Effects of postpolymerization time and temperature on the flexural properties and hardness profile of three-dimensional printed provisional resin.

Background/purpose: Three-dimensional (3D) printing technique was widely used for provisional restorations in clinical use. However, the effects of post-polymerization temperature and time on the flexural properties and hardness profile were not fully elucidated yet. The purpose of this study is to investigate the effects of post-polymerization temperature and time on the flexural properties and hardness profile of the provisional restoration. Materials and methods: 3D-printing provisional resin was printed and post-polymerized at various temperatures (room temperature, 40 °C, 60 °C and 80 °C) and periods (0, 15, 30, 60, 90 and 120 min of photopolymerization). Afterwards, the flexural strength, flexural modulus, surface hardness, and internal hardness at different depth were evaluated. Results: The group post-polymerized without concurrent heating had significantly shallow depth of cure comparing to the heating counterparts. The surface hardness of the groups post-polymerized at different temperatures did not show any difference. All groups with post-polymerization temperature at 40 °C, 60 °C and 80 °C and post-polymerization time ranged between 15 and 90 min, had curing depth between 3 and 4 mm. Group post-polymerized without concurrent heating has significantly shallow depth of cure comparing to the heating counterparts. Conclusion: Post-polymerization at an elevated temperature, preferably 60 °C, is suggested. The wall thickness of the 3D-printing provisional prosthesis thinner than 3-4 mm is recommended.

Synthesis of Silanized Bioactive Glass/Gelatin Methacrylate (GelMA/Si-BG) composite hydrogel for Bone Tissue Engineering Application.

Bioactive glass (BG) has been widely employed in the field of bone tissue engineering owing to its osteoconductive properties. These properties increase the stiffness and bioactivity of polymeric hydrogels, making them ideal for the repair, replacement, and regeneration of damaged bones. In this study, we investigated the effects of incorporating silanized 45S5 bioactive glass (Si-BG) into gelatin methacrylate (GelMA) hydrogel (GelMA/Si-BG) for potential bone tissue engineering. Our findings revealed that crosslinking GelMA with Si-BG had a striking increase in bioactivity with and without osteogenic induction of human mesenchymal stem cells (hMSCs) when compared to GelMA/BG hydrogels. Meanwhile, both GelMA/Si-BG and GelMA/BG hydrogels were able to maintain the cell viability of hMSC for up to 14 days. Additionally, GelMA/Si-BG hydrogels were shown to have a significantly higher compressive modulus than GelMA/BG hydrogels. This study has demonstrated the introduction of silanized 45S5 BG into GelMA hydrogel bioactivity and mechanical properties of GelMA hydrogels, exemplifying the potential application of silanization of BG in bone tissue engineering.

Automatic recognition of teeth and periodontal bone loss measurement in digital radiographs using deep-learning artificial intelligence.

Background/purpose: Artificial Intelligence (AI) can optimize treatment approaches in dental healthcare due to its high level of accuracy and wide range of applications. This study seeks to propose a new deep learning (DL) ensemble model based on deep Convolutional Neural Network (CNN) algorithms to predict tooth position, detect shape, detect remaining interproximal bone level, and detect radiographic bone loss (RBL) using periapical and bitewing radiographs. Materials and methods: 270 patients from January 2015 to December 2020, and all images were deidentified without private information for this study. A total of 8000 periapical radiographs with 27,964 teeth were included for our model. AI algorithms utilizing the YOLOv5 model and VIA labeling platform, including VGG-16 and U-Net architecture, were created as a novel ensemble model. Results of AI analysis were compared with clinicians' assessments. Results: DL-trained ensemble model accuracy was approximately 90% for periapical radiographs. Accuracy for tooth position detection was 88.8%, tooth shape detection 86.3%, periodontal bone level detection 92.61% and radiographic bone loss detection 97.0%. AI models were superior to mean accuracy values from 76% to 78% when detection was performed by dentists. Conclusion: The proposed DL-trained ensemble model provides a critical cornerstone for radiographic detection and a valuable adjunct to periodontal diagnosis. High accuracy and reliability indicate model's strong potential to enhance clinical professional performance and build more efficient dental health services.

Biofabrication of Cell-Laden Gelatin Methacryloyl Hydrogels with Incorporation of Silanized Hydroxyapatite by Visible Light Projection.

Gelatin methacryloyl (GelMA) hydrogel is a photopolymerizable biomaterial widely used for three-dimensional (3D) cell culture due to its high biocompatibility. However, the drawback of GelMA hydrogel is its poor mechanical properties, which may compromise the feasibility of biofabrication techniques. In this study, a cell-laden GelMA composite hydrogel with a combination incorporating silanized hydroxyapatite (Si-HAp) and a simple and harmless visible light crosslinking system for this hydrogel were developed. The incorporation of Si-HAp into the GelMA hydrogel enhanced the mechanical properties of the composite hydrogel. Moreover, the composite hydrogel exhibited low cytotoxicity and promoted the osteogenic gene expression of embedded MG63 cells and Human bone marrow mesenchymal stem cells (hBMSCs). We also established a maskless lithographic method to fabricate a defined 3D structure under visible light by using a digital light processing projector, and the incorporation of Si-HAp increased the resolution of photolithographic hydrogels. The GelMA-Si-HAp composite hydrogel system can serve as an effective biomaterial in bone regeneration.

Comparison of flexural properties and cytotoxicity of interim materials printed from mono-LCD and DLP 3D printers.

STATEMENT OF PROBLEM: Because few 3D-printing resins have been specifically developed for liquid crystal display (LCD) 3D printers, mono-LCD users may use digital light processing (DLP) 3D-printing resins. However, the suitability of these resins requires evaluation. PURPOSE: The purpose of this in vitro study was to evaluate whether 3D-printing resins designed for DLP 3D printers can be used successfully in a mono-LCD 3D printer. MATERIAL AND METHODS: 3D printers based on photopolymerization and 3D-printing resin for interim restorations were used in this study. Enlighten AA temp and NextDent C&B MFH were printed from both the MiiCraft Ultra 125 and Phrozen Sonic printers, followed by postpolymerization by using the FormCure and PhrozenCure units for different times. The flexural strength and cytotoxicity of the specimens were evaluated. RESULTS: After postpolymerization treatment, the flexural strength of Enlighten AA temp and NextDent C&B MFH from both 3D printers was over the 50-MPa minimal requirement for the flexural strength of interim resins specified in the International Standards Organization (ISO) 10477 standard. With 15 minutes of FormCure and 1 minute of PhrozenCure postpolymerization, 4 material-printer combinations reached nearly 100% in cell viability. CONCLUSIONS: Interim resins designed for DLP 3D printers can be successfully used in mono-LCD 3D printers if the printed specimens are postpolymerized in a more powerful postpolymerization unit or in a less powerful postpolymerization unit for a longer time.

Synthesis and Formulation of PCL-Based Urethane Acrylates for DLP 3D Printers.

In this study, three PCL-based polyurethane acrylates were synthesized and further formulated into twelve resins for digital light processing (DLP) 3D printing. Three PCL diols with different molecular weights were synthesized via ring-opening reaction of ε-caprolactone on diethylene glycol, with the catalyst stannous octoate. Isophorone diisocyanate (IPDI) was reacted with 2-hydroxyethyl acrylate (2-HEA) and the PCL diols form PCL-based polyurethane acrylates. Twelve resins composed of different percentages of PCL-based polyurethane acrylates, poly (ethylene glycol) diacrylate (PEGDA), propylene glycol (PPG) and photo-initiator were further printed from a DLP 3D printer. The viscosities of twelve resins decreased by 10 times and became printable after adding 30% of PEGDA. The degree of conversion for the twelve resins can reach more than 80% after the post-curing process. By changing the amount of PEGDA and PPG, the mechanical properties of the twelve resins could be adjusted. PUA530-PEG-PPG (70:30:0), PUA800-PEG-PPG (70:30:0), and PUA1000-PEG-PPG (70:30:0) were successfully printed into customized tissue scaffolds. Twelve PCL-based polyurethane photo-curable resins with tunable mechanical properties, cytotoxicity, and degradability were successfully prepared. With the DLP 3D printing technique, a complex structure could be achieved. These resins have great potential for customized tissue engineering and other biomedical application.

Mechanical properties, accuracy, and cytotoxicity of UV-polymerized 3D printing resins composed of Bis-EMA, UDMA, and TEGDMA.

STATEMENT OF PROBLEM: Three-dimensional printing has the potential for clinical applications, and additive manufacturing materials for dental use merit further investigation. PURPOSE: The purpose of this in vitro study was to evaluate the properties of materials formulated with ethoxylated bisphenol A-dimethacrylate (Bis-EMA), urethane dimethacrylate (UDMA), and triethylene glycol dimethacrylate (TEGDMA) as 3D printing resins for ultraviolet digital light processing (UV-DLP) 3D printers and to characterize the mechanical and biological properties and accuracy of the printed objects. MATERIAL AND METHODS: Ten different light-polymerized resins were formulated using Bis-EMA, UDMA, and TEGDMA. Their viscosities were measured, and only 7 resins with viscosities lower than 1500 centipoise (cP) were selected for 3D printing and further material characterization. The light-polymerized resins were printed into representative shapes using a custom-made 3D printer equipped with a 405-nm UV-DLP projector as the light source. The printed specimens were subjected to biologic, mechanical, and accuracy tests, and the data were submitted to 1-way ANOVA and Tukey post hoc comparisons (α=.05). RESULTS: Photopolymerizable resins made of Bis-EMA, UDMA, and TEGDMA were successfully formulated for 3D printing to fabricate objects of various shapes and sizes. TEGDMA served as the diluent to reduce the viscosity and increase the degree of conversion, while UDMA and Bis-EMA provided strength as demonstrated by the mechanical testing. All the printed objects passed cytotoxicity testing. The flexural strengths of the printed specimens ranged between 60 MPa and 90 MPa; flexural modulus ranged between 1.7 GPa and 2.1 GPa; and surface hardness ranged between 14.5 HV and 24.6 HV. These represent similar mechanical properties to those of currently used clinical resin materials. In the accuracy test, the resin mixture composed of 80% Bis-EMA, 10% UDMA, and 10% TEGDMA had the highest accuracy, with a 0.051-mm deviation from the original design. CONCLUSIONS: Bis-EMA, UDMA, and TEGDMA are good candidates for the formulation of 3D printing resins for dental use. The printed objects demonstrated favorable biological and mechanical properties. Further, the accuracy of the printed specimens showed potential for clinical application.

Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells.

For stem cell differentiation, the microenvironment can play an important role, and hydrogels can provide a three-dimensional microenvironment to allow native cell growth in vitro. A challenge is that the stem cell's differentiation can be influenced by the matrix stiffness. We demonstrate a low-toxicity method to create different stiffness matrices, by using a photopolymerizable gelatin methacrylate (GelMA) hydrogel cross-linked by blue light (440 nm). The stiffness and porosity of GelMA hydrogel is easily modified by altering its concentration. We used human bone marrow mesenchymal stem cells (MSCs) as a cell source and cultured the GelMA-encapsulated cells with EGM-2 medium to induce endothelial differentiation. In our GelMA blue light hydrogel system, we found that MSCs can be differentiated into both endothelial-like and osteogenic-like cells. The mRNA expressions of endothelial cell markers CD31, von Willebrand factor, vascular endothelial growth factor receptor-2, and CD34 were significantly increased in softer GelMA hydrogels (7.5% and 10%) compared with stiffer matrices (15% GelMA). On the other hand, the enhancements of osteogenic markers mRNA expressions (Alkaline phosphatase (ALP), Runx2, osteocalcin, and osteopontin) were highest in 10% GelMA. We also found that 10% GelMA hydrogel offered optimal conditions for MSCs to form capillary-like structures. These results suggest that the mechanical properties of the GelMA hydrogel can influence both endothelial and osteogenic differentiation of MSCs and sequent capillary-like formation.

Synthesis, Characterization, and Visible Light Curing Capacity of Polycaprolactone Acrylate.

Polycaprolactone (PCL) is drawing increasing attention in the field of medical 3D printing and tissue engineering because of its biodegradability. This study developed polycaprolactone prepolymers that can be cured using visible light. Three PCL acrylates were synthesized: polycaprolactone-530 diacrylate (PCL530DA), glycerol-3 caprolactone triacrylate (Glycerol-3CL-TA), and glycerol-6 caprolactone triacrylate (Glycerol-6CL-TA). PCL530DA has two acrylates, whereas Glycerol-3CL-TA and Glycerol-6CL-TA have three acrylates. The Fourier transform infrared and nuclear magnetic resonance spectra suggested successful synthesis of all PCL acrylates. All are liquid at room temperature and can be photopolymerized into a transparent solid after exposure to 470 nm blue LED light using 1% camphorquinone as photoinitiator and 2% dimethylaminoethyl methacrylate as coinitiator. The degree of conversion for all PCL acrylates can reach more than 80% after 1 min of curing. The compressive modulus of PCL530DA, Glycerol-3CL-TA, and Glycerol-6CL-TA is 65.7 ± 12.7, 80.9 ± 6.1, and 32.1 ± 4.1 MPa, respectively, and their compressive strength is 5.3 ± 0.29, 8.3 ± 0.18, and 3.0 ± 0.53 MPa, respectively. Thus, all PCL acrylates synthesized in this study can be photopolymerized and because of their solid structure and low viscosity, they are applicable to soft tissue engineering and medical 3D printing.

Synthesis and Characterization of Polycaprolactone-Based Polyurethanes for the Fabrication of Elastic Guided Bone Regeneration Membrane.

The aim of this research is to synthesize polycaprolactone-based polyurethanes (PCL-based PUs) that can be further used for the fabrication of guided bone regeneration (GBR) membranes with higher tensile strength and elongation at break than collagen and PTFE membranes. The PCL-based PUs were prepared by the polymerization of polycaprolactone (PCL) diol with 1,6-hexamethylene diisocyanate (HDI) at different ratios using either polyethylene glycol (PEG) or ethylenediamine (EDA) as chain extenders. The chemical, mechanical, and thermal properties of the synthesized polymers were determined using NMR, FTIR, GPC, DSC, and tensile tester. The PCL and polyurethanes were fabricated as nanofiber membranes by electrospinning, and their mechanical properties and SEM morphology were also investigated. In vitro tests, including WST-1 assay, SEM of cells, and phalloidin cytoskeleton staining, were also performed. It was shown that electrospun membranes made of PCL and PCL-HDI-PEG (2 : 3 : 1) possessed tensile strength of 19.84 MPa and 11.72 MPa and elongation at break of 627% and 362%, respectively. These numbers are equivalent or higher than most of the commercially available collagen and PTFE membrane. As a result, these membranes may have potential for future GBR applications.

Dental implant customization using numerical optimization design and 3-dimensional printing fabrication of zirconia ceramic.

This study proposes a new methodology for dental implant customization consisting of numerical geometric optimization and 3-dimensional printing fabrication of zirconia ceramic. In the numerical modeling, exogenous factors for implant shape include the thread pitch, thread depth, maximal diameter of implant neck, and body size. Endogenous factors are bone density, cortical bone thickness, and non-osseointegration. An integration procedure, including uniform design method, Kriging interpolation and genetic algorithm, is applied to optimize the geometry of dental implants. The threshold of minimal micromotion for optimization evaluation was 100 µm. The optimized model is imported to the 3-dimensional slurry printer to fabricate the zirconia green body (powder is bonded by polymer weakly) of the implant. The sintered implant is obtained using a 2-stage sintering process. Twelve models are constructed according to uniform design method and simulated the micromotion behavior using finite element modeling. The result of uniform design models yields a set of exogenous factors that can provide the minimal micromotion (30.61 µm), as a suitable model. Kriging interpolation and genetic algorithm modified the exogenous factor of the suitable model, resulting in 27.11 µm as an optimization model. Experimental results show that the 3-dimensional slurry printer successfully fabricated the green body of the optimization model, but the accuracy of sintered part still needs to be improved. In addition, the scanning electron microscopy morphology is a stabilized t-phase microstructure, and the average compressive strength of the sintered part is 632.1 MPa.

Differential left-to-right atria gene expression ratio in human sinus rhythm and atrial fibrillation: Implications for arrhythmogenesis and thrombogenesis.

BACKGROUND: Atrial fibrillation (AF) causes atrial remodeling, and the left atrium (LA) is the favored substrate for maintaining AF. It remains unclear if AF remodels both atria differently and contributes to LA arrhythmogenesis and thrombogenesis. Therefore, we wished to characterize the transcript profiles in the LA and right atrium (RA) in sinus rhythm (SR) and AF respectively. METHODS: Paired LA and RA appendages acquired from patients receiving cardiac surgery were used for ion-channel- and whole-exome-based transcriptome analysis. The ultrastructure was evaluated by immunohistochemistry. RESULTS: Twenty-two and twenty ion-channels and transporters were differentially expressed between the LA and RA in AF and SR, respectively. Among these, 15 genes were differentially expressed in parallel between AF and SR. AF was associated with increased LA/RA expression ratio in 9 ion channel-related genes, including genes related to calcium handling. In microarray, AF was associated with a differential LA/RA gene expression ratio in 309 genes, and was involved in atherosclerosis-related signaling. AF was associated with the upregulation of thrombogenesis-related genes in the LA appendage, including P2Y12, CD 36 and ApoE. Immunohistochemistry showed higher expressions of collagen-1, oxidative stress and TGF-ß1 in the RA compared to the LA. CONCLUSIONS: AF was associated with differential LA-to-RA gene expression related to specific ion channels and pathways as well as upregulation of thrombogenesis-related genes in the LA appendage. Targeting the molecular mechanisms underlying the LA-to-RA difference and AF-related remodeling in the LA appendage may help provide new therapeutic options in treating AF and preventing thromboembolism in AF.

Antioxidant N-Acetylcysteine and Glutathione Increase the Viability and Proliferation of MG63 Cells Encapsulated in the Gelatin Methacrylate/VA-086/Blue Light Hydrogel System.

Photoencapsulation of cells inside a hydrogel system can provide a suitable path to establish a gel in situ for soft tissue regeneration applications. However, the presence of photoinitiators and blue or UV light irradiation can result in cell damage and an increase of reactive oxygen species. We here evaluate the benefits of an antioxidant pretreatment on the photoencapsulated cells. We study this by evaluating proliferation and viability of MG63 cells, which we combined with a gelatin methacrylate (GelMA) hydrogel system, using the photoinitiator, VA-086, cured with 440 nm blue light. We found that blue light irradiation as well as the presence of 1% VA-086 reduced MG63 cell proliferation rates. Adding a short pretreatment step to the MG63 cells, consisting of the antioxidant molecules N-acetylcysteine (NAC) and reduced glutathione (GSH), and optimizing the GelMA encapsulation steps, we found that both NAC and GSH pretreatments of MG63 cells significantly increased both proliferation and viability of the cells, when using a 15% GelMA hydrogel, 1% VA-086, and 1-min blue light exposure. These findings suggest that the use of antioxidant pretreatment can counteract the negative presence of the photoinitiators and blue light exposure and result in a suitable environment for photoencapsulating cells in situ for tissue engineering and soft tissue applications.

Proinflammatory gene expression in patients undergoing mitral valve surgery and maze ablation for atrial fibrillation.

OBJECTIVE: It is difficult to achieve rhythm control in patients with long-standing persistent atrial fibrillation (AF). The radiofrequency maze procedure is an effective means in curing AF with a variable recurrence rate depending on patient characteristics and AF duration. In these patients, the characteristics of the atrial substrate have not been well investigated. Because the inflammatory process has been shown to be important in the pathogenesis of AF, we sought to characterize the proinflammatory gene expression in left atria obtained from patients with AF undergoing mitral valve surgery combined with the maze procedure to distinguish the changes associated with AF and its recurrence after the surgical ablation. METHODS: Left atrial appendages from 35 patients receiving mitral valve surgery were used for study. Ten patients had sinus rhythm (SR) and 25 patients had persistent AF for more than 1 year and underwent the maze procedure. Among the AF patients, 13 patients remained in SR (AF-SR) and 12 patients had recurrent AF during the 1-year clinical follow-up (AF-AF). The nCounter Human Inflammation Array (NanoString Technologies, Seattle, Wash) was used for evaluating proinflammatory gene expression. Quantitative polymerase chain reaction, Western blot, and immunohistochemistry were applied for studying messenger RNA and protein expression. RESULTS: Of 144 expressed proinflammatory genes, the inflammation array analysis revealed that 32 genes were differentially expressed between AF (including AF-SR and AF-AF) and SR. Thirteen genes were differentially expressed between AF-SR and AF-AF. The array and quantitative polymerase chain reaction produced parallel results in analyzing the expression of particular genes. Concordant with the gene expression difference between AF and SR patients, rapid pacing increased the expressions of SHC1, RHOA, PDGFA, and TRAF2 in HL-1 myocytes, implicating a causative effect of tachyarrhythmia on these genes. Compared with AF-SR, AF-AF expressed more intense oxidative stress, upregulations of collagen, transforming growth factor beta 1, and intranuclear nuclear factor of activated T-cells. Regression analysis showed that increased left atrial diameter was associated with the expression of RHOA and STAT1. CONCLUSIONS: Differential expression profiles of proflammatory genes were presented between SR and AF and between maintained SR and recurrent AF after the maze procedure. The identified inflammatory molecules associated with AF and failed surgical ablation may provide clues for developing new potential therapeutic targets to improve AF rhythm control.

Region-specific gene expression profiles in the left atria of patients with valvular atrial fibrillation.

BACKGROUND: Previous studies have demonstrated that atrial regions contribute differently to atrial fibrillation (AF) substrate. Pulmonary vein and the surrounding left atrial junction (LA-PV junction) are crucial areas in AF substrates and important ablation targets. OBJECTIVE: To identify regional differences in the left atria of patients with AF by using a genome-wide approach. METHODS: Paired LA-PV junction and left atrial appendage (LAA) specimens were obtained from 16 patients with persistent AF and 3 with sinus rhythm who underwent valvular surgery. The paired specimens were sent for microarray comparison. RESULTS: Of 54,675 expressed sequence tags, microarray analysis in patients with AF revealed that 391 genes were differentially expressed between the LA-PV junction and the LAA, including genes related to arrhythmia, cell death, fibrosis, hypertrophy, and inflammation. Microarray and real time-polymerase chain reaction produced parallel results in analyzing the expression of particular genes. In both patients with AF and sinus rhythm, the LA-PV junction exhibited greater paired-like homeodomain-2 and its target protein (short stature homeobox-2) expression than the LAA, which might contribute to arrhythmogenesis. Five genes related to thrombogenesis were upregulated in the LAA of patients with AF, which might implicate for the preferential thrombus formation in the LAA. Genes related to fibrosis were highly expressed in the LAA of patients with AF, which was reflected by intense ultrastructural changes in this region. CONCLUSIONS: We used a genome-wide approach to investigate region-specific gene expression in the left atria. Our findings provide important information relevant to region-specific arrhythmogenesis and thrombogenesis in AF pathogenesis.

Effects of fibroblast growth factors on the differentiation of the pulmonary progenitors from murine embryonic stem cells.

The fibroblast growth factors (FGFs) play an important role in the development of embryonic lung. In this study, we investigated the effects of mainly FGF 1, 2, and 10 at concentrations selected on the basis of data obtained from previous in vitro culture on the derivation of the pulmonary progenitors from murine embryonic stem cells cultured on gelatin or Matrigel-coated plates. For cells cultured on a gelatin-coated plate, high concentrations of FGF1 were found to enhance the expression of mRNAs for SPC and CC10, markers of distal airway epithelium, while high levels of FGF2 decreased the expression of RNAs for not only SPC, CC10 but also for the additional markers SPD and aquaporin 5. FGF10 at all tested concentrations was found to have no effect on the differentiation of pneumocytes when ESCs were grown on gelatin-coated plates. However, when differentiation was performed on Matrigel-coated plates, the addition of 60 ng/ml FGF10 enhanced the expression of pneumocyte markers, suggesting a synergic effect of FGF10 and extracellular matrix. In conclusion, growth factors were proven to be effective in the differentiation of pulmonary progenitors from mESCs. The need of signals from extracellular matrix proteins depends on the growth factors supplemented.

Tissue engineering of lung: the effect of extracellular matrix on the differentiation of embryonic stem cells to pneumocytes.

We have previously differentiated lung epithelium from human and murine embryonic stem cells (mESCs) and are now exploring the potential applications of these cells, including in the engineering of lung tissue constructs. In this study, we hypothesized that the differentiation and maintenance of lung epithelium derived from mESCs can be enhanced by extracellular matrix (ECM) proteins. Our established differentiation protocol was applied to mESCs grown on a range of ECMs: collagen I, laminin 332, fibronectin, Matrigel, and, as an experimental control, gelatin. The ECMs were coated onto tissue culture plastic (TCP) and poly-DL-lactic acid (PDLLA), a biodegradable polymer we have previously shown to support the growth of mature pneumocytes. Matrigel or Laminin-332 coating of either TCP or PDLLA film resulted in enhanced surfactant protein C gene expression in differentiating mESCs, a direct indication of the upregulation of lung epithelial differentiation. For each combination, changes in the contact angle and zeta potential of protein-coated TCP and PDLLA film confirmed protein adsorption. We conclude that the choice of the coating protein can greatly affect the differentiation of ESCs, and laminin-332-coated PDLLA provided an ECM-degradable scaffold combination that is suitable for engineering of lung tissue constructs.

Molecular dynamics simulations of human cystatin C and its L68Q varient to investigate the domain swapping mechanism.

Human cystatin C variant (L68Q), one of the amyloidgenic proteins, has been shown to form dimeric structure spontaneously via domain swapping and easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. The monomeric L68Q and wild-type (wt) HCCs share similar structural feature consisting of a core with a five-stranded anti-parallel beta-sheet (beta-region) wrapped around a central helix. In this study, various molecular dynamics simulations were conducted to investigate the conformational fluctuations of the monomeric L68Q and wt HCCs at various combinations of temperature (300 and 500K) and pH (2 and 7) to gain insights into the domain swapping mechanism. The results show that elevated temperature accelerates the disruption of the hydrophobic core and acidic condition promotes the destruction of three salt bridges between beta2 and beta3 in both HCCs. The results also indicate that the interior hydrophobic core of the L68Q variant is relatively unstable, leading to domain swapping more readily comparing to wt HCC under conditions favoring this process. However, these two monomeric HCCs adopt the same mechanism of domain swapping as follows: (i) first, the interior hydrophobic core is disrupted; (ii) subsequently, the central helix departs from the beta-region; (iii) then, the beta2-L1-beta3 hairpin structure unfolds following the so-called "zip-up" mechanism; and (iv) finally, the open form HCC is generated.

Molecular dynamics simulations to investigate the domain swapping mechanism of human cystatin C.

Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form a dimeric structure via a domain swapping process and then cause amyloid deposits in the brains of patients suffering from Alzheimer's disease. HCC monomer consists of a core with a five-stranded antiparallel beta-sheet (beta region) wrapped around a central helix. The connectivity of these secondary structures is: (N)-beta1-alpha-beta2-L1-beta3-AS-beta4-L2-beta5-(C). In this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500 K) and pH levels (2, 4, and 7) to gain insight into the domain swapping mechanism. The results show that high temperature (500 K) and low pH (pH 2) will trigger the domain swapping process of HCC. We further proposed that the domain swapping mechanism of HCC follows four steps: (1) the alpha-helix moves away from the beta region; (2) the contacts between beta2 and beta3-AS disappear; (3) the beta2-L1-beta3 hairpin unfolds following the so-called "zip-up" mechanism; and finally (4) the HCC dimer is formed. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of the alpha-helix from the beta-region, especially at low pH value. This is attributed to the fact that that low pH results in the protonation of the side chains of Asp, Glu, and His residues, which further disrupts the following four salt-bridge interactions stabilizing the alpha-beta interface of the native structure: Asp15-Arg53 (beta1-beta2), Glu21/20-Lys54 (helix-beta2), Asp40-Arg70 (helix-AS), and His43-Asp81 (beta2-AS).