Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis.

BACKGROUND & AIMS: A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesized that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis. METHODS: In vivo investigations were performed in a bile duct-ligated mouse model. Actomyosin contractility was assessed using sandwich-cultured rat hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility. RESULTS: Actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8µm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi. CONCLUSION: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis. LAY SUMMARY: Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.

Effects of Pore Size on the Osteoconductivity and Mechanical Properties of Calcium Phosphate Cement in a Rabbit Model.

Calcium phosphate cement (CPC) porous scaffold is widely used as a suitable bone substitute to repair bone defect, but the optimal pore size is unclear yet. The current study aimed to evaluate the effect of different pore sizes on the processing of bone formation in repairing segmental bone defect of rabbits using CPC porous scaffolds. Three kinds of CPC porous scaffolds with 5 mm diameters and 12 mm length were prepared with the same porosity but different pore sizes (Group A: 200-300 µm, Group B: 300-450 µm, Group C: 450-600 µm, respectively). Twelve millimeter segmental bone defects were created in the middle of the radius bone and filled with different kinds of CPC cylindrical scaffolds. After 4, 12, and 24 weeks, alkaline phosphatase (ALP), histological assessment, and mechanical properties evaluation were performed in all three groups. After 4 weeks, ALP activity increased in all groups but was highest in Group A with smallest pore size. The new bone formation within the scaffolds was not obvious in all groups. After 12 weeks, the new bone formation within the scaffolds was obvious in each group and highest in Group A. At 24 weeks, no significant difference in new bone formation was observed among different groups. Besides the osteoconductive effect, Group A with smallest pore size also had the best mechanical properties in vivo at 12 weeks. We demonstrate that pore size has a significant effect on the osteoconductivity and mechanical properties of calcium phosphate cement porous scaffold in vivo. Small pore size favors the bone formation in the early stage and may be more suitable for repairing segmental bone defect in vivo.

The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells.

Icariin had been reported as a potential agent for osteogenesis, but the dose-effect relationship needed further research to realize the clinical application of icariin. We isolated and purified human bone mesenchymal stem cells (hBMSCs) and stimulated them with different concentrations of icariin. The cytotoxicity of icariin was evaluated by the methylthiazolytetrazolium (MTT) assay method. The proliferation and osteogenic differentiation of such hBMSCs were investigated for different concentrations of icariin. We found that icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of hBMSCs in a suitable concentration range from 10(-9) M to 10(-6) M, but at concentrations above 10(-5) M, the cytotoxicity limited its use. The extremely low cost of icariin and its high abundance make it appealing for bone regeneration.

Effect of co-inoculation with arbuscular mycorrhizal fungi and phosphate solubilizing fungi on nutrient uptake and photosynthesis of beach palm under salt stress environment.

Beach plum (Prunus maritima) is an ornamental plant, famous for its strong salt and drought stress tolerance. However, the poor growth rate of transplanted seedlings has seriously restricted its application in salinized soil. This study investigated the effects of inoculation with arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, and phosphate-solubilizing fungus (PSF), Apophysomyces spartima, on the growth, nutrient (N, P, and K) uptake, and photosynthesis of beach plum under saline (170 mM NaCl) and non-saline (0 mM NaCl) conditions. We aimed to find measures to increase the growth rate of beach plum in saline-alkali land and to understand the reasons for this increase. The results showed that salinization adversely affected colonization by AMF but positively increased PSF populations (increased by 33.9-93.3% over non-NaCl treatment). The dual application of AMF and PSF mitigated the effects of salt stress on all growth parameters and nutrient uptake, significantly for roots (dry weight and P and N contents increased by 91.0%, 68.9%, and 40%, respectively, over non-NaCl treatment). Salinization caused significant reductions in net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), and intercellular CO2 concentration (Ci) value, while inoculation with AMF and PSF inoculations significantly abated such reductions. The maximum efficiency of photosystem II (PSII) (Fv/Fm), the photochemical quenching coefficient (qP), and the nonphotochemical quenching (NPQ) values were affected little by inoculation with AMF, PSF, or both under non-NaCl treatments. However, plants inoculated with AMF and/or PSF had higher Fv/Fm, qP, and ФPSII values (increased by 72.5-188.1%) than the control under NaCl treatment, but not a higher NPQ value. We concluded that inoculation with AMF or PSF increased nutrient uptake and improved the gas-exchange and Chl fluorescence parameters of beach plum under salt stress environment. These effects could be strengthened by the combination of AMF and PSF, especially for nutrient uptake, root growth, and Pn, thereby alleviating the deleterious effects of NaCl stress on beach plum growth.

Metabolic Remodeling during Liver Regeneration.

Liver disease is linked to a decreased capacity of hepatocytes to divide. In addition, cellular metabolism is important for tissue homeostasis and regeneration. Since metabolic changes are a hallmark of liver disease, we investigated the connections between metabolism and cell division. We determined global metabolic changes at different stages of liver regeneration using a combination of integrated transcriptomic and metabolomic analyses with advanced functional redox in vivo imaging. Our data indicate that blocking hepatocyte division during regeneration leads to mitochondrial dysfunction and downregulation of oxidative pathways. This resulted in an increased redox ratio and hyperactivity of alanine transaminase allowing the production of alanine and α-ketoglutarate from pyruvate when mitochondrial functions are impaired. Our data suggests that during liver regeneration, cell division leads to hepatic metabolic remodeling. Moreover, we demonstrate that hepatocytes are equipped with a flexible metabolic machinery able to adapt dynamically to changes during tissue regeneration.

Study on relationship between pollen exine ornamentation pattern and germplasm evolution in flowering crabapple.

Pollen ornamentation patterns are important in the study of plant genetic evolution and systematic taxonomy. However, they are normally difficult to quantify. Based on observations of pollen exine ornamentation characteristics of 128 flowering crabapple germplasms (44 natural species and 84 varieties), three qualitative variables with binary properties (Xi: regularity of pollen exine ornamentation; Yi: scope of ornamentation arrangement regularity; Zi: ornamentation arrangement patterns) were extracted to establish a binary three-dimensional data matrix (Xi Yi Zi) and the matrix data were converted to decimal data through weight assignment, which facilitated the unification of qualitative analysis and quantitative analysis. The result indicates that from species population to variety population and from parent population to variety population, the exine ornamentation of all three dimensions present the evolutionary trend of regular → irregular, wholly regular → partially regular, and single pattern → multiple patterns. Regarding the evolutionary degree, the regularity of ornamentation was significantly lower in both the variety population and progeny population, with a degree of decrease 0.82-1.27 times that of the regularity range of R-type ornamentation. In addition, the evolutionary degree significantly increased along Xi → Yi → Zi. The result also has certain reference values for defining the taxonomic status of Malus species.

Synergistic Effect of Mesoporous Silica and Hydroxyapatite in Loaded Poly(DL-lactic-co-glycolic acid) Microspheres on the Regeneration of Bone Defects.

A microsphere composite made of poly(DL-lactic-co-glycolic acid) (PLGA), mesoporous silica nanoparticle (MSN), and nanohydroxyapatite (nHA) (PLGA-MSN/nHA) was prepared and evaluated as bone tissue engineering materials. The objective of this study was to investigate the synergistic effect of MSN/nHA on biocompatibility as well as its potential ability for bone formation. First, we found that this PLGA-MSN/nHA composite performed good characteristics on microstructure, mechanical strength, and wettability. By cell culture experiments, the adhesion and proliferation rate of the cells seeded on PLGA-MSN/nHA composite was higher than those of the controls and high levels of osteogenetic factors such as ALP and Runx-2 were detected by reverse transcriptase polymerase chain reaction. Finally, this PLGA-MSN/nHA composite was implanted into the femur bone defect in a rabbit model, and its ability to induce bone regeneration was observed by histological examinations. Twelve weeks after implantation, the bone defects had significantly more formation of mature bone and less residual materials than in the controls. These results demonstrate that this PLGA-MSN/nHA composite, introducing both MSN and nHA into PLGA microspheres, can improve the biocompatibility and osteoinductivity of composite in vitro and in vivo and had potential application in bone regeneration.

Different effects of implanting sensory nerve or blood vessel on the vascularization, neurotization, and osteogenesis of tissue-engineered bone in vivo.

To compare the different effects of implanting sensory nerve tracts or blood vessel on the osteogenesis, vascularization, and neurotization of the tissue-engineered bone in vivo, we constructed the tissue engineered bone and implanted the sensory nerve tracts (group SN), blood vessel (group VB), or nothing (group Blank) to the side channel of the bone graft to repair the femur defect in the rabbit. Better osteogenesis was observed in groups SN and VB than in group Blank, and no significant difference was found between groups SN and VB at 4, 8, and 12 weeks postoperatively. The neuropeptides expression and the number of new blood vessels in the bone tissues were increased at 8 weeks and then decreased at 12 weeks in all groups and were highest in group VB and lowest in group Blank at all three time points. We conclude that implanting either blood vessel or sensory nerve tract into the tissue-engineered bone can significantly enhance both the vascularization and neurotization simultaneously to get a better osteogenesis effect than TEB alone, and the method of implanting blood vessel has a little better effect of vascularization and neurotization but almost the same osteogenesis effect as implanting sensory nerve.

Repairing the osteochondral defect in goat with the tissue-engineered osteochondral graft preconstructed in a double-chamber stirring bioreactor.

To investigate the reparative efficacy of tissue-engineered osteochondral (TEO) graft for repairing the osteochondral defect in goat, we designed a double-chamber stirring bioreactor to construct the bone and cartilage composites simultaneously in one ß-TCP scaffold and observed the reparative effect in vivo. The osteochondral defects were created in goats and all the animals were divided into 3 groups randomly. In groups A, the defect was treated with the TEO which was cultured with mechanical stimulation of stir; in group B, the defect was treated with TEO which was cultured without mechanical stimulation of stir; in groups C, the defect was treated without TEO. At 12 weeks and 24 weeks after operation, the reparative effects in different groups were assessed and compared. The results indicated that the reparative effect of the TEO cultured in the bioreactor was better than the control group, and mechanical stimulation of stir could further improve the reparative effect. We provided a feasible and effective method to construct the TEO for treatment of osteochondral defect using autologous BMSCs and the double-chamber bioreactor.

The potential use of allogeneic platelet-rich plasma for large bone defect treatment: immunogenicity and defect healing efficacy.

Autologous platelet-rich plasma (PRP) has been extensively investigated for large bone defect treatment, but its clinical application is harassed by controversial outcome, due to highly variable PRP quality among patients. Alternatively, allogeneic PRP from well-characterized donors cannot only generate more consistent and reliable therapeutic effect but also avoid harvesting large quantities of blood, an additional health burdens to patients. However, the use of allogeneic PRP for bone defect treatment is generally less investigated, especially for its immunogenicity in such application. Here, we meticulously investigated the immunogenicity of allogeneic PRP and evaluated its healing efficacy for critical-sized defect treatment. Allogeneic PRP contained 4.1-fold and 2.7- to 4.9-fold higher amount of platelets and growth factors than whole blood, respectively. The intramuscular injection of allogeneic PRP to rabbits did not trigger severe and chronic immunoresponse, evidenced by little change in muscular tissue microstructure and CD4⁺/CD8⁺ T lymphocyte subpopulation in peripheral blood. The implantation of allogeneic PRP/deproteinized bone matrix (DPB) constructs (PRP+DPB) successfully bridged 1.5-cm segmental radial defects in rabbits, achieving similar healing capacity as autologous MSC/DPB constructs (MSC+DPB), with greater bone formation (1.1-1.5×, p<0.05) and vascularization (1.3-1.6×, p<0.05) than DPB alone, shown by histomorphometric analysis, bone mineral density measurement, and radionuclide bone imaging. Furthermore, the implantation of both allogeneic PRP- and autologous MSC-mediated DPB constructs (PRP + MSC + DPB) resulted in the most robust bone regeneration (1.2-2.1×, p<0.05) and vascularization (1.3-2.0×, p<0.05) than others (PRP+DPB, MSC+DPB, or DPB alone). This study has demonstrated the promising use of allogeneic PRP for bone defect treatment with negligible immunogenicity, great healing efficacy, potentially more consistent quality, and no additional health burden to patients; additionally, the synergetic enhancing effect found between allogeneic PRP and autologous MSCs may shed a light on developing new therapeutic strategies for large bone defect treatment.

Enhancing the bioactivity of Poly(lactic-co-glycolic acid) scaffold with a nano-hydroxyapatite coating for the treatment of segmental bone defect in a rabbit model.

PURPOSE: Poly(lactic-co-glycolic acid) (PLGA) is excellent as a scaffolding matrix due to feasibility of processing and tunable biodegradability, yet the virgin scaffolds lack osteoconduction and osteoinduction. In this study, nano-hydroxyapatite (nHA) was coated on the interior surfaces of PLGA scaffolds in order to facilitate in vivo bone defect restoration using biomimetic ceramics while keeping the polyester skeleton of the scaffolds. METHODS: PLGA porous scaffolds were prepared and surface modification was carried out by incubation in modified simulated body fluids. The nHA coated PLGA scaffolds were compared to the virgin PLGA scaffolds both in vitro and in vivo. Viability and proliferation rate of bone marrow stromal cells of rabbits were examined. The constructs of scaffolds and autogenous bone marrow stromal cells were implanted into the segmental bone defect in the rabbit model, and the bone regeneration effects were observed. RESULTS: In contrast to the relative smooth pore surface of the virgin PLGA scaffold, a biomimetic hierarchical nanostructure was found on the surface of the interior pores of the nHA coated PLGA scaffolds by scanning electron microscopy. Both the viability and proliferation rate of the cells seeded in nHA coated PLGA scaffolds were higher than those in PLGA scaffolds. For bone defect repairing, the radius defects had, after 12 weeks implantation of nHA coated PLGA scaffolds, completely recuperated with significantly better bone formation than in the group of virgin PLGA scaffolds, as shown by X-ray, Micro-computerized tomography and histological examinations. CONCLUSION: nHA coating on the interior pore surfaces can significantly improve the bioactivity of PLGA porous scaffolds.