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Understanding piezoelectricity, the linear electromechanical transduction, in bone and tendon and its potential role in mechanoelectric transduction leading to their growth and remodeling remains a challenging subject. With high-resolution piezoresponse force microscopy, we probed piezoelectric behavior in relevant biological samples at different scale levels: from the subfibrillar structures of single isolated collagen fibrils to bone. We revealed that, beyond the general understanding of collagen fibril being a piezoelectric material, there existed an intrinsic piezoelectric heterogeneity within a collagen fibril coinciding with the periodic variation of its gap and overlap regions. This piezoelectric heterogeneity persisted even for the collagen fibrils embedded in bone, bringing about new implications for its possible roles in structural formation and remodeling of bone.
ACS Nano 2009 Jul 28
PMID:Uncovering nanoscale electromechanical heterogeneity in the subfibrillar structure of collagen fibrils responsible for the piezoelectricity of bone. 1950 15

The objective of the following case reports was to assess whether mineralized bone replacement grafts (eg, xenografts and allografts) could be added to recombinant human bone morphogenetic protein-2/acellular collagen sponge (rhBMP-2/ACS) in an effective manner that would: (1) reduce the graft shrinkage observed when using rhBMP-2/ACS alone, (2) reduce the volume and dose of rhBMP-2 required, and (3) preserve the osteoinductivity that rhBMP-2/ACS has shown when used alone. The primary outcome measures were histomorphometric analysis of vital bone production and analysis of serial computed tomographic scans to determine changes in bone graft density and stability. Over the 6-month course of this investigation, bone graft densities tended to increase (moreso with the xenograft than the allograft). The increased density in allograft cases was likely the result of both compression of the mineralized bone replacement graft and vital bone formation, seen histologically. Loss of volume was greater with the four-sponge dose than the two-sponge dose because of compression and resorption of the sponges. Vital bone formation in the allograft cases ranged from 36% to 53% but, because of the small sample size, it was not possible to determine any significant difference between the 5.6 mL (four-sponge) dose and the 2.8 mL (two-sponge) dose. Histology revealed robust new woven bone formation with only minimal traces of residual allograft, which appeared to have undergone accelerated remodeling or rhBMP-2-mediated resorption.
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PMID:Maxillary sinus augmentation using recombinant bone morphogenetic protein-2/acellular collagen sponge in combination with a mineralized bone replacement graft: a report of three cases. 2022 73

Collagen, as the major structural protein of the extracellular matrix in animals, is a versatile biomaterial of great interest in various engineering applications. Electrospun nanofibers of collagen are regarded as very promising materials for tissue engineering applications because they can reproduce the morphology of the natural bone but have as a drawback a poor structural consistency in wet conditions. In this paper, a comparative study between the performance of different cross-linking methods such as a milder enzymatic treatment procedure using transglutaminase, the use of N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide, and genipin, and the use of a physical method based on exposure to ultraviolet light was carried out. The chemical and enzymatic treatments provided, in this order, excellent consistency, morphology, cross-linking degree, and osteoblast viability for the collagen nanofibers. Interestingly, the enzymatically cross-linked collagen mats, which are considered to be a more biological treatment, promoted adequate cell adhesion, making the biomaterial biocompatible and with an adequate degree of porosity for cell seeding and in-growth.
ACS Appl Mater Interfaces 2009 Jan
PMID:Comparative performance of electrospun collagen nanofibers cross-linked by means of different methods. 2035 75

To make polycaprolactone (PCL) more suitable for tissue engineering, PCL in the form of electrospun fibrous scaffolds was first modified with 1,6-hexamethylenediamine to introduce amino groups on their surface. Various biomolecules, i.e., collagen, chitosan, and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, were then immobilized on their surface, with N,N'-disuccinimidylcarbonate being used as the coupling agent. Dynamic water contact angle measurement indicated that the scaffold surface became more hydrophilic after the aminolytic treatment and the subsequent immobilization of the biomolecules. The appropriateness of these PCL fibrous scaffolds for the tissue/cell culture was evaluated in vitro with three different cell lines, e.g., mouse fibroblasts (L929), human epidermal keratinocytes (HEK001), and mouse calvaria-derived preosteoblastic cells (MC3T3-E1). Both the neat and the modified PCL fibrous scaffolds released no substances in the levels that were harmful to these cells. Among the various biomolecule-immobilized PCL fibrous scaffolds, the ones that had been immobilized with type I collagen, a Arg-Gly-Asp-containing protein, showed the greatest ability to support both the attachment and the proliferation of all of the investigated cell types, followed by those that had been immobilized with GRGDS peptide.
ACS Appl Mater Interfaces 2009 May
PMID:Immobilization of biomolecules on the surface of electrospun polycaprolactone fibrous scaffolds for tissue engineering. 2035 94

This paper describes a novel surface engineering approach that combines oxygen plasma treatment and electrochemical activation to create micropatterned cocultures on indium tin oxide (ITO) substrates. In this approach, photoresist was patterned onto an ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surface to oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagen regions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry (ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagen and PEG molecules on the same surface. Imaging ellipsometry and atomic force microscopy (AFM) were employed to further investigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selective attachment of mammalian cells on the ITO substrate. Importantly, after seeding the first cell type, the ITO surfaces could be activated by applying negative voltage (-1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another cell type onto the same surface and to create micropatterned cocultures. Micropatterned cocultures of primary hepatocytes and fibroblasts created by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineering strategy described here may be used to pattern multiple cell types on an optically transparent and conductive substrate and is envisioned to have applications in tissue engineering and biosensing.
ACS Appl Mater Interfaces 2009 Nov
PMID:Micropatterning of proteins and mammalian cells on indium tin oxide. 2035 32

Although it has been demonstrated that carbon nanotubes (CNTs) may have potentials for tissue engineering applications because of their unparalleled physical properties, little has been known on the cell adhesion mechanisms on model CNT monolayer pertaining to the design of novel cell therapeutics device. In this study, the adhesion dynamics of primary porcine esophageal fibroblasts (PEFs) on CNT monolayer were elucidated with confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy. Moreover, CNT monolayer (CNT-ML) was functionalized with two typical extracellular matrix (ECM) proteins including collagen type I (COL) and fibronectin (FN) in order to promote its biocompatibility. First, it is shown by atomic force microscopy that the topographical features of CNT-ML were dependent on the types of immobilized ECM protein. Second, significant time lag in adhesion contact evolution (around 10 min) for PEFs was found on both CNT-ML and CNT-COL compared to the negligible time lag on CNT-FN. It was found that adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces reached steady state at 60 and 30 min after cell seeding compared to 70 min on CNT-ML surface. At steady state, the adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces was about twice as much than that on the CNT-ML surface. Moreover, immobilization of collagen or fibronectin on CNT monolayer led to an increase in seeding efficiency and proliferation rate of PEFs. Scanning electron microscopy and immunostaining together demonstrated that PEFs displayed an elongated morphology and highly polarized actin network on both CNT-COL and CNT-FN surfaces, whereas PEFs displayed nonuniform cell morphology and actin organization on the CNT-ML surface. Overall, our results demonstrated that the biophysical responses and biological behavior of PEFs on unmodified or functionalized CNT monolayer were different. Functionalization of CNT through extracellular matrix protein immobilization effectively promotes cell adhesion and proliferation, which may provide information for designing CNT-based biomaterials or novel cell therapeutics devices in biomedical engineering.
ACS Appl Mater Interfaces 2010 Apr
PMID:Modulating cell adhesion dynamics on carbon nanotube monolayer engineered with extracellular matrix proteins. 2042 24

A few percent of transition metals impregnated inside some biological organisms in nature remarkably improve such organisms' mechanical stability. Although the lure to emulate them for development of new biomimetic structural materials has been great, the practical advances have been rare because of the lack of proper synthetic approaches. Multiple pulsed vapor phase infiltration proved successful for the preparation of such transition metal impregnated materials with highly improved mechanical stability. The artificially infiltrated metals (Al, Ti, or Zn) from gas phase lead to around 3 times increase of toughness (in terms of breaking energy) of natural collagen in a dried state. In addition, the infiltrated metals apparently induce considerable crystallographic changes in the natural collagen structures. This infiltration approach can be used as guide for the synthesis of bioinspired structural materials related to metal infiltration.
ACS Appl Mater Interfaces 2010 Aug
PMID:Improved mechanical stability of dried collagen membrane after metal infiltration. 2067 30

Three-dimensional (3D) cellular assays closely mimic the in vivo milieu, providing a rapid, inexpensive system for screening drug candidates for toxicity or efficacy in the early stages of drug discovery. However, 3D culture systems may suffer from mass transfer limitations, particularly in delivery of large polypeptide or nucleic acid compounds. Nucleic acids (e.g., genes, silencing RNA) are of particular interest both as potential therapeutics and due to a desire to modulate the gene-expression patterns of cells exposed to small-molecule pharmacological agents. In the present study, polyethylenimine (PEI)-coated superparamagnetic nanoparticles (SPMNs) were designed to deliver interfering RNA and green fluorescent protein (GFP) plasmids through a collagen-gel matrix into 3D cell cultures driven by an external magnetic field. The highest transfection efficiency achieved was 64% for siRNA and 77% for GFP plasmids. Delivery of an shRNA plasmid against GFP by PEI-coated SPMNs silenced the GFP expression with 82% efficiency. We further demonstrated that this delivery approach could be used for screening interfering RNA constructs for therapeutic or toxic effects for cells grown in 3D cultures. Four known toxic shRNA plasmids were delivered by PEI-coated SPMNs into 3D cell cultures, and significant toxicities (41-51% cell death) were obtained.
ACS Nano 2010 Aug 24
PMID:Gene delivery in three-dimensional cell cultures by superparamagnetic nanoparticles. 2073 51

Cell migration plays a critical role in numerous physiological processes, such as wound healing, response to inflammation, and cancer metastasis. In recent years, accumulating evidence indicates that cell movement is regulated not only by chemical signals but also by mechanical stimuli. In this study, the primary goal is to identify whether a chemical or mechanical stimulus plays the decisive role in directing cell migration. Measuring the motility of cells when they are presented with a combination of chemical and mechanical cues will provide insight into the complex physiological phenomena that guide and direct migration. A novel polyacrylamide hydrogel was designed with an interfacial region where the chemical and mechanical properties varied in opposing directions. One side of the interface was stiff (high Young's modulus) with a low protein concentration, whereas the other side of the interface was compliant (low Young's modulus) with a high protein concentration. The chemical gradient was created by varying the collagen (type I) concentration and the mechanical gradient was introduced by changing the extent of cross-linking in the polymer. The length of the interface with opposing chemical-mechanical profiles was found to be approximately 100 mum. Our results demonstrate that when Balb/c 3T3 fibroblasts were presented with a choice, they either migrated preferentially toward the high-collagen-compliant (low Young's modulus) side of the interfacial region or remained on the high-collagen region, suggesting a more dominant role for chemical stimuli in directing fibroblast locomotion.
ACS Appl Mater Interfaces 2010 Aug
PMID:Cell migration at the interface of a dual chemical-mechanical gradient. 2073 3

Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.
ACS Chem Biol 2011 Apr 15
PMID:Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli. 2121 Jun 82


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