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Although (E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenolate anion (DCPO- ) has recently emerged as a potential near infrared (NIR) biosensor signaling unit, the pKa value of its conjugate acid is relatively high (â¼9); this will lead to relatively low concentrations of DCPO- under physiological conditions and, hence, unsatisfactory sensitivity of DCPO- -based bio-probes. By difluoro-substitution on DCPO- , we have exploited a new fluorophore of o-FDCPO- whose conjugate acid has a much lower pKa value of 7.42. Meanwhile, o-FDCPO- is NIR emissive with λem =693â nm and has a 0.76-fold higher fluorescence efficiency than DCPO- . The significant superiority of o-FDCPO- over DCPO- in sensitivity for NIR biosensor applications was confirmed by comparative studies on two HNO probes, namely o-FDCPO-P and DCPO-P, which bear signaling units of o-FDCPO- and DCPO- , respectively. Moreover, o-FDCPO-P has been demonstrated to be a high-performance HNO probe with high selectivity, high sensitivity (detection limit: 50â nm), and a rapid response, together with a two-photon NIR-excitation imaging capability.
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Técnicas Biossensoriais , Corantes Fluorescentes , Ionóforos/química , Fenóis/química , FótonsRESUMO
A novel fluorescent probe (CA-N) was designed and synthesized for detection of biothiols. CA-N displayed a strong fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the Michael addition reaction of a thiol group to α,ß-unsaturated ketones. CA-N showed low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15 µM, respectively. At the same time, CA-N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved that CA-N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols.
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Cumarínicos/química , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Compostos de Sulfidrila/análise , Linhagem Celular , Células/química , Células/metabolismo , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Compostos de Sulfidrila/metabolismoRESUMO
A hybrid material possessing both componential and structural imitation of bone tissue is the preferable composites for bone defect repair. Inspired by the microarchitecture of native bone, this work synthesized in vitro a functional mineralized collagen fibril (MCF) material by utilizing the method of in situ co-precipitation, which was designed to proceed in the presence of Astragalus polysaccharide (APS), thus achieving APS load within the biomineralized collagen-Astragalus polysaccharide (MCAPS) fibrils. Transmission electron microscope (TEM), selected area electron diffraction (SAED) and scanning electronic microscopy (SEM) identified the details of the intrafibrillar mineralization of the MCAPS fibrils, almost mimicking the secondary level of bone tissue microstructure. A relatively uniform and continuous mineral layer formed on and within all collagen fibrils and the mineral phase was identified as typical weak-crystalline hydroxyapatite (HA) with a Ca/P ratio of about 1.53. The proliferation of bone marrow-derived mesenchymal stem cells (BMSC) and mouse embryo osteoblast precursor cells (MC3T3-E1) obtained a significant promotion by MCAPS. As for the osteogenic properties of MCAPS, a distinct increase in the alkaline phosphatase (ALP) activity and the number of calcium nodules (CN) in BMSC and MC3T3-E1 was detected. The up-regulation of three osteogenic-related genes of RUNX-2, BMP-2 and OCN were confirmed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to further verify the osteogenic performance promotion of MCAPS. A period of 14 days of culture demonstrated that MCAPS-L exhibited a preferable efficacy in enhancing ALP activity and CN quantity, as well as in promoting the expression of osteogenic-related genes over MCAPS-M and MCAPS-H, indicating that a lower dose of APS within the material of MCAPS is more appropriate for its osteogenesis promotion properties.
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Osteoblasts and osteoclasts are two of the most important types of cells in bone repair, and their bone-forming and bone-resorbing activities influence the process of bone repair. In this study, we proposed a physicochemical bidirectional regulation strategy via ration by physically utilizing hydroxyapatite nanopatterning to recruit and induce MSCs osteogenic differentiation and by chemically inhibiting osteolysis activity through the loaded zoledronate. The nanorod-like hydroxyapatite coating was fabricated via a modified hydrothermal process while the zoledronic acid was loaded through the chelation within the calcium ions. The fabrication of a hydroxyapatite/zoledronic acid composite biomaterial. This biomaterial promotes bone tissue regeneration by physically utilizing hydroxyapatite nanopatterning to recruit and induce MSCs osteogenic differentiation and by chemically inhibiting osteolysis activity through the loaded zoledronate. The nanorod-like hydroxyapatite coating was fabricated via a modified hydrothermal process while the zoledronic acid was loaded through the chelation within the calcium ions. The in vitro results tested on MSCs and RAW 246.7 indicated that the hydroxyapatite enhanced cells' physical sensing system, therefore enhancing the osteogenesis. At the same time the zoledronic acid inhibited osteolysis by downregulating the RANK-related genes. This research provides a promising strategy for enhancing bone regeneration and contributes to the field of orthopedic implants.
Assuntos
Regeneração Óssea , Fosfatos de Cálcio , Células-Tronco Mesenquimais , Osteogênese , Impressão Tridimensional , Ácido Zoledrônico , Regeneração Óssea/efeitos dos fármacos , Animais , Osteogênese/efeitos dos fármacos , Camundongos , Fosfatos de Cálcio/química , Fosfatos de Cálcio/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Ácido Zoledrônico/farmacologia , Ácido Zoledrônico/química , Osteólise/tratamento farmacológico , Durapatita/química , Durapatita/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células RAW 264.7RESUMO
Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dodecylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO2), was synthesized for labeling the hypoxic cells specially in this paper. BTTD-NO2 showed no cytotoxicity to MG63 cells by MTT method. When MG63 cells were cultured with BTTD-NO2 under hypoxic condition for 24h, strong red fluorescence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Furthermore, Real time RT-PCR proved that BTTD-NO2 could stimulate high gene expression of the nitroreductase in the cells which could improve the conversion rate of BTTD-NO2 to BTTD-NH2 in turn. It proved that the fluorescence of BTTD-NO2 was quenched by its two nitro groups, however, strong red fluorescence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor cells under hypoxic condition. These results suggested that BTTD-NO2 had the potential as a superior fluorescent probe for tumor detection.
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Neoplasias Ósseas/química , Hipóxia Celular , Corantes Fluorescentes/química , Osteossarcoma/química , Tiadiazóis/química , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Humanos , Estrutura Molecular , Nitrorredutases/genética , Nitrorredutases/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Espectrometria de Fluorescência , Tiadiazóis/síntese químicaRESUMO
Thyroid cancer is the most common form of endocrine cancer around the world, and among which papillary thyroid carcinoma (PTC) is the most ubiquitous pathological sub-kind. Sushi repeat-containing protein X-linked 2 (SRPX2) was reported to be an independent prognostic factor and significantly overexpressed in advanced PTC patients. However, the biological functions of SRPX2 remain ambiguous in PTC. Here, we explored SRPX2 expression profiles and functions in PTC, finding that SRPX2 expression was remarkably upregulated in PTC tissues and cell lines. Further colony formation, CCK-8, as well as transwell assay, suggested that SRPX2 silencing remarkably dampened PTC growth and migration. Mouse xenograft models were established to find that SRPX2 silence remarkably suppressed PTC proliferation and migration in vivo. Following mechanism studies revealed that SRPX2 realized its functions in the PTC process partially through activating the Focal adhesion kinase (FAK) phosphorylation. In conclusion, this study investigated the functions and mechanisms of the SRPX2/FAK pathway in PTC progression. SRPX2 could act as a prospective biologic signature and therapeutic target molecule for PTC.
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Cytoplasmic lipid droplets (LDs) can store neutral lipids as an energy source when needed and also regulate the key metabolic processes of intracellular lipid accumulation, which is associated with several metabolic diseases. The perilipins (Plins) are a family of proteins that associate with the surface of LDs. As a member of Plins superfamily, perilipin 5 (Plin5) coats LDs in cardiomyocytes, which is significantly related to reactive oxygen species (ROS) production originated from mitochondria in the heart, consequently determining the progression of diabetic cardiomyopathy. Plin5 may play a bidirectional function in lipid metabolism which is in a state of dynamic balance. In the basic state, Plin5 inhibited the binding of comparative gene identification-58 (CGI-58) to adipose triglyceride lipase (ATGL) by binding CGI-58, thus inhibiting lipolysis. However, when the body is under stress (such as cold, fasting, exercise, and other stimuli), protein kinase A (PKA) phosphorylates and activates Plin5, which then causes Plin5 to release the binding site of CGI-58 and ATGL, prompting CGI-58 to bind to ATGL and activate ATGL activity, thus accelerating the lipolysis process, revealing the indispensable role of Plin5 in lipid turnover. Here, the purpose of this review is to summarize the present understanding of the bidirectional regulation role of Plin5 in oxidative tissues and to reveal its potential role in diabetic cardiomyopathy protection.
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Cardiomiopatias Diabéticas , Metabolismo dos Lipídeos , Estresse Oxidativo , Perilipina-5 , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , LipídeosRESUMO
In alcoholic liver disease (ALD) research, animal models, as one of the most popular methods to explore pathology and therapeutic drug screening, show the limitations of expensive cost and ethic, as well as long modeling time. To minimize the use of animal models in ALD research, an artificial liver model has been developed by incorporating HepG2 cells into hydrogel matrix based on difunctional hyaluronan and collagen. And on this basis an alcohol-induced ALD model in vitro by adding alcohol in the engineering process has been established. Results showed that the construct exhibited a simulated synthetic and metabolic liver function thanks to the bionic fibrillar and viscoelastic characteristics of hydrogels. And the in vitro alcohol-induced ALD model was also proved to be successfully established, even presenting equal results with ALD mice. Furthermore, epigallocatechin gallate (EGCG) as an intervention on ALD was confirmed in both in vitro and in vivo model. The findings indicate our simple artificial liver model is not only highly predictive but also easy to apply to drug screening and implantation studies, suggesting a promising alternative to animal models. Moreover, as the main active ingredient of tea, EGCG's effective intervention and reversal effect on fatty liver provides support for the theory that green tea could prevent alcoholic fatty liver.
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Catequina , Hepatopatias Alcoólicas , Fígado Artificial , Animais , Catequina/análogos & derivados , Catequina/farmacologia , Colágeno , Ácido Hialurônico , Hidrogéis , Fígado , CamundongosRESUMO
Colorectal cancer (CRC) is a major contributor to cancer-associated mortality in China and remains a vast challenge worldwide. Although the genetic basis of CRC has been investigated, the uncommonly mutated genes in CRC remain unknown, in particular in the Asian population. In the present study, targeted region sequencing on 22 CRC and 10 paired non-cancerous tissues was performed to determine the genetic pattern of CRC samples in the Chinese population. Driver genes were detected by three distinct softwares, including MutSigCV, oncodriveFM and iCAGES. A total of 1,335 reliable somatic mutations were identified in tumour samples compared with normal samples. Furthermore, mismatch repair (MMR) mutant patients presented significantly higher mutation density compared with MMR wild-type patients. The results from MutSigCV, oncodriveFM and iCAGES analyses simultaneously detected 29 unique driver genes. In addition, the genes APC regulator of WNT signaling pathway, SMAD family member 4, neurofibromin 1, AT-rich interaction domain 5B and nuclear receptor corepressor 1 were the top five most frequently mutated genes in CRC samples, with mutation rates of 68, 36, 36, 32 and 27%, respectively. The findings from the present study may therefore serve as a basis for future investigation on the diagnosis and oncogenesis of CRC.
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Collagen (Col) has been intensively exploited as a biomaterial for its excellent biocompatibility, biodegradation and bioactivity. However, the poor mechanical properties and rapid biodegradation of reconstituted collagen hydrogels have always been the bottlenecks for their further development especially for vascular tissue engineering. Herein, based on the self-assembly characteristics of collagen, a ternary hydrogel scaffold, comprising rigid collagen molecules, flexible konjac glucomannan (KGM) chains and biocompatible crosslinkers of proanthocyanidin (PA), has been designed to achieve a synergistic interaction for essentially optimizing the mechanical properties of the so-obtained Col/KGM/PA hydrogel, which possesses not only substantially improved strength but also good elasticity. PA endows these scaffolds with controllable biodegradation and anti-calcification and antioxidant activities. TEM discovered the co-existence of two types of fibrils with distinctly different arrangement patterns, explaining the contribution of KGM macromolecules to elasticity generation. The in vivo variations of Col/KGM/PA implants are visualized in real-time by magnetic resonance imaging (MRI). Moreover, a quantitative technique of MRI T2-mapping combined with histology is designed to visualize the in vivo biodegradation mechanism of layer-by-layer erosion for these hydrogels. Simultaneously, three different relationships between the respective processes of in vivo degradation and in vivo dehydration of these controlled hydrogel implants were clearly revealed by this technique. Such a designed Col/KGM/PA composite hydrogel realizes the essential integration of good biocompatibility, controllable biodegradation and improved mechanical properties for developing a desired scaffold material for tissue engineering applications.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Colágeno/uso terapêutico , Hidrogéis/uso terapêutico , Mananas/uso terapêutico , Proantocianidinas/uso terapêutico , Engenharia Tecidual/métodos , Animais , Linhagem Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Alicerces TeciduaisRESUMO
BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. lncRNAs are demonstrated to improve the DN by changing the expression of miRNAs. This study was aimed to investigate the effect of lncRNA GAS5/miR-452-5p on the inflammation, oxidative stress and pyroptosis of high-glucose-induced renal tubular cells. METHODS: HK-2 cells were induced by HG to simulate DN cells. RT-qPCR analysis confirmed the transfection effects and detected the expression of GAS5, NLRP3, caspase1, IL-1ß, pro-caspase1, pro-IL-1ß, GSDMD-N and miR-452-5p. Western blot analysis determined the protein expression of NLRP3, caspase1, IL-1ß, pro-caspase1, pro-IL-1ß and GSDMD-N. The expression of GSDMD-N was also verified by immunofluorescence. The levels of TNF-α, IL-6, MCP-1, ROS, MDA and SOD were measured by commercial assay kits, respectively. Dual-luciferase reporter assay indicated that GAS5 could combine with miR-452-5p. RESULTS: GAS5 expression was decreased in HG-induced HK-2 cells. GAS5 overexpression could decrease the levels of TNF-α, IL-6, MCP-1, ROS and MDA and increase the levels of SOD. Moreover, GAS5 overexpression suppressed the expression of NLRP3, caspase1, IL-1ß and GSDMD-N, and the results of immunofluorescence verified the above results. miR-452-5p interference could cause the same changes as GAS5 overexpression for HG-induced HK-2 cells, and GAS5 inhibition could reverse the effect of miR-452-5p interference. CONCLUSION: GAS5 overexpression inhibited the inflammation, oxidative stress and pyroptosis of HG-induced renal tubular cells by downregulating the expression of miR-452-5p.
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The discovery of osteoinductivity of calcium phosphate (Ca-P) ceramics has set an enduring paradigm of conferring biological regenerative activity to materials with carefully designed structural characteristics. The unique phase composition and porous structural features of osteoinductive Ca-P ceramics allow it to interact with signaling molecules and extracellular matrices in the host system, creating a local environment conducive to new bone formation. Mounting evidence now indicate that the osteoinductive activity of Ca-P ceramics is linked to their physicochemical and three-dimensional structural properties. Inspired by this conceptual breakthrough, many laboratories have shown that other materials can be also enticed to join the rank of tissue-inducing biomaterials, and besides the bones, other tissues such as cartilage, nerves and blood vessels were also regenerated with the assistance of biomaterials. Here, we give a brief historical recount about the discovery of the osteoinductivity of Ca-P ceramics, summarize the underlying material factors and biological characteristics, and discuss the mechanism of osteoinduction concerning protein adsorption, and the interaction with different types of cells, and the involvement of the vascular and immune systems.
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For an ideal biomimetic microenvironment to realize reliable cartilage regeneration, the ability to induce mesenchymal stem cell (MSCs) differentiation along the chondrogenic lineage and prevent further dedifferentiation is expected. With native bioactivity, collagen has been proved to be preferential for inducing the chondrogenic differentiation of MSCs. However, the phenotypic maintenance of differentiated chondrocytes in a collagen matrix is still a challenge. Actin traction, which causes drastic contraction of the collagen matrix, is frequently observed and might be an important factor that affects cell fates including chondrogenic differentiation and phenotypic maintenance. In this study, photochemical modification was applied to acquire collagen hydrogels with improved mechanical strength and creep behavior. Accompanied by inherited bioactivity, the photo-crosslinked collagen hydrogel well supported the actin cytoskeleton functionalization while resisting the actin-mediated matrix contraction. Benefitting from this, the hydrogel system promoted MSCs proliferation and chondrogenic differentiation, and more importantly, prevented further dedifferentiation. By exploring the mesenchymal development-related signal transduction markers, it was revealed that the promoted chondrogenesis was achieved through inhibiting the over-expression of MAPK and Wnt/ß-catenin signaling pathways that up-regulated dedifferentiated gene expression. The strategy of applying the hydrogel system to cartilage regeneration is foreseeable based on the positive heterotopic and orthotopic chondrogenic differentiation.
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The in vivo osteoinductive capacity of porous calcium phosphate ceramics (Ca/P ceramics) with special structure and phase composition had been found for almost decades. The mechanism of the osteoinductivity of porous calcium phosphate is studied by C2C12 cells culture in this paper. C2C12 cells were cocultured with four kinds of porous Ca/P ceramics for 2 and 5 days, without adding other growth factors. The four kinds of Ca/P ceramics were pure HA sintered at 1250 degrees C and HA/TCP with a ratio of 60/40 sintered at 1100, 1200, and 1250 degrees C respectively. RT-PCR analysis found that the Ca/P ceramics induced the expression of Cbfa1, collagen type I, bone sialoprotein, and osteocalcin in C2C12 cells, while they did not induce mRNA expression of Indian hedgehog (IHH) that regulate chondrocyte differentiation. Our results showed that Ca/P ceramics alone were sufficient to induce C2C12 cells differentiation. The induction of bone-related markers expression by Ca/P ceramics in osteoprogenitor cells suggested that the osteogenesis induced by the ceramics was intramembranous and the osteoinductivity was their intrinsic property.
Assuntos
Materiais Biocompatíveis , Fosfatos de Cálcio , Cerâmica , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Osteoblastos/metabolismo , Animais , Sequência de Bases , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Colágeno Tipo I/genética , Primers do DNA/genética , Expressão Gênica , Técnicas In Vitro , Sialoproteína de Ligação à Integrina , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Osteocalcina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sialoglicoproteínas/genéticaRESUMO
Phase composition played a key role in the biodegradation of calcium phosphate ceramics (CaP), which in turn influences the osteoinductive ability. The in vivo biological mechanism is still poorly understood. In this study, four types of porous CaP ceramics were investigated, namely, hydroxyapatite (HA), ß-tricalcium phosphate (TCP), and biphasic calcium phosphates BCP1 and BCP2, with HA to ß-TCP ratios of 70/30 and 30/70, respectively. The four types of ceramics were implanted into thigh muscle of mice for 16weeks. Longitudinal ectopic bone formation process at gene, protein, and tissue level induced by the material was assessed. Histological analysis revealed that BCP2 was the only group that had promoted new bone formation after 16weeks. In micro-CT analysis of biodegradation, the BCP2 group had the least increment of porosity due to the new bone formation, resulting in a significant elevation in material density. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression patterns. The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Among the target genes, Osterix and type I collagen were activated successively. The osteoinductive BCP2 group showed earlier and significantly higher peaks in BMP2, BMPR1A, and OPG expressions than non-bone forming groups. These findings revealed that the occurrence time and magnitude of these osteogenetic gene expression peaks can be crucial in the osteoinduction process.
Assuntos
Fosfatos de Cálcio/farmacologia , Cerâmica/farmacologia , Coristoma/patologia , Osteogênese/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Implantes Experimentais , Masculino , Camundongos Endogâmicos BALB C , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Porosidade , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Difração de Raios X , Microtomografia por Raio-XRESUMO
Stimuli-responsive targeted polymeric micelles as drug delivery systems have recently attracted significant attention for the treatment of various cancers, which could improve delivery efficiency by tumor-specific recognition via active targeting strategies. In this research, DOX-incorporated bioreducible polymeric micelles based on PAA-g-PEG/PEG-Fa conjugated polymers were prepared and characterized as nanocarriers for promoting intracellular anti-tumor drug delivery efficiency via folate receptor-mediated endocytosis. The anti-proliferative activity on cancer cells, biodistribution, active-/passive-targeting efficiency, tumor growth inhibition efficiency, and biological toxicity were evaluated in vitro and in vivo. The MTT assay demonstrated that the DOX-encapsulated active-targeting PAA-g-PEG/PEG-Fa micelles had much greater growth inhibition effect against 4T1 and KB than passive-targeting micelles. These active-targeting micelles showed superior tumor accumulation and excellent tumor growth inhibition effect as revealed by the fluorescence optical imaging technique and tumor volume change investigation, as well as the survival study of the tumor-bearing Balb/c mice. Furthermore, the active targeting micelles greatly decreased the toxicity of DOX on the heart and other organs. These potential results encouraged us to further optimize the molecular structure to achieve more excellent targeted therapeutic effect.
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Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.
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Materiais Biocompatíveis/química , Proteínas Morfogenéticas Ósseas/metabolismo , Fosfatos de Cálcio/química , Cerâmica/química , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Proteínas Smad/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Durapatita/química , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Difração de Raios XRESUMO
In this study, two methods--the supernatant hemoglobin spectrophotometry, i.e. hemolysis percentage measurement(according to ISO/TR 7405) and the hemiglobincyanide measurement(according to ISO 10993-4)--were used to assay the hemolytic properties of hydroxyapatite(bioceramics) and collagen (polymer). The results showed that the conclusions drawn from using the two methods were basically consistent, and the latter was more sensitive, stable and comparable. However, some of the procedures in the hemiglobincyanide method were not defined in details. So based on our experiments we have offered some suggestions and improvements, which do not deviate from ISO and ASTM standards, for hiher practicability of usig it in standardizing the evaluation of the hemolytic properties of biomaterials. Hemiglobincyanide measurement is worthy of wider application.
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Hemoglobinas , Hemólise , Teste de Materiais/métodos , Metemoglobina/análogos & derivados , Animais , Colágeno , Durapatita , Humanos , CoelhosRESUMO
A three-level hierarchical calcium phosphate/collagen/hydroxyapatite (CaP/Col/HAp) scaffold for bone tissue engineering was developed using biomimetic synthesis. Porous CaP ceramics were first prepared as substrate materials to mimic the porous bone structure. A second-level Col network was then composited into porous CaP ceramics by vacuum infusion. Finally, a third-level HAp layer was achieved by biomimetic mineralization. The three-level hierarchical biomimetic scaffold was characterized using scanning electron microscopy, energy-dispersive x-ray spectra, x-ray diffraction and Fourier transform infrared spectroscopy, and the mechanical properties of the scaffold were evaluated using dynamic mechanical analysis. The results show that this scaffold exhibits a similar structure and composition to natural bone tissues. Furthermore, this three-level hierarchical biomimetic scaffold showed enhanced mechanical strength compared with pure porous CaP scaffolds. The biocompatibility and osteoinductivity of the biomimetic scaffolds were evaluated using in vitro and in vivo tests. Cell culture results indicated the good biocompatibility of this biomimetic scaffold. Faster and increased bone formation was observed in these scaffolds following a six-month implantation in the dorsal muscles of rabbits, indicating that this biomimetic scaffold exhibits better osteoinductivity than common CaP scaffolds.
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Materiais Biocompatíveis/química , Materiais Biomiméticos/química , Células da Medula Óssea/citologia , Fosfatos de Cálcio/química , Colágeno/química , Durapatita/química , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/síntese química , Fenômenos Biomecânicos , Materiais Biomiméticos/síntese química , Humanos , Masculino , Porosidade , CoelhosRESUMO
The purpose of this study was to investigate the effect of phase compositions of porous calcium phosphate (CaP) ceramics on their protein adsorption behaviors in vitro and osteoinductive potentials in vivo in mice. Under competitive conditions, a high adsorption of bone morphogenetic protein 2 (BMP-2) was observed at a high initial concentration of BMP-2 in the multi-protein solution on all the four types of ceramics, indicating their strong affinity for BMP-2. No significant difference in BMP-2 adsorption between the ceramics was noted, indicating that phase composition could have little influence on BMP-2 adsorption. After implantation into the thigh muscles of mice for 45 and 90 days, the histological and histomorphometric analyses showed that porous biphasic calcium phosphate (BCP) ceramic consisting of 30% hydroxyapatite HA and 70% tricalcium phosphate (ß-TCP), i.e. BCP-2 had stronger osteoinductive ability than the other three groups of ceramics. The immunohistochemical staining showed the highest expression of BMP-2 and osteocalcin (OCN) in BCP-2 group. Osteoinduction of porous CaP ceramics might be influenced by the amount of BMP-2 present in the local microenvironment in the implant, which was regulated by the phase composition of the ceramics. BCP-2 promoted the highest expression of BMP-2 and then showed the strongest osteoinduction in mice.