidification poses a chronic threat to coral reefs by reducing the saturation state of the aragonite mineral of which coral skeletons are primarily composed, and lowering the concentration of carbonate ions required to maintain the carbonate reef. Reduced calcification, coupled with increased bioerosion and dissolution, may drive reefs into a state of net loss this century. Our ability to predict changes in ecosystem function and associated services ultimately hinges on our understanding of community- and ecosystem-scale responses. Past research has primarily focused on the responses of individual species rather than evaluating more complex, community-level responses. Here we use an in situ carbon dioxide enrichment experiment to quantify the net calcification response of a coral reef flat to acidification. We present an estimate of community-scale calcification sensitivity to ocean acidification that is, to our knowledge, the first to be based on a controlled experiment in the natural environment. This estimate provides evidence that near-future reductions in the aragonite saturation state will compromise the ecosystem function of coral reefs.

Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts.

Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography-mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients.

Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum.

Arterial calcification is a common feature of pseudoxanthoma elasticum (PXE), a disease characterized by ABCC6 mutations, inducing a deficiency in pyrophosphate, a key inhibitor of calcium phosphate crystallization in arteries. METHODS: we analyzed whether long-term exposure of Abcc6-/- mice (a murine model of PXE) to a mild vitamin D supplementation, with or without calcium, would impact the development of vascular calcification. Eight groups of mice (including Abcc6-/- and wild-type) received vitamin D supplementation every 2 weeks, a calcium-enriched diet alone (calcium in drinking water), both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Aorta and kidney artery calcification was assessed by 3D-micro-computed tomography, Optical PhotoThermal IR (OPTIR) spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and Yasue staining. RESULTS: at 6 months, although vitamin D and/or calcium did not significantly increase serum calcium levels, vitamin D and calcium supplementation significantly worsened aorta and renal artery calcification in Abcc6-/- mice. CONCLUSIONS: vitamin D and/or calcium supplementation accelerate vascular calcification in a murine model of PXE. These results sound a warning regarding the use of these supplementations in PXE patients and, to a larger extent, patients with low systemic pyrophosphate levels.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Immunomodulatory Properties and Osteogenic Activity of Polyetheretherketone Coated with Titanate Nanonetwork Structures.

Polyetheretherketone (PEEK) is a potential substitute for conventional metallic biomedical implants owing to its superior mechanical and chemical properties, as well as biocompatibility. However, its inherent bio-inertness and poor osseointegration limit its use in clinical applications. Herein, thin titanium films were deposited on the PEEK substrate by plasma sputtering, and porous nanonetwork structures were incorporated on the PEEK surface by alkali treatment (PEEK-TNS). Changes in the physical and chemical characteristics of the PEEK surface were analyzed to establish the interactions with cell behaviors. The osteoimmunomodulatory properties were evaluated using macrophage cells and osteoblast lineage cells. The functionalized nanostructured surface of PEEK-TNS effectively promoted initial cell adhesion and proliferation, suppressed inflammatory responses, and induced macrophages to anti-inflammatory M2 polarization. Compared with PEEK, PEEK-TNS provided a more beneficial osteoimmune environment, including increased levels of osteogenic, angiogenic, and fibrogenic gene expression, and balanced osteoclast activities. Furthermore, the crosstalk between macrophages and osteoblast cells showed that PEEK-TNS could provide favorable osteoimmunodulatory environment for bone regeneration. PEEK-TNS exhibited high osteogenic activity, as indicated by alkaline phosphatase activity, osteogenic factor production, and the osteogenesis/osteoclastogenesis-related gene expression of osteoblasts. The study establishes that the fabrication of titanate nanonetwork structures on PEEK surfaces could extract an adequate immune response and favorable osteogenesis for functional bone regeneration. Furthermore, it indicates the potential of PEEK-TNS in implant applications.

New calcification model for intact murine aortic valves.

Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, ß-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

ß-Catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats.

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the ß-catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once-daily injections, 0.75 mg/kg) with/without ß-catenin inhibitor indocyanine green (ICG)-001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX-treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX-induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis-related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX-induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of ß-catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c-Kit+  hematopoietic progenitor cell contents. Thus, ß-catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy.

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

Puerarin facilitates osteogenesis in steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation.

The present study investigated the effect of puerarin on promoting the osteogenesis in steroid-induced necrosis of the femoral head (SONFH). New Zealand rabbits were administrated with horse serum and methylprednisolone (MPS) for establishing SONFH in vivo model, which was then treated with puerarin treatment. Histo-morphological changes in the femoral head were examined by hematoxylin-eosin staining. Osteoblasts were isolated from healthy rabbits and treated by individual or combined administration of dexamethasone and puerarin. Osteoblast viability was measured by CCK-8 assay. Mineralized nodule formation was evaluated by alizarin red assay. Expressions of RUNX family transcription factor 2 (RUNX2), Type-I collagen α 1 (COL1A1), ALP and miR-34a in the femoral head were determined by qRT-PCR and Western blot. Puerarin attenuated the effect of SONFH on promoting histopathological abnormalities and counteracted SONFH inhibition on the expressions of ALP, RUNX2, COL1A1 and miR-34a in the rabbits. Rabbit osteoblasts were successfully isolated, as they showed red mineralized nodules. Dexamethasone exposure decreased osteoblast viability, which was increased by puerarin treatment. Furthermore, puerarin treatment attenuated dexamethasone-induced inhibition on the viability, osteoblastic differentiation, and the expressions of ALP, RUNX2, COL1A1 and miR-34a in the osteoblasts. Puerarin facilitated osteogenesis of steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation.

Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro.

INTRODUCTION: Glucocorticoids are widely used to treat various diseases including rheumatoid arthritis (RA); however, one of the most frequent and severe adverse effects is glucocorticoid-induced osteoporosis (GIOP). Iguratimod (IGU) is a novel conventional synthetic disease-modifying anti-rheumatic drug developed in Japan. The aim of this study is to investigate the effects of IGU on glucocorticoid-induced disorder of bone metabolism in vitro. MATERIALS AND METHODS: In osteoclastogenesis of mouse bone marrow-derived cells, tartrate-resistant acid phosphatase staining, resorption pit assay, western blotting, real-time polymerase chain reaction (PCR), and mRNA sequencing were performed. In osteoblastogenesis of MC3T3-E1 cells, alkaline phosphatase (ALP) staining and activity, alizarin red staining, and mRNA sequencing were performed, and real-time PCR and western blotting were conducted in MC3T3-E1 cells and murine osteocyte-like cell line MLO-Y4 cells. RESULTS: IGU significantly suppressed a dexamethasone-induced increase in osteoclasts, differentiation, and bone resorption activity by inhibition of the receptor activator of the nuclear factor kappa-B (RANK)/tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)/nuclear factor kappa-B (NFκB)-p52 pathway. In MC3T3-E1 cells, IGU significantly upregulated dexamethasone-induced downregulation of ALP activity, bone mineralization, and osteoblast-related gene and protein expression. In MLO-Y4 cells, IGU significantly upregulated dexamethasone-induced downregulation of the gene expression of ALP and osteocalcin, and also downregulated receptor activator of NFκB ligand (RANKL)/osteoprotegerin gene expression ratio without dexamethasone. CONCLUSION: These results suggest that IGU may improve glucocorticoid-induced disorder of bone metabolism and may exhibit positive effects against GIOP associated with RA.

Effect of teriparatide on ligamentum flavum mesenchymal stem cells isolated from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) within the spinal canal sometimes leads to severe myelopathy. Teriparatide (TPD) is a recombinant human parathyroid hormone (PTH) (1-34), which promotes osteogenesis of mesenchymal stem cells (MSCs) via PTH 1 receptor (PTH1R). Although ligamentum flavum (LF)-MSCs from patients with OPLL have a high osteogenic potency, the effect of TPD on them remains unknown. In this study, we determined PTH1R expression in LF-MSCs from patients with OPLL and investigated whether TPD promotes osteogenic differentiation in them. First, LF-MSCs were isolated from patients with OPLL and cervical spondylotic myelopathy (CSM) (controls). Cultured LF-MSCs were treated with different concentrations of TPD on days 0, 7, and 14. On day 21, osteogenic gene expression was quantified. Mineralization was measured based on optical density after Alizarin Red S staining. LF-MSCs from both groups expressed PTH1R at the same level. TPD did not enhance osteogenic gene expression and mineralization in LF-MSCs from both groups. TPD did not promote the osteogenic differentiation of LF-MSCs from patients with OPLL. Thus, it may be safe for patients with OPLL. However, further confirmation of our results with in vivo studies is necessary.

Melatonin attenuates vascular calcification by activating autophagy via an AMPK/mTOR/ULK1 signaling pathway.

Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. Autophagy may affect vascular calcification by inhibiting apoptosis and the transdifferentiation process. This study sought to explore whether melatonin attenuates vascular calcification by regulating autophagy via the AMP-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2, an osteogenic transcription factor), light chain 3 (LC3) II/I, and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were downregulated, whereas LC3 II/I was increased in response to melatonin, and was accompanied by decreased apoptosis. An immunofluorescence assay revealed that melatonin treatment markedly decreased Runx2 expression and upregulated LC3 expression. Treatment with the autophagy inhibitor 3-methyladenine reversed this phenomenon. Melatonin significantly increased expression of p-AMPK and p-ULK1, and decreased mTOR expression. Treatment with compound C (an inhibitor of AMPK) or MHY1485 (an agonist of mTOR) ablated the observed benefits of melatonin treatment. Melatonin protects VSMCs against calcification by activating autophagy via the AMPK/mTOR/ULK1 pathway.

A novel 3D printed bioactive scaffolds with enhanced osteogenic inspired by ancient Chinese medicine HYSA for bone repair.

Some traditional Chinese medicine (TCM) has been applied in bone repair, however, hydroxy-safflower yellow A (HYSA), one composition of safflower of the typical invigorating the circulation of TCM, has little been studied in orthopedics field for osteogenesis and angiogenesis clinically. Herein, we hypothetically speculated that the synthetic bioactive glasses (BG, 1393) scaffolds carried HYSA by a 3D print technique could enhance osteogenic repair properties. Notably, scaffolds coating chitosan/sodium alginate endowed with excellent drug control release ability, and significantly improved the BG mechanical strength. HYSA was loaded into BG scaffolds by coating chitosan/sodium alginate film, and the osteogenesis and angiogenesis of the HYSA/scaffolds were evaluated in vitro and in vivo. In vitro the cell culture results exhibited that the high dose of HYSA (0.5 mg/ml) loaded scaffolds can promote the proliferation of bone marrow stromal cells (rBMSCs) and migration, tubule formation of human umbilical vein endothelial cells (HUVECs). The active alkaline phosphatase (ALP) of rBMSCs can also be improved by the high dose of HYSA/scaffolds. Results of qRT-PCR and Western blot indicated that the high dose of HYSA/scaffolds can up-regulate ALP, OCN, OPN and RUNX-2 expression and relative protein secretion of the HIF-1α and BMP-2. In the animal experiment, the high dose of HYSA/scaffolds has a significantly better capacity to promote new bone formation than the undoped scaffolds at 8 weeks post-surgery. Thus, our results claimed that the novel HYSA/scaffolds hold the substantial potential to be further developed as effective and safe bone tissue engineering biomaterials for bone regeneration by combining enhanced osteogenesis and angiogenesis.

Protective Effects of Fucoxanthin on Hydrogen Peroxide-Induced Calcification of Heart Valve Interstitial Cells.

Cardiovascular diseases such as atherosclerosis and aortic valve sclerosis involve inflammatory reactions triggered by various stimuli, causing increased oxidative stress. This increased oxidative stress causes damage to the heart cells, with subsequent cell apoptosis or calcification. Currently, heart valve damage or heart valve diseases are treated by drugs or surgery. Natural antioxidant products are being investigated in related research, such as fucoxanthin (Fx), which is a marine carotenoid extracted from seaweed, with strong antioxidant, anti-inflammatory, and anti-tumor properties. This study aimed to explore the protective effect of Fx on heart valves under high oxidative stress, as well as the underlying mechanism of action. Rat heart valve interstitial cells under H2O2-induced oxidative stress were treated with Fx. Fx improved cell survival and reduced oxidative stress-induced DNA damage, which was assessed by cell viability analysis and staining with propidium iodide. Alizarin Red-S analysis indicated that Fx has a protective effect against calcification. Furthermore, Western blotting revealed that Fx abrogates oxidative stress-induced apoptosis via reducing the expression of apoptosis-related proteins as well as modulate Akt/ERK-related protein expression. Notably, in vivo experiments using 26 dogs treated with 60 mg/kg of Fx in combination with medical treatment for 0.5 to 2 years showed significant recovery in their echocardiographic parameters. Collectively, these in vitro and in vivo results highlight the potential of Fx to protect heart valve cells from high oxidative stress-induced damage.

Hydrogel contained valproic acid accelerates bone-defect repair via activating Notch signaling pathway in ovariectomized rats.

The purpose was to observe whether valproic acid (VPA) has a positive effect on bone-defect repair via activating the Notch signaling pathway in an OVX rat model. The MC3T3-E1 cells were cocultured with VPA and induced to osteogenesis, and the osteogenic activity was observed by alkaline phosphatase (ALP) staining, Alizarin Red (RES) staining and Western blotting (WB). Then the hydrogel-containing VPA was implanted into the femoral epiphysis bone-defect model of ovariectomized (OVX) rats for 12 weeks. Micro-CT, biomechanical testing, histology, immunofluorescence, RT-qPCR, and WB analysis were used to observe the therapeutic effect and explore the possible mechanism. ALP and ARS staining and WB results show that the cell mineralization, osteogenic activity, and protein expression of ALP, OPN, RUNX-2, OC, Notch 1, HES1, HEY1, and JAG1 of VPA group is significantly higher than the control group. Micro-CT, biomechanical testing, histology, immunofluorescence, and RT-qPCR evaluation show that group VPA presented the stronger effect on bone strength, bone regeneration, bone mineralization, higher expression of VEGFA, BMP-2, ALP, OPN, RUNX-2, OC, Notch 1, HES1, HEY1, and JAG1 of VPA when compared with OVX group. Our current study demonstrated that local treatment with VPA could stimulate repair of femoral condyle defects, and these effects may be achieved by activating Notch signaling pathway and acceleration of blood vessel and bone formation.

Biomimetic mineralisation systems for in situ enamel restoration inspired by amelogenesis.

Caries and dental erosion are common oral diseases. Traditional treatments involve the mechanical removal of decay and filling but these methods are not suitable for cases involving large-scale enamel erosion, such as hypoplasia. To develop a noninvasive treatment, promoting remineralisation in the early stage of caries is of considerable clinical significance. Therefore, biomimetic mineralisation is an ideal approach for restoring enamel. Biomimetic mineralisation forms a new mineral layer that is tightly attached to the surface of the enamel. This review details the state-of-art achievements on the application of amelogenin and non-amelogenin, amorphous calcium phosphate, ions flow and other techniques in the biomimetic mineralisation of enamel. The ultimate goal of this review was to shed light on the requirements for enamel biomineralisation. Hence, herein, we summarise two strategies of biological minimisation systems for in situ enamel restoration inspired by amelogenesis that have been developed in recent years and compare their advantages and disadvantages.