Deep Learning Image Recognition-assisted Atomic Force Microscopy for Precise and Efficient Detection of Single-cell Mechanical Properties / 生物化学与生物物理进展

ObjectiveThe advent of atomic force microscope (AFM) provides a powerful tool for the studies of life sciences. Particularly, AFM-based indentation assay has become an important method for the detection of cellular mechanics, yielding numerous novel insights into the physiological and pathological activities from the single-cell level and considerably complementing traditional biochemical ensemble-averaged assays. However, current AFM indentation technology is mainly dependent on manual operation with low efficiency, seriously restricting its practical applications in the field of life sciences. Here, a method based on the combination of deep learning image recognition and AFM is developed for precisely and efficiently detecting the mechanical properties of single isolated cells and clustered cells. MethodsThe YOLO deep learning algorithm was used to recognize the central region of the cell in the optical image, the dual UNet neural network with an embedded vision transformer (ViT) module was used to recognize the peripheral regions of cell, and the template matching algorithm was used to recognize the tip of spherical probe. Based on the automatic determination of the positional relationships between the microsphere tip and the different parts of cell, the AFM tip was accurately moved to the central and peripheral regions of the target cell for rapid measurements of cell mechanical properties. Two types of cells, including HEK 293 cell (human embryonic kidney cell) and HGC-27 cell (human undifferentiated gastric cancer cell), were used for the experiments. The Hertz model was applied to analyze the force curves obtained on cells to obtain cellular Young’s modulus. ResultsAFM probe can be precisely moved to the different parts (central areas and peripheral areas) of cells to perform mechanical measurements under the guidance of deep learning-based optical image automatic recognition. The experimental results show that the proposed method is not only suitable for single isolated cells, but also suitable for clustered cells. ConclusionThe research demonstrates the great potential of deep learning image recognition to aid AFM in the precise and efficient detection of cellular mechanical properties mechanics, and combining deep learning-based image recognition with AFM will benefit the development of high-throughput AFM-based methodology to measure the mechanical properties of cells.

Applications of atomic force microscopy in immunology / 医学前沿

Cellular mechanics, a major regulating factor of cellular architecture and biological functions, responds to intrinsic stresses and extrinsic forces exerted by other cells and the extracellular matrix in the microenvironment. Cellular mechanics also acts as a fundamental mediator in complicated immune responses, such as cell migration, immune cell activation, and pathogen clearance. The principle of atomic force microscopy (AFM) and its three running modes are introduced for the mechanical characterization of living cells. The peak force tapping mode provides the most delicate and desirable virtues to collect high-resolution images of morphology and force curves. For a concrete description of AFM capabilities, three AFM applications are discussed. These applications include the dynamic progress of a neutrophil-extracellular-trap release by neutrophils, the immunological functions of macrophages, and the membrane pore formation mediated by perforin, streptolysin O, gasdermin D, or membrane attack complex.

Effects of graphene on the proliferation, migration and morphology of dental pulp stem cells / 口腔疾病防治

Objective@# To investigate the effects of graphene on the proliferation, migration and cell morphology of dental pulp stem cells (DPSCs).@*Methods@#Graphene powder was prepared by the oxidation-reduction method, and a 0.5 mg/mL graphene dispersion was prepared. Raman spectroscopy and atomic force microscopy were used to characterize the structure and surface morphology of graphene. DPSCs were isolated and cultured in vitro. MTT assay was used to detect the effects of different concentrations of graphene dispersions (0, 1, 5, 10, 20, 50, 100 μg/mL) on the proliferation and wound healing assay was used to detected the migration abilities of DPSCs. The effects of graphene on the morphology of DPSCs were observed by immunofluorescence staining. @*Results @# In the present study, compared with the control group (0 μg/mL), the proliferation of DPSCs in the 100 μg/mL group was inhibited at 72 h (P < 0.05), and the proliferation of DPSCs in the other groups was not significantly affected (P > 0.05). Graphene dispersions at 10 and 20 μg/mL promoted the migration of DPSCs (P < 0.05). After being cultured in 20 μg/mL graphene dispersions for 3 days, the DPSCs showed a large and orderly cytoskeletal structure, and the spread area of cells was not significantly different from that of the control group (0 μg/mL) (P > 0.05), while some cells had the morphological characteristics of nerve cells.@* Conclusion @# Graphene has good biocompatibility and is expected to be a suitable material for tissue engineering within fitting concentration.

Effects of High-Fat Diet on Rat Aortic Endothelial Cell Morphology and Stiffness / 医用生物力学

Objective To investigate the high-fat diet effect on morphology and stiffness of endothelial cells. Methods SD rats were randomly divided into high-fat diet group (AS group, n=3) and control group (CON group, n=3). Rat aortic endothelial cells were obtained from rat thoracic aorta by explant method. Cell morphology was observed under inverted microscopy. The mean fluorescent intensity of F-actin in two groups was calculated by immunofluorescence staining. Cell stiffness was measured by atomic force microscopy (AFM). Results The endothelial cells migrated from tissue plant on the 7th day and formed confluence after cultivation for 14 days. Endothelial cells were identified by factor Ⅷ immunofluorescence staining. Cells in AS group showed enhanced perimeter (P＜0.01), aspect ratio (P＜0.01), and area (P＞0.05), while less circularity (P＜0.01) compared with the cells in control group. The mean fluorescence intensity of F-actin in AS group was significantly higher than that in control group (P＜0.01). AFM showed that the cell stiffness of AS group was significantly higher than that of control group (P＜0.01). Conclusions High-fat diet would change the morphology and stiffness of endothelial cells, which might subsequently affect their normal function and become an important incentive to AS.

Mechanical properties and cytoskeletal protein changes after anln deficiency / 中国组织工程研究

BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported. OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells. METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy. RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.

Micro and nanocrystalline cellulose based oral dispersible film; preparation and evaluation of in vitro/in vivo rapid release studies for donepezil

Oral fast-dispersible film was prepared by utlizing donepezil hydrochloride (drug) and various cellulose derivatives such as hydroxypropyl methyl cellulose (hypermellose) (HPMC), microcrystalline cellulose (MCC) and nanocrystalline cellulose (NCC) to treat Alzheimer's disease. NCC was synthesized by ultra-sonication method using MCC and this was converted to thinfilm formulation (NCC-F) using solvent casting technique. The interaction between the polymer and the drug was investigated by spectral analysis such as UV, FTIR, and 1H- NMR. FTIR confirmed that the compatibility of drug and polymer in ODF formulation. NCC-F has shown an average surface roughness of 77.04 nm from AFM and the average particle size of 300 nm from SEM analysis. Nano sized particle of NCC-F leads faster in vitro dissolution rate (94.53%) when compared with MCC-F and F3 formulation. Animal model (in vivo) studies of NCC-F formulation has reached peak plasma concentration (Cmax) up to 19.018 ng/mL in the span of (tmax) 4 h with greater relative bioavailability of 143.1%. These results suggested that high surface roughness with nanosized NCC-F formulation attained extended drug availability up to (t1/2) 70 h.

Effects of different toothpastes on the prevention of erosion in composite resin and glass ionomer cement enamel and dentin restorations

Abstract Objective This study aimed to evaluate the effects of different toothpastes on the surface wear of enamel, dentin, composite resin (CR), and resin-modified glass ionomer cement (RMGIC), and to perform a topographic analysis of the surfaces, based on representative images generated by atomic force microscopy (AFM) after erosion-abrasion cycles. Methodology One hundred and forty bovine incisors were collected and divided into two groups: 72 enamel and 72 dentin blocks (4×4 mm). Half of the specimens were restored with CR (Filtek Z350 XT) and the other half with RMGIC (Fuji II LC). Then, samples were submitted to a demineralization cycle (5 days, 4×2 min/day, 1% citric acid, pH 3.2) and exposed to three different toothpastes (2×15 s/day): without fluoride (WF, n=12), sodium fluoride-based (NaF, n=12), and stannous fluoride-based (SnF2, n=12). Surface wear, as well as restoration interfaces wear, were investigated by profilometry of the dental substrates and restorative materials. All representative surfaces underwent AFM analysis. Data were analyzed by two-way analysis of variance and Tukey's tests (α=0.05). Results NaF-based toothpaste caused the greater dentin surface wear (p<0.05). Toothpastes affected only enamel-restoration interfaces. AFM analysis showed precipitate formation in dentinal tubules caused by the use of fluoride toothpastes. Conclusions NaF-based toothpastes had no protective effect on enamel adjacent to CR and RMGIC against erosion-abrasion challenges, nor on dentin adjacent to RMGIC material. SnF2-based toothpastes caused more damage to interfaces between enamel and RMGIC.

Effect of lactobacillus postbiotics on entamoeba histolytica trophozoites

ABSTRACT Background Amebiasis is an infectious disease caused by Entamoeba histolytica. It represents one of the three worldwide leading causes of death by parasites and a public health problem due to its frequency, morbidity, mortality, and easy dispersion. Objective The study was aimed to evaluate the in vitro effect of Lactobacillus spp. postbiotics on E. histolytica trophozoites (HM1-IMSS strain) and to determine morphometric changes in trophozoite membrane by atomic force microscopy (AFM). Methods Bioassays on trophozoites were conducted with lyophilized postbiotics at 0.1, 0.3, and 0.5 mg/mL concentrations, and trophozoite samples were obtained for AFM analysis Results Results indicated postbiotic inhibitory activity; the highest percentage inhibition was 89.63% at 0.5 mg/mL. Trophozoites nanomechanical analysis showed 28.32% increase in ruggedness and 56% decrease in size with treatments compared to the control. Conclusion Our study showed that the synergy of Lactobacillus postbiotics inhibited E. histolytica HM1-IMSS in vitro growth under axenic conditions, inducing morphometric alterations in trophozoites’ cell membrane. These results would allow designing strategies or treatments aimed at E. histolytica control in the future.

In vitro biological activities of silver nanoparticles synthesized from Scedosporium sp. isolated from soil

One of the important fields in nanotechnology is the development of an environment friendly method for the synthesis of nanoparticles. Many approaches show that microorganisms are the most reliable tools for biosynthesis of nanoparticles compared to physical and chemical methods. In our study, fungi have been exploited for extracellular production of metal nanoparticles. It was observed that in Scedosporium, silver ions are reduced to silver nanoparticles, which was confirmed by UV-visible spectrophotometry and AFM. Optimization studies showed that as the concentration of AgNO3 used for synthesis increased, particles' size also increased. Size of the particles at different concentrations of AgNO3 was observed to be 79-107 nm with particles being ellipsoidal to spherical in shape. Silver nanoparticles synthesized from 2.0 mM silver nitrate, showed maximum antimicrobial activity compared to all antibiotics tested including synergistic effects. In vitro cytotoxicity of silver nanoparticles against MCF 7 and PC 3 showed that as the concentration of silver nanoparticles increased, a decrease in the percentage cell viability was observed with IC50 values being 60.09 and 57.43 µg/ml respectively. Therefore, through this study, it could be said that extracellular synthesis of silver nanoparticles from Scedosporium was simple, ecofriendly, proving excellent antimicrobial and anticancer agents.

Using prodigiosin against some gram-positive and gram-negative bacteria and Trypanosoma cruzi

This work aimed to explore the action of natural prodigiosin on both bacterial organisms and Trypanosoma cruzi cells. Methods: Natural prodigiosin pigment was extracted and purified from cultures of Serratia marcescens. Two media, peanut broth and peptone glycerol broth, both recommended in the literature for prodigiosin production, were compared. The prodigiosin obtained was employed to explore its antimicrobial properties against both bacteria and Trypanosoma cruzi cells. Results: Peanut broth yielded four times more prodigiosin. The prodigiosin showed remarkable activity (minimal inhibitory concentrations in the range of 2-8 µM for bacteria and half maximal inhibitory concentration of 0.6 µM for Trypanosoma cruzi). In fact, the prodigiosin concentration required to inhibit parasite growth was as low as 0.25 mg/l versus 4.9 mg/l of benznidazole required. Furthermore, atomic force microscopy revealed marked morphological alterations in treated epimastigote forms, although no pore-formation activity was detected in protein-free environments. Conclusions: This work demonstrates the potential usefulness of prodigiosin against some gram-positive and gram-negative bacteria and Trypanosoma cruzi although further studies must be done in order to assess its value as a candidate molecule.(AU)

Adhesion to a lithium disilicate glass ceramic etched with hydrofluoric acid at distinct concentrations

Abstract This study evaluated the effect of different hydrofluoric acid (HF) concentrations on the bond strength between a lithium disilicate-based glass ceramic and a resin cement. Eighty ceramic-blocks (12×7×2 mm) of IPS e.Max CAD (Ivoclar Vivadent) were produced and randomly assigned to 8 groups, considering 2 study factors: HF concentration in 4 levels, i.e., 1% (HF1), 3% (HF3), 5% (HF5), and 10% (HF10), and storage in 2 levels, i.e., baseline (tests were performed 24 h after cementation), and aged (storage for 150 days + 12,000 thermal-cycles at 5°C and 55°C). Acid etching (20 s) was performed, followed by washing, drying, and silanization. Four resin cement cylinders (ϕ= 0.96 mm) were built-up from starch matrices on each ceramic sample (n= 40). Additional ceramic samples were etched and analyzed for contact angle, micro-morphology, and roughness. In baseline condition (without aging), the HF3, HF5, and HF10 groups showed similar bond strength values (13.9 - 15.9 MPa), and HF1 (11.2 MPa) presented lower values than HF5, being that statistically different (p= 0.012). After aging, all the mean bond strengths statistically decreased, being that HF3, HF5, and HF10 (7.8 - 11 MPa) were similar and higher than HF1 (1.8 MPa) (p= 0.0001). For contact angle, HF3, HF5, and HF10 presented similar values (7.8 - 10.4°), lower than HF1 and CTRL groups. HF5 and HF10 presented rougher surfaces than other conditions. For better bond strength results, the tested ceramic may be etched by HF acid in concentrations of 3%, 5%, and 10%.

Resumo Este estudo avaliou o efeito de diferentes concentrações de ácido fluorídrico (HF) na resistência de união entre uma cerâmica vítrea à base de dissilicato de lítio e um cimento resinoso. Oitenta blocos cerâmicos (12×7×2 mm) de IPS e.Max CAD (Ivoclar Vivadent) foram produzidos e distribuídos aleatoriamente em 8 grupos, considerando 2 fatores de estudo: concentração de HF em 4 níveis, isto é, 1% (HF1), 3% (HF3), 5% (HF5), e 10% (HF10), e armazenamento em 2 níveis, isto é, condição inicial (testes foram realizados 24 h após a cimentação), e envelhecidos (150 dias de armazenamento + 12.000 ciclos térmicos a 5°C e 55°C). Condicionamento ácido (20 s) foi realizado, seguido por lavagem, secagem e silanização. Quatro cilindros de cimento resinoso (ϕ= 0.96 mm) foram construídos a partir de matrizes de amido em cada amostra cerâmica (n= 40). Amostras cerâmicas adicionais foram condicionadas e analisadas quanto ao ângulo de contato, micro-morfologia e rugosidade. Na condição inicial (sem envelhecimento), os grupos HF3, HF5, e HF10 mostraram valores de resistência de união similares (13.9 - 15.9 MPa), e HF1 apresentou valores menores que HF5, sendo estatisticamente diferente (p= 0.012). Após o envelhecimento, todas as médias de resistência de união diminuíram estatisticamente, sendo que HF3, HF5 e HF10 foram similares e maiores que HF1 (p= 0.0001). Para o ângulo de contato, HF3, HF5 e HF10 apresentaram valores similares (7.8 - 10.4°), menores que os grupos HF1 e CTRL. HF5 e HF10 apresentaram superfícies mais rugosas que as outras condições. Para melhores resultados de resistência de união, a cerâmica testada pode ser condicionada com ácido fluorídrico nas concentrações de 3%, 5% e 10%.

Effectiveness of Dioscorea hispida Dennst as antibacterial and antibiofilm agent

Aims@#Antimicrobial resistance (AMR) is a growing threat to public health, where treatments using conventional drugs are becoming ineffective. One viable but underexplored alternative is through the use of Dioscorea hispida, a wild plant that exhibits antimicrobial properties. This study aims to explore D. hispida effectiveness as an antibacterial and antibiofilm agent against selected pathogenic and non-pathogenic bacteria. @*Methodology and results@#Different concentrations of D. hispida crude extracts (0 – 2.5 mg/mL) were tested against the growth of planktonic bacterial cells over 24 h incubation, and the half maximal effective concentration (EC50) obtained was used in the antibiofilm test over 24 and 48 h. All bacteria treated with D. hispida showed significant (P<0.05) reduction in planktonic cell and biofilm densities against the negative control starting at 0.3 mg/mL. However, in comparison to the antibiotic, only certain bacteria were significantly affected by D. hispida, implying the plant has a ‘moderate’ biocidal activity in general. Furthermore, Atomic Force Microscopy imaging of S. aureus biofilm with D. hispida revealed increased height and width of cell clusters despite reduction in volume compared to the negative control, suggesting unique biofilm resistance behaviour against the plant. @*Conclusion, significance and impact of study@#This study demonstrated D. hispida capability as a natural antimicrobial and antibiofilm agent. The plant could complement current antimicrobials to maximise killing efficiency and minimise occurrences of resistance. Unique biofilm behaviour against D. hispida also warrants further investigation on the effect of biocides towards biofilm structure. Overall, this research provides new insights into a traditional plant-based antimicrobial activity in combating infectious diseases and AMR.

The effect of root canal preparation on the surface roughness of WaveOne and WaveOne Gold files: atomic force microscopy study

OBJECTIVES: To examine the surface topography of intact WaveOne (WO; Dentsply Sirona Endodontics) and WaveOne Gold (WOG; Dentsply Sirona Endodontics) nickel-titanium rotary files and to evaluate the presence of alterations to the surface topography after root canal preparations of severely curved root canals in molar teeth. MATERIALS AND METHODS: Forty-eight severely curved canals of extracted molar teeth were divided into 2 groups (n = 24/each group). In group 1, the canals were prepared using WO and in group 2, the canals were prepared using WOG files. After the preparation of 3 root canals, instruments were subjected to atomic force microscopy analysis. Average roughness and root mean square values were chosen to investigate the surface features of endodontic files. The data was analyzed using one-way analysis of variance and post hoc Tamhane's tests at 5% significant level. RESULTS: The surface roughness values of WO and WOG files significantly changed after use in root canals (p < 0.05). The used WOG files exhibited higher surface roughness change when compared with the used WO files (p < 0.05). CONCLUSIONS: Using WO and WOG Primary files in 3 root canals affected the surface topography of the files. After being used in root canals, the WOG files showed a higher level of surface porosity value than the WO files.

Application of atomic force microscopy-based single molecule force spectroscopy in G-quadruplex studies / 南方医科大学学报

Telomere plays a crucial role in the physiological and pathological processes of cells. At the end of the telomere, the single-stranded DNA repeat sequence rich in guanine (G) folds in the presence of monovalent metal ions such as Na or K to form a G-quadruplex structure. This structure can not be extended by telomerase and inhibits the activity of telomerase, thus becoming a potential anticancer target. Stabilizing the formation of DNA G-quadruplex structures by small molecule ligands has become a new strategy for designing many anticancer drugs, and studying the interaction strength of these small molecule ligands with G-quadruplex is thus of particular importance for screening highly effective anticancer drugs. Single molecule force spectroscopy enables direct measurement of the interaction between small molecule ligands and G-quadruplexes. This review highlights the advances of single-molecule force spectroscopy based on atomic force microscopy in the study of the G quadruplex structure and its interaction with small molecule ligands, and summarizes the application and development trend of single molecule force spectrum technology in G quadruplex.

Progress in the applications of high-speed atomic force microscopy in cell biology / 南方医科大学学报

Without losing its high resolution, high-speed atomic force microscope (HS-AFM) represents a perfect combinationof scanning speed and precision and allows real-time and observation of the dynamic processes in a biological system atboth the cellular and molecular levels. By combining the extremely high temporal resolution with the spatial resolution andcoupling with other advanced technologies, HS-AFM shows promising prospects for applications in life sciences such as cellbiology. In this review, we summarize the latest progress of HS-AFM in the field of cell biology, and discuss the impact ofenvironmental factors on conformation dynamics of DNA, the binding processes between DNA and protein, the domainchanges of membrane proteins, motility of myosin, and surface structure changes of living cells.

Mechanical Alteration Associated With Chemotherapeutic Resistance of Breast Cancer Cells

BACKGROUND: The mechanical deformability of cancer cells has attracted particular attention as an emerging biomarker for the prediction of anti-cancer drug sensitivity. Nevertheless, it has not been possible to establish a general rubric for the identification of drug susceptibility in breast cancer cells from a mechanical perspective. In the present study, we investigated the mechanical alteration associated with resistance to adjuvant therapy in breast cancer cells. METHODS: We performed an ‘atomic force microscopy (AFM)-based nanomechanical study’ on ‘drug-sensitive (MCF-7)’ and ‘drug-resistant (MCF-7/ADR)’ breast cancer cells. We also conducted cell viability tests to evaluate the difference in doxorubicin responsiveness between two breast cancer cell lines. We carried out a wound closure experiment to investigate the motility changes associated with chemotherapeutic resistance. To elucidate the changes in molecular alteration that accompany chemotherapeutic resistance, we investigated the expression of vinculin and integrin-linked kinase-1–which are proteins involved in substrate adhesion and the actin cytoskeleton–using Western blotting analysis. RESULTS: A MTT assay confirmed that the dose-dependent efficacy of doxorubicin was reduced in MCF-7/ADR cells compared to that in MCF-7 cells. The wound assay revealed enhanced two-dimensional motility in the MCF-7/ADR cells. The AFM mechanical assay showed evidence that the drug-resistant breast cancer cells exhibited a significant decrease in mechanical deformability compared to their drug-sensitive counterparts. The mechanical alteration in the MCF-7/ADR cells was accompanied by upregulated vinculin expression. CONCLUSIONS: The obtained results manifestly showed that the altered mechanical signatures–including mechanical deformability and motility–were closely related with drug resistance in the breast cancer cells. We believe that this investigation has improved our understanding of the chemotherapeutic susceptibility of breast cancer cells.

Impacto de um método de polimento adicional em propriedades físicas, micromorfológicas e microtopográficas de compósitos convencionais e Bulk Fill / Impact of an additional polishing method on physical, micromorphological and microtopographical properties of conventional and Bulk Fill composites

O objetivo deste trabalho foi avaliar a rugosidade superficial, a molhabilidade, a disposição em profundidade das partículas de carga, o mapeamento de elementos químicos, a microtopografia em 3D e a micromorfologia de compósitos convencionais e bulk fill após polimento adicional. Éspécimes foram preparados de cada compósito testado, sendo quatro do tipo bulk fill (Filtek Bulk, Fill Tetric N-Ceram Bulk Fill, Opus Bulk Fill e X-tra Fil) e quatro convencionais (Filtek Z250 XT, Grandioso, Tetric NCeram, Vittra APS), de acordo com três técnicas de acabamento/polimento/polimento adicional (n = 10): sem acabamento e polimento, acabamento e polimento com borrachas abrasivas (Astropol), acabamento e polimento com Astropol mais polimento adicional com escova de carbeto de silício. A rugosidade superficial (Ra) e o ângulo de contato foram medidos usando-se um perfilômetro e goniômetro adaptado, respectivamente. A microtopografia 3D foi avaliada utilizando microscopia de força atômica (MFA), enquanto a micromorfologia e a disposição em profundidade das partículas de carga, através da microscopia eletrônica de varredura (MEV). O mapeamento de elementos químicos foi avaliado por meio de Espectroscopia por energia dispersiva de raio-X (EDS). A rugosidade e o ângulo de contato foram analisados pelo ANOVA-dois fatores e teste de Tukey (p <0,05); os demais dados foram analisados descritivamente. A disposição das partículas de carga em profundidade de todas as resinas envolvidas neste estudo apresentou uma camada superficial rica em matriz orgânica e uma camada subsuperficial rica em partículas de dimensões mais diminutas. O polimento adicional: diminuiu a rugosidade superficial das resinas Filtek Bulk Fill, Vittra APS, Tetric N-ceram Bulk Fill e X-tra fil; aumentou o valor do ângulo de contato da X-tra Fil e diminuiu da Filtek Z250 XT. Nas análises para microtopografia em 3D e a micromorfologia, superfícies mais lisas e uniformes foram observadas em todas as resinas. Os elementos: carbono (C), cxigênio (O), silício (Si), zircônia (Zr) e alumínio (Al) foram presentes em todas as resinas compostas. O bário (Ba) foi ausente na Filtek Z250 XT, Filtek Bulk Fil e Vittra APS. O carbono foi predominante em todas as resinas. Após polimento adicional, houve um aumento na detecção de oxigênio para todas as resinas, exceto para Tetric N-Ceram e Xtra Fil e uma diminuição de carbono, exceto para a Tetric N-Ceram Bulk Fil. O silício diminuiu apenas nas resinas Z250 XT, Tetric N-Ceram e Tetric N-Ceram Bulk Fill. A zircônia diminuiu para a Tetric N-Ceram Bulk Fill e o alumínio para Z250 XT e Tetric N-Ceram Bulk Fill. O bário aumentou para Opus Bulk fill e X-tra Fil. O titânio foi ausente para todas as resinas. Portanto, o polimento adicional melhorou as propriedades de superfície das resinas estudadas (AU).

The objective of this study was to evaluate the surface roughness, wettability, the depth distribution of the charge particles, the mapping of chemical elements, the 3D microtopography and the micromorphology of the composites of the conventional and bulk fill after additional polishing. The specimens were prepared from each of the composites tested, four of them being bulk fillers (Filtek Bulk Fill Tetric N-Ceram Bulk Fill Opus Bulk Fill X-tra Fil) and four conventional ones (Filtek Z250 XT, Grandioso, Tetric N-Ceram, Vittra APS ), according to three additional finishing / polishing / polishing techniques (n = 10): without finishing and polishing, finishing and polishing with abrasive rubbers (Astropol), finishing and polishing with Astropol plus additional polishing with silicon carbide brush. The surface roughness (Ra) and contact angles were measured using a profilometer and adapted goniometer, respectively. The 3D microtopography was evaluated using atomic force microscopy (AFM); while the micromorphology and the in-depth arrangement of the charge particles by scanning electron microscopy (SEM). The mapping of chemical elements was evaluated by means of X-ray Dispersive Energy Spectroscopy (EDS). The roughness and the contact angle were analyzed by ANOVA- two factors and Tukey test (p <0.05); the other data were analyzed descriptively. The arrangement of the in-depth charge particles of all the resins involved in this study had an organic matrix rich surface layer and a particulate rich subsurface layer of smaller dimensions. Addicional polishing: reduced surface roughness of Filtek Bulk Fill resins, Vittra APS, Tetric N-ceram Bulk Fill and X-trafil resins; increased the contact angle value of the X-tra Fil and decreased the Filtek Z250 XT. In the analyzes for 3D microtopography and micromorphology, smoother and more uniform surfaces were observed in all a resins. The elements: carbon (C), oxygen (O), silicon (Si), zirconia (Zr) and aluminum (Al) were present in all composite resins. Barium (Ba) was absent on Filtek Z250 XT, Filtek Bulk Fil and Vittra APS. Carbon was predominant in all resins. After additional polishing, there was an increase in oxygen detection for all resins except for Tetric N-Ceram and X-tra Fil and a decrease in carbon except for Tetric N-Ceram Bulk Fil. Silicon decreased only in the Z250 XT, Tetric N-Ceram and Tetric NCeram Bulk Fill resins. Zirconia decreased for Tetric N-Ceram Bulk Fill and aluminum for Z250 XT and Tetric N-Ceram Bulk Fill. Barium increased for Opus Bulk fill and X-tra Fil. Titanium was absent for all resins. Therefore, additional polishing improved the surface properties of the resins studied (AU).

The In Situ Effect of Titanium Tetrafluoride Gel on Erosion/Abrasion Progression in Human Dentin

Abstract Erosion incidence is increasing and its control is still a challenge in clinical practice. This study evaluated 4% TiF4-gel effects on eroded human dentin subjected to in situ erosive/abrasive episodes. Seventy-two previously eroded dentin slabs (0.05 M citric acid, pH 2.3, 20 min) were allocated to 6 groups (n=12) according to the treatment to be performed during the in situ phase and number of erosive/abrasive cycles, as follows: 4% TiF4-gel applied once (TiF41), twice (TiF42) or three times (TiF43) followed by 1, 2 and 3 erosive/abrasive cycles, respectively. Gel was applied before the beginning of the next cycle. Control groups were subjected to 1 (C1), 2 (C2) and 3 (C3) erosive/abrasive cycles only. A seventh group (n=12) comprised in vitro uneroded samples (UN) subjected to 3 erosive/abrasive cycles. Each cycle corresponded to 2 days of erosive (citric acid 0.5%, pH 2.6, 6x/day) and abrasive (electric toothbrush, 10 s/sample, 1 x/day) challenges. Samples were evaluated under profilometry and environmental scanning electron microscopy (ESEM). Atomic force microscopy images (AFM) were also made (n=3). Repeated measures 2-way ANOVA and Tukey test (p<0.001) showed that TiF42, which did not differ from TiF41 and TiF43, revealed a significant reduction in surface loss compared to all control groups. TiF41 and TiF43 showed no significant difference from C1, but both groups demonstrated significantly smaller surface loss than C2 and C3. ESEM and AFM micrographs suggested alterations on treated surfaces compared to samples from control groups, showing reduced diameters of dentinal tubules lumens. Therefore, TiF4 was able to reduce the progression of erosive/abrasive lesions.

Resumo A incidência da erosão tem aumentado e o seu controle ainda é um desafio na prática clínica. Este estudo avaliou os efeitos do gel de TiF4 a 4% sobre a dentina humana erodida submetida a episódios erosivos/abrasivos in situ. Setenta e dois fragmentos de dentina previamente erodida (ácido cítrico 0,05 M, pH 2,3, 20 min) foram distribuídas em 6 grupos (n=12) de acordo com o tratamento a ser realizado durante a fase in situ e o número de ciclos erosivos/abrasivos, como descrito a seguir: gel de TiF4 a 4% aplicado uma (TiF41), duas (TiF42) ou três vezes (TiF43) seguido de 1, 2 e 3 ciclos erosivos/abrasivos, respectivamente. As aplicações dos géis foram realizadas antes do início do ciclo erosivo seguinte. Grupos controle foram submetidos a 1 (C1), 2 (C2) e 3 (C3) ciclos erosivos/abrasivos apenas. Um sétimo grupo (n=12) compreendia amostras sem erosão in vitro (UN) submetidas a 3 ciclos erosivos/abrasivos. Cada ciclo correspondia a 2 dias de desafios erosivos (ácido cítrico a 0,5%, pH 2,6, 6x/dia) e abrasivos (escova de dentes elétrica, 10 s/amostra, 1x/dia). As amostras foram avaliadas em perfilômetro e Microscopia Eletrônica de Varredura Ambiental (MEV). Imagens de microscopia de força atômica (AFM) também foram capturadas (n=3). ANOVA a 2-fatores para medidas repetidas e o teste de Tukey (p<0,001) demonstraram que TiF42, que não diferiu do TiF41 e TiF43, revelou redução significativa na perda de superfície quando comparado a todos os grupos controle. TiF41 e TiF43 não apresentaram diferença estatisticamente significativa em relação ao C1, mas ambos os grupos demonstraram perda de superfície significativamente menor que C2 e C3. Micrografias de MEV e AFM sugeriram alterações nas superfícies tratadas quando comparadas a amostras dos grupos controle, apresentando redução no diâmetro das luzes dos túbulos dentinários. Portanto, o TiF4 foi capaz de reduzir a progressão das lesões erosivas/abrasivas.

Differentiation Potential of Mesenchymal Stem Cells Is Related to Their Intrinsic Mechanical Properties / 대한배뇨장애요실금학회지

PURPOSE: The differentiation properties of stem cells are not yet fully understood due to their close association with multiple environmental and extrinsic factors. This study investigates the differentiation properties of mesenchymal stem cells (MSCs) and correlates them with their intrinsic mechanical properties. METHODS: A total of 3 different types of MSCs, namely bone marrow-derived MSCs (BMSCs), umbilical cord-derived MSCs (UCSCs), and adipose-derived MSCs (ADSCs) were evaluated. These 3 MSCs were individually differentiated into adipocytes and osteoblasts for 3 weeks. The mechanical properties of the MSCs and differentiated cells were determined by atomic force microscopy. RESULTS: ADSCs showed the greatest ability to differentiate into adipocytes, followed by BMSCs and UCSCs. While UCSCs differentiated readily into osteoblasts, BMSCs and ADSCs were less likely to undergo this differentiation. UCSCs were the “hardest” cells, while ADSCs were the “softest.” The cells differentiated from “hard” MSCs were stiffer than the cells differentiated from “soft” MSCs, irrespective of lineage specification. CONCLUSIONS: The differentiation ability of MSCs and the mechanical properties of the differentiated cells were closely linked. However, there were no significant correlations regarding changes in the mechanical properties between the nuclear region and the cytoplasm during differentiation.

Application of atomic force microscopy (AFM) to study bacterial biofilms / 生物工程学报

Because of the nanometre resolution, piconewton force sensitivity, label-free technique and the ability to operate in liquid environments, atomic force microscopy (AFM) has emerged as a powerful tool to explore the biofilm development processes. AFM provides three-dimensional topography and structural details of biofilm surfaces under in-situ conditions. It also helps to generate key information on the mechanical properties of biofilm surfaces, such as elasticity and stickiness. Additionally, single-molecule and single-cell force spectroscopies can be applied to measure the strength of adhesion, attraction, and repulsion forces between cell-solid and cell-cell surfaces. This paper outlined the basic principle of AFM technique and introduced recent advances in the application of AFM for the investigation of ultra-morphological, mechanical and interactive properties of biofilms. Furthermore, the existing problems and future prospects were discussed.