Silk Protein Assisted Ex Vivo Remineralization of Enamel-Like Microstructure

2011 ◽  
Vol 66-68 ◽  
pp. 1682-1687
Author(s):  
Kun Tian ◽  
Min Peng ◽  
Ping Wu ◽  
Chu Hang Liao ◽  
Fa Yin Huang
Keyword(s):  
Ex Vivo ◽  
Three Dimensional ◽  
Silk Protein ◽  
Molar Ratio ◽  
Dentinal Tubule ◽  
Dental Tissue ◽  
Parallel Direction ◽  
Protein Monolayer ◽  
Dental Tissue Engineering

Based on the basic theory of molecular recognition, we designed an organic molecules model that spontaneously form three-dimensional fibrillar scaffolds to induce the crystallization of hydroxyapatite to synthesized enamel-like calcium phosphate/hydroxyapatite under a controllable way in vitro. Cross-linking of collagen on the dentin surface and silk fibroin with N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimized by varying the NHS/EDC molar ratio at constant EDC concentration. CaCl2 and Na3PO4-12H2O solution was added with Ca: P odd as 1.67:1 after conjugated. The results showed that the dentinal tubule were blocked by neonatal hydroxyapatite layer which has a continuous structure of columns crystal with size of 10-40nm. Furthermore, there were column crystal with parallel direction inside, similar to the crystal array in the top of enamel rod. The results suggest that silk protein monolayer may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Biomineralization of the Hydroxyapatite with 3D-Structure for Enamel Reconstruction

2011 ◽  
Vol 391-392 ◽  
pp. 633-637
Author(s):  
Kun Tian ◽  
Min Peng ◽  
Ping Wu ◽  
Chu Hang Liao ◽  
Fa Yin Huang
Keyword(s):  
3D Structure ◽  
Oral Epithelium ◽  
Dentinal Tubule ◽  
Dental Tissue ◽  
Reciprocal Interactions ◽  
Mineralized Tissues ◽  
Parallel Direction ◽  
Dental Tissue Engineering

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. Based on these basic theory, we design a organic molecules model to induced the crystallization of hydroxyapatite to synthesized tooth-like calcium phosphate/hydroxyapatite with 3D-structure in a controllable way in vitro. We observed that hydroxyapatite nanorods can be controlled followed by in situ phosphorylation process and triggered by conditions of pH and ionic strength. The results showed that he dentinal tubule were blocked by neonatal hydroxyapatite layer and this composite a continuous structure of columns crystal with size of 30-80nm. At the same time, XRD showed that the precipitation was calcium fluoride phosphate and Ca:P was 1.6. Furthermore, there were column crystal with parallel direction inside, as same as the crystal array in the top of enamel rod. The results suggest that collagen monolayer may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Induced Synthesis of Hydroxyapatite by Chitosan for Enamel Remineralization

2012 ◽  
Vol 530 ◽  
pp. 40-45 ◽  
Author(s):  
Kun Tian ◽  
Min Peng ◽  
Wei Fei ◽  
Chu Hang Liao ◽  
Xiao Hua Ren
Keyword(s):  
Calcium Fluoride ◽  
Dentinal Tubule ◽  
Dental Tissue ◽  
Crosslinking Process ◽  
Parallel Direction ◽  
Biomimetic Method ◽  
Dental Tissue Engineering ◽  
In Situ Crosslinking

The formation of organized nanocrystals that resemble tooth-like hydroxyapatite is crucial for successful enamel remineralization. Based on the principles of biomineralization , spindle - shaped hydroxyapatites (HA) were synthesized through biomimetic method with chitosan as template under a controllable way in vitro. We observed that hydroxyapatite nanorods can be controlled followed by in situ crosslinking process and triggered by conditions of pH and ionic strength. The dentinal tubule were blocked by neonatal hydroxyapatite layer and this composite a continuous structure of columns crystal with size of 10-40nm. At the same time, XRD showed that the precipitation was calcium fluoride phosphate and Ca:P was 1.6. Furthermore, there were column crystal with parallel direction inside, as same as the crystal array in the top of enamel rod. The results suggest that chitosan monolayer may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Biomimetic Synthesis of Tooth-Like Hydroxyapatite under Organic Polymer Monolayer

2009 ◽  
Vol 610-613 ◽  
pp. 1054-1058
Author(s):  
Kun Tian ◽  
Hui Min Shuai ◽  
Xiao Min Yang
Keyword(s):  
Biomimetic Synthesis ◽  
Organic Polymer ◽  
Molar Ratio ◽  
Dentinal Tubule ◽  
Ftir Analysis ◽  
X Ray ◽  
Dentin Surface ◽  
Crosslinking Process ◽  
Protein Monolayer

Based on the basic theory of molecular recognition , we design a organic molecules model to induce the crystallization of hydroxyapatite to synthesized tooth-like calcium phosphate/hydroxyapatite under a controllable way in vitro. The cross-linking of collagen on the dentin surface and extraneous collagen was optimized by varying the molar ratio of N,N-(3-dimethylaminopropyl)- N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a constant EDC concentration. CaCl2 and Na3PO4-12H2O solutions were added after the crosslinking process. X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared spectroscopy (FTIR) analysis of organic protein monolayer for samples. The obtained composite were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) as well as energy dispersive X-ray (EDX). XPS and FTIR analysis showed the surface organic compositions in experimental group is higher than that of normal dentin and decalcified dentin surface. The results showed that the dentinal tubule were blocked by neonatal hydroxyapatite layer which has a continuous structure of columns crystal with size of 10-40nm. Furthermore, there were column crystal with parallel direction inside, similar to the crystal array in the top of enamel rod. This study showed that the specific organic molecule model can be used as a potential effective crystal growth modifier.

Controlled Crystallization of Tooth-Like Hydroxyapatite under Gelatin Monolayer

2007 ◽  
Vol 330-332 ◽  
pp. 663-666
Author(s):  
Kun Tian ◽  
Dong Hua Guan ◽  
Ping Wu ◽  
Chun Peng Huang ◽  
Lin Niu ◽  
...  
Keyword(s):  
Photoelectron Spectroscopy ◽  
Molar Ratio ◽  
Dentinal Tubule ◽  
X Ray Diffraction ◽  
X Ray ◽  
Whole Process ◽  
Crosslinking Process ◽  
Recognition Theory ◽  
Parallel Direction

Based on the molecular recognition theory, an organic molecules model was designed to induce the hydroxyapatite crystallization, to build a tooth-like calcium phosphate/hydroxyapatite under a controllable way in vitro. The cross-linking of collagen on the dentin surface and gelatin was optimized by varying the molar ratio of N,N-(3-dimethylaminopropyl)- N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a constant EDC concentration. CaCl2 and Na3PO4-12H2O solutions were added after the crosslinking process. The whole process requires repeating the crosslinking and mineralization process for five times. The obtained composite were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) as well as energy dispersive X-ray photoelectron spectroscopy (XPS). The results showed that the dentinal tubule were blocked by neonatal hydroxyapatite layer which has a continuous structure of columns crystal with size of 10-40nm. Furthermore, there was column crystal with parallel direction inside, similar to the crystal array in the top of enamel rod. This study showed that the specific organic molecule model can be used as a potential effective crystal growth modifier.

scholarly journals Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer

2021 ◽  
Vol 13 ◽  
pp. 175883592110598
Author(s):  
Inken Flörkemeier ◽  
Tamara N. Steinhauer ◽  
Nina Hedemann ◽  
Magnus Ölander ◽  
Per Artursson ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Topoisomerase I ◽  
Ex Vivo ◽  
Chemotherapy Resistance ◽  
Membrane Integrity ◽  
Three Dimensional ◽  
New Drugs ◽  
Highly Active ◽  
Gynaecologic Neoplasms

Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[ c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor. Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected. Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.

scholarly journals Development of A Three-Dimensional Tissue Construct from Dental Human Ectomesenchymal Stem Cells: In Vitro and In Vivo Study

2012 ◽  
Vol 6 (1) ◽  
pp. 226-234 ◽  
Author(s):  
Daniela Guzmán-Uribe ◽  
Keila Neri Alvarado Estrada ◽  
Amaury de Jesús Pozos Guillén ◽  
Silvia Martín Pérez ◽  
Raúl Rosales Ibáñez
Keyword(s):  
Three Dimensional ◽  
Human Tooth ◽  
Animal Cells ◽  
Dental Tissue ◽  
Membrane Markers ◽  
Tissue Organization ◽  
Specific Membrane ◽  
Tissue Construct

Application of regenerative medicine technology provides treatment for patients with several clinical problems, like loss of tissue and its function. The investigation of biological tooth replacement, dental tissue engineering and cell culture, scaffolds and growth factors are considered essential. Currently, studies reported on the making of threedimensional tissue constructs focused on the use of animal cells in the early stages of embryogenesis applied to young biomodels. The purpose of this study was the development and characterization of a three-dimensional tissue construct from human dental cells. The construct was detached, cultured and characterized in mesenchymal and epithelial cells of a human tooth germ of a 12 year old patient. The cells were characterized by specific membrane markers (STRO1, CD44), making a biocomplex using Pura Matrix as a scaffold, and it was incubated for four days and transplanted into 30 adult immunosuppressed male Wistar rats. They were evaluated at 6 days, 10 days and 2 months, obtaining histological sections stained with hematoxylin and eosin. Cell cultures were positive for specific membrane markers, showing evident deviations in morphology under phase contrast microscope. Differentiation and organization were noted at 10 days, while the constructs at 2 months showed a clear difference in morphology, organization and cell type. It was possible to obtain a three-dimensional tissue construct from human dental ectomesenchymal cells achieving a degree of tissue organization that corresponds to the presence of cellular stratification and extracellular matrix.

Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

2006 ◽  
Vol 96 (11) ◽  
pp. 671-684 ◽  
Author(s):  
Alexandre Fontayne ◽  
Karen Vanhoorelbeke ◽  
Inge Pareyn ◽  
Isabel Van Rompaey ◽  
Muriel Meiring ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Three Dimensional ◽  
Vector System ◽  
Dimensional Structure ◽  
Fab Fragment ◽  
Variable Regions ◽  
Significant Prolongation ◽  
Von Willebrand ◽  
First Time

SummaryFab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

scholarly journals MT1-MMP–dependent neovessel formation within the confines of the three-dimensional extracellular matrix

2004 ◽  
Vol 167 (4) ◽  
pp. 757-767 ◽  
Author(s):  
Tae-Hwa Chun ◽  
Farideh Sabeh ◽  
Ichiro Ota ◽  
Hedwig Murphy ◽  
Kevin T. McDonagh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Collagenolytic Activity ◽  
Type I ◽  
Ex Vivo Model

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

scholarly journals Intercellular coupling between peripheral circadian oscillators by TGF-β signaling

2021 ◽  
Vol 7 (30) ◽  
pp. eabg5174
Author(s):  
Anna-Marie Finger ◽  
Sebastian Jäschke ◽  
Marta del Olmo ◽  
Robert Hurwitz ◽  
Adrián E. Granada ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Clock Genes ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Transforming Growth Factor Β ◽  
Molecular Clocks ◽  
Peripheral Oscillators ◽  
Circadian Oscillators

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor–β (TGF-β) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-β signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

scholarly journals A vascularized 3D model of the human pancreatic islet for ex vivo study of immune cell-islet interaction

2021 ◽  
Author(s):  
R. Hugh F. Bender ◽  
Benjamen T O'Donnell ◽  
Bhupinder Shergill ◽  
Brittany Q Pham ◽  
Damie J Juat ◽  
...  
Keyword(s):  
Immune Cell ◽  
Ex Vivo ◽  
Cell Damage ◽  
Three Dimensional ◽  
Human Islets ◽  
Culture Conditions ◽  
Β Cells ◽  
Standard Culture ◽  
Key Steps

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively by β cells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of islet β cells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlying β cell damage in diabetes rely on in vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing -- key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel, ex vivo platform for studying human islet biology in both health and disease.

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