Somatic Mutation Profiling of Papillary Thyroid Carcinomas by Whole-exome Sequencing and Its Relationship with Clinical Characteristics.

The incidence of papillary thyroid carcinomas (PTCs) has increased rapidly during the past several decades. Until now, the mechanisms underlying the tumorigenesis of PTCs have remained largely unknown. Next-generation-sequencing (NGS) provides new ways to investigate the molecular pathogenesis of PTCs. To characterize the somatic alterations associated with PTCs, we performed whole-exome sequencing (WES) of PTCs from 23 Chinese patients. This study revealed somatic mutations in genes with relevant functions for tumorigenesis, such as BRAF, BCR, CREB3L2, DNMT1, IRS2, MSH6, and TP53. We also identified novel somatic gene alterations which may be potentially involved in PTC progression. Gene set enrichment analysis revealed that the cellular response to hormone stimulus, epigenetic modifications, such as protein/histone methylation and protein alkylation, as well as MAPK, PI3K-AKT, and FoxO/mTOR signaling pathways, were significantly altered in the PTCs studied here. Moreover, Protein-Protein Interaction (PPI) network analysis of our mutated gene selection highlighted EP300, KRAS, PTEN, and TP53 as major core genes. The correlation between gene mutations and clinicopathologic features of the PTCs defined by conventional ultrasonography (US) and contrast-enhanced ultrasonography (CEUS) were assessed. These analyses established significant associations between subgroups of mutations and respectively taller-than-wide, calcified, and peak time iso- or hypo-enhanced and metastatic PTCs. In conclusion, our study supplements the genomic landscape of PTCs and identifies new actionable target candidates and clinicopathology-associated mutations. Extension of this study to larger cohorts will help define comprehensive genomic aberrations in PTCs and validate target candidates. These new targets may open methods of individualized treatments adapted to the clinicopathologic specifics of the patients.

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP.

Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of SaOS-2 cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells.

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.

Noninvasive Continuous Monitoring of Adipocyte Differentiation: From Macro to Micro Scales.

3T3-L1 cells serve as model systems for studying adipogenesis and research of adipose tissue-related diseases, e.g. obesity and diabetes. Here, we present two novel and complementary nondestructive methods for adipogenesis analysis of living cells which facilitate continuous monitoring of the same culture over extended periods of time, and are applied in parallel at the macro- and micro-scales. At the macro-scale, we developed visual differences mapping (VDM), a novel method which allows to determine level of adipogenesis (LOA)-a numerical index which quantitatively describes the extent of differentiation in the whole culture, and percentage area populated by adipocytes (PAPBA) across a whole culture, based on the apparent morphological differences between preadipocytes and adipocytes. At the micro-scale, we developed an improved version of our previously published image-processing algorithm, which now provides data regarding single-cell morphology and lipid contents. Both methods were applied here synergistically for measuring differentiation levels in cultures over multiple weeks. VDM revealed that the mean LOA value reached 1.11 ± 0.06 and the mean PAPBA value reached >60%. Micro-scale analysis revealed that during differentiation, the cells transformed from a fibroblast-like shape to a circular shape with a build-up of lipid droplets. We predict a vast potential for implementation of these methods in adipose-related pharmacological research, such as in metabolic-syndrome studies.

Amorphous, Smart, and Bioinspired Polyphosphate Nano/Microparticles: A Biomaterial for Regeneration and Repair of Osteo-Articular Impairments In-Situ.

Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The results revealed that the amorphous Ca-polyP particles promote the growth/viability of mesenchymal stem cells, as well as the osteogenic and chondrogenic differentiation of the bone marrow cells in rat femur explants, as revealed by an upregulation of the expression of the transcription factors SOX9 (differentiation towards osteoblasts) and RUNX2 (chondrocyte differentiation). In parallel to this bone anabolic effect, incubation of the femur explants with these particles significantly reduced the expression of the gene encoding the osteoclast bone-catabolic enzyme, cathepsin-K, while the expression of the tartrate-resistant acid phosphatase remained unaffected. The gene expression data were supported by the finding of an increased mineralization of the cells in the femur explants in response to the Ca-polyP particles. Finally, we show that the hybrid particles of polyP complexed with zoledronic acid exhibit both the cytotoxic effect of the bisphosphonate and the morphogenetic and mineralization inducing activity of polyP. Our results suggest that the Ca-polyP nano/microparticles are not only a promising scaffold material for repairing long bone osteo-articular damages but can also be applied, as a hybrid with zoledronic acid, as a drug delivery system for treatment of bone metastases. The polyP particles are highlighted as genuine, smart, bioinspired nano/micro biomaterials.

Amorphous Ca²âº polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells.

Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca(2+), that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alkaline phosphatase to the cell surface. If SaOS-2 cells are activated by the mineralization activation cocktail (comprising ß-glycerophosphate, ascorbic acid and dexamethasone) and additionally incubated with polyP, a tenfold intracellular increase of the ATP level occurs. Even more, in those cells, an intensified release of ATP into the extracellular space is also seen. We propose and conclude that polyP acts as metabolic fuel after the hydrolytic cleavage of the phosphoanhydride linkages, which contributes to hydroxyapatite formation on the plasma membranes of osteoblasts.

Rebalancing ß-Amyloid-Induced Decrease of ATP Level by Amorphous Nano/Micro Polyphosphate: Suppression of the Neurotoxic Effect of Amyloid ß-Protein Fragment 25-35.

Morbus Alzheimer neuropathology is characterized by an impaired energy homeostasis of brain tissue. We present an approach towards a potential therapy of Alzheimer disease based on the high-energy polymer inorganic polyphosphate (polyP), which physiologically occurs both in the extracellular and in the intracellular space. Rat pheochromocytoma (PC) 12 cells, as well as rat primary cortical neurons were exposed to the Alzheimer peptide Aß25-35. They were incubated in vitro with polyphosphate (polyP); ortho-phosphate was used as a control. The polymer remained as Na⁺ salt; or complexed in a stoichiometric ratio to Ca2+ (Na-polyP[Ca2+]); or was processed as amorphous Ca-polyP microparticles (Ca-polyP-MP). Ortho-phosphate was fabricated as crystalline Ca-phosphate nanoparticles (Ca-phosphate-NP). We show that the pre-incubation of PC12 cells and primary cortical neurons with polyP protects the cells against the neurotoxic effect of the Alzheimer peptide Aß25-35. The strongest effect was observed with amorphous polyP microparticles (Ca-polyP-MP). The effect of the soluble sodium salt; Na-polyP (Na-polyP[Ca2+]) was lower; while crystalline orthophosphate nanoparticles (Ca-phosphate-NP) were ineffective. Ca-polyP-MP microparticles and Na-polyP[Ca2+] were found to markedly enhance the intracellular ATP level. Pre-incubation of Aß25-35 during aggregate formation, with the polyP preparation before exposure of the cells, had a small effect on neurotoxicity. We conclude that recovery of the compromised energy status in neuronal cells by administration of nontoxic biodegradable Ca-salts of polyP reverse the ß-amyloid-induced decrease of adenosine triphosphate (ATP) level. This study contributes to a new routes for a potential therapeutic intervention in Alzheimer's disease pathophysiology.

In Vitro Uptake of Silver Nanoparticles and Their Toxicity in Human Mesenchymal Stem Cells Derived from Bone Marrow.

During the last decade, the usage of silver nanoparticles in biomedical fields has increased rapidly, mainly due to their excellent antibacterial effects. They are used in many medical products such as wound dressings, catheters, bone cement and artificial cardiac valves. In tissue engineering, silver nanoparticles are often loaded as a filler for fabrication of nanocomposite scaffolds which subsequently are seeded with human mesenchymal stem cells. Thus, possible adverse effects of silver nanoparticles on human stem cells should be investigated carefully to ensure a safe usage. In this study, silver nanoparticles with a mean diameter of ~30 nm were prepared and their toxicity in human mesenchymal stem cells was investigated. Transmission electron microscopic images reveal the uptake and localization of the silver nanoparticles in the cytoplasm. Upon internalization of Ag NPs inside the cells, an increase in the release of lactate dehydrogenase and the production of reactive oxygen species was quantified. Furthermore, they caused a reduction in both cell viability and mitochondrial membrane potential in a dose-dependent manner. Annexin V-FITC/PI staining implied that silver nanoparticles did not only induce apoptosis but also cause necrosis. Based on cell cycle analysis, G2/M arrest was detected in cells treated with silver nanoparticles, implicating DNA damage. The high level of reactive oxygen species induced by nanoparticles is considered to be the main cause of their toxicity.

Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation.

Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3 (-) ) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non-enzymatic exchange process.

Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate.

Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO3 is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO3 formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO3 (-)) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca²âº salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO3 deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive-negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects.

Identifying novel clinical phenotypes of acute respiratory distress syndrome using trajectories of daily fluid balance: a secondary analysis of randomized controlled trials.

BACKGROUND: Previously identified phenotypes of acute respiratory distress syndrome (ARDS) could not reveal the dynamic change of phenotypes over time. We aimed to identify novel clinical phenotypes in ARDS using trajectories of fluid balance, to test whether phenotypes respond differently to different treatment, and to develop a simplified model for phenotype identification. METHODS: FACTT (conservative vs liberal fluid management) trial was classified as a development cohort, joint latent class mixed models (JLCMMs) were employed to identify trajectories of fluid balance. Heterogeneity of treatment effect (HTE) for fluid management strategy across phenotypes was investigated. We also constructed a parsimonious probabilistic model using baseline data to predict the fluid trajectories in the development cohort. The trajectory groups and the probabilistic model were externally validated in EDEN (initial trophic vs full enteral feeding) trial. RESULTS: Using JLCMM, we identified two trajectory groups in the development cohort: Class 1 (n = 758, 76.4% of the cohort) had an early positive fluid balance, but achieved negative fluid balance rapidly, and Class 2 (n = 234, 24.6% of the cohort) was characterized by persistent positive fluid balance. Compared to Class 1 patients, patients in Class 2 had significantly higher 60-day mortality (53.5% vs. 17.8%, p < 0.001), and fewer ventilator-free days (0 vs. 20, p < 0.001). A significant HTE between phenotypes and fluid management strategies was observed in the FACTT. An 8-variables model was derived for phenotype assignment. CONCLUSIONS: We identified and validated two novel clinical trajectories for ARDS patients, with both prognostic and predictive enrichment. The trajectories of ARDS can be identified with simple classifier models.

Inorganic polymers: morphogenic inorganic biopolymers for rapid prototyping chain.

In recent years, considerable progress has been achieved towards the development of customized scaffold materials, in particular for bone tissue engineering and repair, by the introduction of rapid prototyping or solid freeform fabrication techniques. These new fabrication techniques allow to overcome many problems associated with conventional bone implants, such as inadequate external morphology and internal architecture, porosity and interconnectivity, and low reproducibility. However, the applicability of these new techniques is still hampered by the fact that high processing temperature or a postsintering is often required to increase the mechanical stability of the generated scaffold, as well as a post-processing, i.e., surface modification/functionalization to enhance the biocompatibility of the scaffold or to bind some bioactive component. A solution might be provided by the introduction of novel inorganic biopolymers, biosilica and polyphosphate, which resist harsh conditions applied in the RP chain and are morphogenetically active and do not need supplementation by growth factors/cytokines to stimulate the growth and the differentiation of bone-forming cells.

The deep-sea natural products, biogenic polyphosphate (Bio-PolyP) and biogenic silica (Bio-Silica), as biomimetic scaffolds for bone tissue engineering: fabrication of a morphogenetically-active polymer.

Bone defects in human, caused by fractures/nonunions or trauma, gain increasing impact and have become a medical challenge in the present-day aging population. Frequently, those fractures require surgical intervention which ideally relies on autografts or suboptimally on allografts. Therefore, it is pressing and likewise challenging to develop bone substitution materials to heal bone defects. During the differentiation of osteoblasts from their mesenchymal progenitor/stem cells and of osteoclasts from their hemopoietic precursor cells, a lineage-specific release of growth factors and a trans-lineage homeostatic cross-talk via signaling molecules take place. Hence, the major hurdle is to fabricate a template that is functioning in a way mimicking the morphogenetic, inductive role(s) of the native extracellular matrix. In the last few years, two naturally occurring polymers that are produced by deep-sea sponges, the biogenic polyphosphate (bio-polyP) and biogenic silica (bio-silica) have also been identified as promoting morphogenetic on both osteoblasts and osteoclasts. These polymers elicit cytokines that affect bone mineralization (hydroxyapatite formation). In this manner, bio-silica and bio-polyP cause an increased release of BMP-2, the key mediator activating the anabolic arm of the hydroxyapatite forming cells, and of RANKL. In addition, bio-polyP inhibits the progression of the pre-osteoclasts to functionally active osteoclasts. Based on these findings, new bioinspired strategies for the fabrication of bone biomimetic templates have been developed applying 3D-printing techniques. Finally, a strategy is outlined by which these two morphogenetically active polymers might be used to develop a novel functionally active polymer.

Pharmacological Activity of Matrine in Inhibiting Colon Cancer Cells VM Formation, Proliferation, and Invasion by Downregulating Claudin-9 Mediated EMT Process and MAPK Signaling Pathway.

Purpose: Matrine (Mat), the main active ingredient of traditional Chinese herbal plant Sophora flavescens Ait, has significant antitumor effects, but its pharmacological mechanism on colon cancer (CC) remains unclear. This study aimed to investigate the therapeutic effect of Mat on CC as well as the potential mechanism. Methods: The vasculogenic mimicry (VM) of CC cells was observed by three-dimensional (3D) Matrigel cell culture. Cell proliferation, apoptosis, migration, invasion, and actin filament integrity were detected by CCK8, flow cytometry, wound healing, Transwell and Phalloidin staining assays. qRT-PCR and Western blotting were applied to detect the expression of EMT factors. RNA-sequencing was conducted to screen differentially expressed genes (DEGs), and the GO and KEGG pathway enrichment analyses were performed. Then, the expression of the key MAPK pathway genes and the target gene Claudin-9 (Cldn9) were analyzed. RNA interference was used to silence Cldn9 expression, and the effects of Cldn9 silencing and simultaneous treatment with Mat on VM formation, proliferation, apoptosis, invasion, and migration were investigated. Finally, the expression of EMT factors and MAPK pathway key genes was detected. Results: CT26 cells formed the most typical VM structure. Mat disrupted the VM of CT26 cells, significantly suppressed their proliferation, migration, invasion, actin filament integrity, induced apoptosis, and inhibited EMT process. RNA-sequencing revealed 163 upregulated genes and 333 downregulated genes in Mat-treated CT26 cells, and the DEGs were significantly enriched in cell adhesion molecules and MAPK signaling pathways. Further confirmed that Mat significantly inhibited the phosphorylation levels of JNK and ERK, and the target gene Cldn9 was significantly upregulated in human CC tissues. Silencing Cldn9 markedly inhibited the VM, proliferative activity, invasiveness, and actin filament integrity of CT26 cells, blocked the EMT process, and downregulated the phosphorylation of JNK and ERK, whereas Mat intervention further strengthened the above trends. Conclusion: This study indicated that Mat may synergistically inhibit the EMT process and MAPK signaling pathway through downregulation Cldn9, thereby exerting pharmacological effects on inhibiting VM formation, proliferation, and invasion of CC cells.

Bifunctional scaffolds for tumor therapy and bone regeneration: Synergistic effect and interplay between therapeutic agents and scaffold materials.

Bone tumor patients often face the problems with cancer cell residues and bone defects after the operation. Therefore, researchers have developed many bifunctional scaffolds with both tumor treatment and bone repair functions. Therapeutic agents are usually combined with bioactive scaffolds to achieve the "bifunctional". However, the synergistic effect of bifunctional scaffolds on tumor therapy and bone repair, as well as the interplay between therapeutic agents and scaffold materials in bifunctional scaffolds, have not been emphasized and discussed. This review proposes a promising design scheme for bifunctional scaffolds: the synergistic effect and interplay between the therapeutic agents and scaffold materials. This review summarizes the latest research progress in bifunctional scaffolds for therapeutic applications and regeneration. In particular, it summarizes the role of tumor therapeutic agents in bone regeneration and the role of scaffold materials in tumor treatment. Finally, a perspective on the future development of bifunctional scaffolds for tumor therapy and bone regeneration is discussed.

Characteristics of frailty phenotype in Chinese nursing home population and significance of motor function indicators in frailty assessment.

The objectives of this study were to analyze the distribution characteristics of frailty phenotypes in older adults of Chinese nursing homes, and to compare some motor function characteristics of older adults in nursing homes between frailty and non-frailty, to determine which motor function and frailty are related. This cross-sectional study included 177 older adults living in nursing homes. Frailty was diagnosed by Fried's phenotype, and motor function assessment characteristics (including muscle tone, ROM, and balance) were also evaluated. Chi-square and logistic regression analyses were performed. Frailty prevalence was 53% in nursing homes in big Chinese cities (average age 82.0 ±â€…6.1). Low levels of physical activity (90.4% in frail elder), decreased handgrip strength (98.9% in frail elder) and slowed walking speed (100% in frail elder) were the 3 main components of the frailty phenotype of frail adults in nursing homes in China. It is worth noting that 74.7% of the non-frail elders also had reduced handgrip strength. Further analysis showed that balance (P < .001), muscle tone (upper, P = .028, lower, P = .001) and the range of motion (P < .001) were associated with frailty in older adults. The frailty of the elders in Chinese nursing homes was characterized by the decline of motor function. And surprisingly, both frail and non-frail elders were found to have poor strength. Frail nursing home seniors also have body muscle tone, range of motion and balance problems. The elderly of China should focus on strength, stretch and balance training to improve motor function, especially strength training, which is important for prevention frailty.

Principles of calcium-based biomineralization.

The chapter provides some basic information on the formation principles of calcium carbonate in biological systems in marine environment in the point of view of materials science in order to provide strategies for biomimetic design and preparation of new functional materials. Many researchers try to explain the principles of biomineralization and get some valuable conclusions. This chapter introduces some calcium-based biominerals in aquatic organisms which mainly include calcium carbonate and calcium phosphate. Then it gives a presentation of the hierarchical structure of calcium carbonate-based and calcium phosphate-based biominerals, e.g., mollusc shell, pearl, carp otolith, tooth, and bone. Moreover, the chapter explains the principles of calcium carbonate mineralization from the aspects of the effects of additives and templates; it also gives some explanations to the principles of calcium phosphate mineralization.

Repair of bone defect in femoral condyle using microencapsulated chitosan, nanohydroxyapatite/collagen and poly(L-lactide)-based microsphere-scaffold delivery system.

Bone repair ability of microencapsulated chitosan, nanohydroxyapatite/collagen (nHAC), and poly(L-lactide) (PLLA)-based microsphere-scaffold delivery system was investigated in present research, with nHAC/PLLA composite scaffold as a control. Chitosan microspheres (CMs) encapsulated with bone morphogenetic protein-2-derived synthetic peptide were incorporated into nHAC and PLLA-based matrix via a thermally induced phase separation method, in which dioxane was used as the solvent for PLLA. Compared with the rapid release from CMs, the synthetic peptide was delivered from CMs/nHAC/PLLA microsphere-scaffold composite in a temporally controlled manner, depending on the degradation of both incorporated CMs and PLLA matrix. MC3T3-E1 osteoblastic cells were seeded into nHAC/PLLA and CMs/nHAC/PLLA scaffolds, respectively, and in vitro cytocompatibility was tested by scanning electron microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results indicated that, with the appearance of CMs in microsphere-scaffold composite, the osteoblasts exhibit better morphology and proliferation ability. In vivo tissue compatibility was evaluated by transplanting the scaffolds into rabbit femoral condyles with a defect 6 mm in diameter. After implanting for 4, 8, and 12 weeks, respectively, radiographic and histological observation revealed that the CMs/nHAC/PLLA composite can accelerate the regeneration of cancellous bone defect as compared with the nHAC/PLLA scaffold. The results demonstrated that the CMs/nHAC/PLLA possesses better biocompatibility, which should be attributed to both the incorporated chitosan component and the encapsulated bioactive synthetic peptide. The promising CMs/nHAC/PLLA microsphere-scaffold composite can be used as delivery system for multiple bioactive factors or as inductive implant scaffold for bone regeneration.

Synergistic Effect of Surface Chemistry and Surface Topography Gradient on Osteogenic/Adipogenic Differentiation of hMSCs.

Much attention has been paid to understanding the individual effects of surface chemistry or topography on cell behavior. However, the synergistic influence of both surface chemistry and surface topography on differentiation of human mesenchymal stem cells (hMSCs) should also be addressed. Here, gold nanoparticles were immobilized in an increasing number density manner to achieve a surface topography gradient; a thin film rich in amine (-NH2) or methyl (-CH3) chemical groups was plasma-polymerized to adjust the surface chemistry of the outermost layer (ppAA and ppOD, respectively). hMSCs were cultured on these model substrates with defined surface chemistry and surface topography gradient. The morphology and focal adhesion (FA) formation of hMSCs were first examined. hMSC differentiation was then co-induced in osteogenic and adipogenic medium, as well as in the presence of extracellular-signal-regulated kinase1/2 (ERK1/2) and RhoA/Rho-associated protein kinase (ROCK) inhibitors. The results show that the introduction of nanotopography could enhance FA formation and osteogenesis but inhibited adipogenesis on both ppAA and ppOD surfaces, indicating that the surface chemistry could regulate hMSC differentiation, in a surface topography-dependent manner. RhoA/ROCK and ERK1/2 signaling pathways may participate in this process. This study demonstrated that surface chemistry and surface topography can jointly affect cell morphology, FA formation, and thus osteogenic/adipogenic differentiation of hMSCs. These findings highlight the importance of the synergistic effect of different material properties on regulation of cell response, which has important implications in designing functional biomaterials.

The relationship between trace elements in fish otoliths of wild carp and hydrochemical conditions.

The trace element composition of the fish otolith is an indicator of biomineralization. In contrast to other skeletal tissue, the otolith retains its entire original structure and does not absorb any elements after the fish dies. Because otoliths in carp degrade very slowly in the dead body, the information it provides on the environment is retained, even in fossil form. Here, we report our analysis of the trace elements in otoliths of carp and of the water in Donghu Lake and Longhupao Lake, Heilongjiang province, China, where the fish lived. The results revealed that the trace elements found in the carp otoliths were clearly correlated with those found in these water bodies. There were high concentrations of Au, Ba, K, Sr and Zn in both the water and otoliths; in contrast there were high levels of As, Na and Se in water, but low concentrations in otoliths. These results indicate that an analysis of the otoliths of carps provides an accurate procedure for studying the surrounding hydrochemistry conditions. The interaction of the elements during deposition was also studied. The correlation coefficients of 13 trace elements identified in the otoliths in both lakes were calculated.