The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation.

Ca2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca2+ uptake which were influenced by the amplitude of Ca2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.

Cell micropatterning reveals the modulatory effect of cell shape on proliferation through intracellular calcium transients.

The mechanism by which cell shape regulates the function of the cell is one of the most important biological issues, but it remains unclear. Here, we investigated the effect of the regulation of cell shape on proliferation by using a micropatterning approach to confine MC3T3-E1 cells into specific shapes. Our results show that the proliferation rate for rectangle-, triangle-, square- and circle-shaped osteoblasts increased sequentially and was related to the nuclear shape index (NSI) but not the cell shape index (CSI). Interestingly, intracellular calcium transients also displayed different patterns, with the number of Ca2+ peaks increasing with the NSI in shaped cells. Further causal investigation revealed that the gene expression levels of the inositol 1,4,5-triphosphate receptor 1 (IP3R1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), two major calcium cycling proteins in the endoplasmic reticulum (ER), were increased with an increase in NSI as a result of nuclear volume changes. Moreover, the down-regulation of IP3R1 and/or SERCA2 using shRNAs in circle-shaped or control osteoblasts resulted in changes in intracellular calcium transient patterns and cell proliferation rates towards that of smaller-NSI-shaped cells. Our results indicate that changes in cell shape changed nuclear morphology and then the gene expression of IP3R1 and SERCA2, which produced different intracellular calcium transient patterns. The patterns of intracellular calcium transients then determined the proliferation rate of the shaped osteoblasts.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc.

A multifunctional bio-patch crosslinked with glutaraldehyde for enhanced mechanical performance, anti-coagulation properties, and anti-calcification properties.

Bio-patches for the treatment of valvular disease have been evaluated in clinical trials. It has been shown that failure of these devices, occurring within a few years of implantation, may be due to cytotoxicity, immune response, calcification and thrombosis. Some of these effects may be due to the glutaraldehyde crosslinking process used in the preparation of the materials. A number of studies have focused on strategies to control calcification, while others have concentrated on the prevention of micro-thrombus formation. In the present work, we have introduced amino-terminated poly(ethylene glycol) (NH2-PEG-NH2) as an intermolecular bridge, which not only eliminates free aldehyde groups to prevent calcification, but also introduces sites for the attachment of anticoagulant molecules. Furthermore, PEG, itself a hydrophilic polymer with good biocompatibility, may effectively prevent protein adsorption in the early stages of blood contact leading to thrombus formation. After further covalent attachment of heparin, modified bovine pericardium (BP) showed strong anti-calcification (calcium content: 39.3 ± 3.1 µg mg-1) and anti-coagulation properties (partial thromboplastin time: >300 s). The biocompatibility and mechanical properties, important for clinical use, were also improved by modification. The strategy used in this work includes new ideas and technologies for the improvement of valve products used in the clinic.

Effects of PEMF and glucocorticoids on proliferation and differentiation of osteoblasts.

Pulsed electromagnetic fields (PEMF) can promote bone healing, while use of dexamethasone induces bone loss and osteoporosis. There is no report available on the combined effects of PEMF and dexamethasone on the activity of osteoblasts. Here, we investigated the effects of PEMF and dexamethasone on the proliferation and differentiation of MC3T3-E1 osteoblasts. Our results showed that PEMF and dexamethasone respectively increased and decreased the proliferation of MC3T3-E1 osteoblasts, meanwhile PEMF eliminated the effect of dexamethasone on MC3T3-E1 osteoblasts. Moreover, we also found that dexamethasone combined with PEMF upregulated the mRNA expression of IGF-1 at the early stage after the stimulation of PEMF and improved the decrease of COX-2 mRNA expression induced by dexamethasone at the late stage after the stimulation of PEMF. PEMF may be beneficial to improve dexamethasone-induced bone loss and osteoporosis.

Improvement of Osteoporosis in Rats With Hind-Limb Unloading Treated With Pulsed Electromagnetic Field and Whole-Body Vibration.

OBJECTIVE: Physical factors have been used to address disuse osteoporosis, but their effects and mechanism remain unclear. The purpose of this study was to determine the effects of pulsed electromagnetic field (PEMF) and whole-body vibration (WBV) on disuse osteoporosis to increase knowledge about treating osteoporosis. METHODS: A disuse osteoporosis rat model was developed by hind-limb unloading (HU) for 6 weeks. Forty 4-month-old female Sprague-Dawley rats were divided into 5 groups and given the following interventions: HU, HU treated with PEMF (HUP), HU treated with WBV (HUW), HU treated with both PEMF and WBV (HUPW), and no intervention (controls). After 8 weeks of intervention, measurements were taken. RESULTS: HU induced a decrease in bone mineral density (BMD), whereas HUP, HUW, and HUPW increased it. Moreover, the bone resorption markers tartrate-resistant acid phosphatase (TRAP) and C-terminal peptide of type 1 collagen in the HU group significantly increased, whereas the osteogenesis markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly decreased. The markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly increased, but TRAP and C-terminal peptide of type 1 collagen significantly decreased in the HUPW, HUP, and HUW groups compared with the HU group. In particular, HUPW effectively increased osteocalcin and decreased TRAP compared with HUP and WBV. Microcomputed tomography analysis of the femur indicated that HUPW improved trabecular number, bone volume over total volume, bone surface over bone volume, trabecular separation, and the structure model index compared with HUP and that it improved bone surface over bone volume, trabecular separation, and structure model index compared with HUW. The HUPW group showed a significant increase in maximum load compared with the HUW group and a significant increase in elastic modulus compared with the HUP group. CONCLUSION: PEMF, WBV, and their combination all attenuated bone resorption and enhanced osteogenesis. WBV and the combination of treatments have great potential to improve osteogenesis compared with PEMF. In addition, HUPW significantly attenuated bone resorption compared with HUW and HUP. IMPACT: The results of this study indicated that HUPW could effectively improve disuse osteoporosis compared with HUP, given that trabecular number and bone volume over total volume are associated with disuse osteoporosis. Moreover, BMD recovered well with HUP, HUW, and HUPW but the bone structure-especially mechanical performance-did not, indicating that osteoporosis should be evaluated with BMD and mechanical performance, not with BMD in isolation.

Blastocyst-Inspired Hydrogels to Maintain Undifferentiation of Mouse Embryonic Stem Cells.

Stem cell fate is determined by specific niches that provide multiple physical, chemical, and biological cues. However, the hierarchy or cascade of impact of these cues remains elusive due to their spatiotemporal complexity. Here, anisotropic silk protein nanofiber-based hydrogels with suitable cell adhesion capacity are developed to mimic the physical microenvironment inside the blastocele. The hydrogels enable mouse embryonic stem cells (mESCs) to maintain stemness in vitro in the absence of both leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs), two critical factors in the standard protocol for mESC maintenance. The mESCs on hydrogels can achieve superior pluripotency, genetic stability, developmental capacity, and germline transmission to those cultured with the standard protocol. Such biomaterials establish an improved dynamic niche through stimulating the secretion of autocrine factors and are sufficient to maintain the pluripotency and propagation of ESCs. The mESCs on hydrogels are distinct in their expression profiles and more resemble ESCs in vivo. The physical cues can thus initiate a self-sustaining stemness-maintaining program. In addition to providing a relatively simple and low-cost option for expansion and utility of ESCs in biological research and therapeutic applications, this biomimetic material helps gain more insights into the underpinnings of early mammalian embryogenesis.

Universal Antifogging and Antimicrobial Thin Coating Based on Dopamine-Containing Glycopolymers.

A novel strategy for preparing universal antifogging and antimicrobial coating is reported by the means of one-step coating and Ag nanoparticle (AgNP) formation in situ. A series of hydrophilic glycopolymers including poly(N-3,4-dihydroxybenzenethyl methacrylamide-co-2-deoxy-2-(methacrylamido)glucopyranose) (P1s) and poly(N-3,4-dihydroxybenzenethyl methacrylamide-co-methacrylic acid-co-2-deoxy-2-(methacrylamido)glucopyranose) (P2s) were synthesized by sunlight-induced reverse addition-fragmentation chain transfer (RAFT) polymerization. With the ability to strongly immobilize onto organic and inorganic surfaces (i.e., glass slide, silicon wafer, and polycarbonate) via catechol groups, P1s are very convenient to form superhydrophilic and transparent thin coatings, which result in a unique antifogging property. Additionally, the antimicrobial property is realized by in situ AgNPs forming P2 coatings, facilitated by the presence of carboxyl groups and catechol groups in the polymer chain, rendering it superior antimicrobial activity against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus microorganisms. This antifogging and antimicrobial thin coating shows strong prospects in medical and optical devices, with the extra benefits of avoiding potential pathogen infection in vitro or while in storage.

Promoting the activation of T cells with glycopolymer-modified dendritic cells by enhancing cell interactions.

Dendritic cell (DC) modification to enhance antigen presentation is a valuable strategy in cancer immune therapy. Other than focusing on regulating interactions between DC and antigens, we intend to promote cell interactions between DC and T cell by cell surface engineering. T cell activation is greatly improved and generates higher tumor toxicity with the aid of the synthetic glycopolymer modified on the DC surface, although the glycopolymer alone shows no effect. The great promotion of DC-T cell attraction is revealed by cell image tracking in terms of both frequency and duration of contacts. Our findings provide a new method of T cell activation by these engineered "sweet DCs." This strategy is beneficial for developing more efficient DC-based vaccines.

Simulation of intracellular Ca²âº transients in osteoblasts induced by fluid shear stress and its application.

Intracellular [Formula: see text] transient induced by fluid shear stress (FSS) plays an important role in mechanical regulation of osteoblasts, but the cellular mechanism remains incompletely understood. Here, we constructed a mathematical model combined with experiments to elucidate it. Our simulated and experimental results showed that it was the delay of membrane potential repolarization to produce the refractory period of FSS-induced intracellular calcium transients in osteoblasts. Moreover, the results also demonstrated that the amplitude of FSS-induced intracellular calcium transient is crucial to the proliferation, while its duration is critical to the differentiation, of osteoblasts. Overall, the present study provides a way to understand the cellular mechanism of intracellular calcium transients in osteoblast induced by FSS and explains some of related physiological events.

Fluid shear stress induces osteoblast differentiation and arrests the cell cycle at the G0 phase via the ERK1/2 pathway.

Numerous studies have demonstrated that fluid shear stress (FSS) may promote the proliferation and differentiation of osteoblast cells. However, proliferation and differentiation are mutually exclusive processes and are unlikely to be promoted by FSS simultaneously. Cell proliferation and differentiation induced by FSS has rarely been reported. In order to provide an insight into this process, the present study investigated the effects of FSS on osteoblast­like MC3T3 cells in the G0/G1 phase, the period during which the fate of a cell is determined. The results of the present study demonstrated that FSS promoted alkaline phosphatase (ALP) activity, and the mRNA expression and protein expression of osteocalcin, collagen type I and runt­related transcription factor 2 (Runx2), while inhibiting DNA synthesis and arresting the cell cycle at the G0/G1 phase. The increase in Runx2 and ALP activity was accompanied by the activation of calcium/calmodulin­dependent protein kinase type II (CaMK II) and extracellular signal­regulated kinases 1/2 (ERK1/2), which was completely abolished by treatment with KN93 and U0126, respectively. In addition, the inhibition of ERK1/2, although not CaMK II, decreased p21Cip/Kip activity, resulting in an increase in cell number and S phase re­entry. The results of the present study indicated that in the G0/G1 phase, FSS promoted osteoblast differentiation via the CaMK II and ERK1/2 signaling pathways, and blocked the cell cycle at the G0/G1 phase via the ERK1/2 pathway only. The present findings provided an increased understanding of osteoblastic mechanobiology.