Fabrication of low-cost micropatterned polydimethyl-siloxane scaffolds to organise cells in a variety of two-dimensioanl biomimetic arrangements for lab-on-chip culture platforms

We present the rapid-prototyping of type I collagen micropatterns on poly-dimethylsiloxane substrates for the biomimetic confinement of cells using the combination of a surface oxidation treatment and 3-aminopropyl triethoxysilane silanisation followed by glutaraldehyde crosslinking. The aim of surface treatment is to stabilise microcontact printing transfer of this natural extracellular matrix protein that usually wears out easily from poly-dimethylsiloxane, which is not suitable for biomimetic cell culture platforms and lab-on-chip applications. A low-cost CD-DVD laser was used to etch biomimetic micropatterns into acrylic sheets that were in turn replicated to poly-dimethylsiloxane slabs with the desired features. These stamps were finally inked with type I collagen for microcontact printing transfer on the culture substrates in a simple manner. Human hepatoma cells (HepG2) and rat primary hepatocytes, which do not adhere to bare poly-dimethylsiloxane, were successfully seeded and showed optimal adhesion and survival on simple protein micropatterns with a hepatic cord geometry in order to validate our technique. HepG2 cells also proliferated on the stamps. Soft and stiff poly-dimethylsiloxane layers were also tested to demonstrate that our cost-effective process is compatible with biomimetic organ-on-chip technology integrating tunable stiffness with a potential application to drug testing probes development where such cells are commonly used.

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Load-induced regulation of tendon homeostasis by SPARC, a genetic predisposition factor for tendon and ligament injuries

Science Translational Medicine ◽

10.1126/scitranslmed.abe5738 ◽

2021 ◽

Vol 13 (582) ◽

pp. eabe5738

Author(s):

Tao Wang ◽

Andrea Wagner ◽

Renate Gehwolf ◽

Wenjin Yan ◽

Fabian S. Passini ◽

...

Keyword(s):

Matrix Protein ◽

Type I Collagen ◽

Botulinum Toxin A ◽

Three Dimensional ◽

Extracellular Matrix Protein ◽

Matricellular Protein ◽

Type I ◽

Ligament Injuries ◽

Ribosomal S6 Kinase

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc−/− mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc−/− mice. Tendon hypoplasia was attenuated in Sparc−/− mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc−/− three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc−/− tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

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The bspA Locus of Lactobacillus fermentum BR11 Encodes an l-Cystine Uptake System

Journal of Bacteriology ◽

10.1128/jb.181.7.2192-2198.1999 ◽

1999 ◽

Vol 181 (7) ◽

pp. 2192-2198 ◽

Cited By ~ 47

Author(s):

Mark S. Turner ◽

Tonia Woodberry ◽

Louise M. Hafner ◽

Philip M. Giffard

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Binding Proteins ◽

Type I Collagen ◽

Lactobacillus Reuteri ◽

Binding Protein ◽

Lactobacillus Fermentum ◽

Extracellular Matrix Protein ◽

Type I ◽

Uptake System

ABSTRACT BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, fromLactobacillus reuteri. Compared with the database ofEscherichia coli proteins, BspA is most similar to thel-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take upl-cystine. Uptake rates of l-glutamine,l-histidine, and l-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with l-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.

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Immunologic characterization of the membrane-bound collagen in normal human fibroblasts: identification of a distinct membrane collagen.

Journal of Experimental Medicine ◽

10.1084/jem.144.1.145 ◽

1976 ◽

Vol 144 (1) ◽

pp. 145-154 ◽

Cited By ~ 16

Author(s):

J R Lichtenstein ◽

E A Bauer ◽

R Hoyt ◽

H J Wedner

Keyword(s):

Cell Membrane ◽

Matrix Protein ◽

Type I Collagen ◽

Proteolytic Enzymes ◽

Human Fibroblasts ◽

Extracellular Matrix Protein ◽

Type Iii Collagen ◽

Type I ◽

Normal Human ◽

Membrane Collagen

Collagen, the major extracellular matrix protein, is also a membrane protein. Two types of collagen are detected on the normal human fibroblast membrane in culture, type I collagen and a new immunologically and chemically distinct collagen, type M (membrane) collagen. Antibodies to type M collagen elicited complement-mediated cytotoxicity, which could be blocked by pretreatment of the cells with bacterial collagenase or the antibody with type M collagen. Pretreatment of the cells with other proteolytic enzymes or the antibody with type I collagen or type III collagen had no effect on this complement-mediated cytotoxicity. Although type I collagen is the major collagen synthesized by normal human fibroblasts type M collagen may be the major cell membrane collagen and may be a major cell membrane component.

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Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility

Journal of Cell Science ◽

10.1242/jcs.114.24.4587 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4587-4598 ◽

Cited By ~ 2

Author(s):

Waleed O. Twal ◽

Andras Czirok ◽

Balazs Hegedus ◽

Christian Knaak ◽

Mastan R. Chintalapudi ◽

...

Keyword(s):

Matrix Protein ◽

Type I Collagen ◽

Cell Attachment ◽

Chinese Hamster ◽

Extracellular Matrix Protein ◽

Endocardial Cushion ◽

Boyden Chamber ◽

Type I ◽

Collagen Gels

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells. These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between α5β1 or α4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.

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The Rise of the OM-LoC: Opto-Microfluidic Enabled Lab-on-Chip

Micromachines ◽

10.3390/mi12121467 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1467

Author(s):

Harry Dawson ◽

Jinane Elias ◽

Pascal Etienne ◽

Sylvie Calas-Etienne

Keyword(s):

Low Cost ◽

Optical Components ◽

New Era ◽

Lab On Chip ◽

Highly Sensitive ◽

Multiple Challenges ◽

On Chip ◽

Optical Circuits ◽

Made In

The integration of optical circuits with microfluidic lab-on-chip (LoC) devices has resulted in a new era of potential in terms of both sample manipulation and detection at the micro-scale. On-chip optical components increase both control and analytical capabilities while reducing reliance on expensive laboratory photonic equipment that has limited microfluidic development. Notably, in-situ LoC devices for bio-chemical applications such as diagnostics and environmental monitoring could provide great value as low-cost, portable and highly sensitive systems. Multiple challenges remain however due to the complexity involved with combining photonics with micro-fabricated systems. Here, we aim to highlight the progress that optical on-chip systems have made in recent years regarding the main LoC applications: (1) sample manipulation and (2) detection. At the same time, we aim to address the constraints that limit industrial scaling of this technology. Through evaluating various fabrication methods, material choices and novel approaches of optic and fluidic integration, we aim to illustrate how optic-enabled LoC approaches are providing new possibilities for both sample analysis and manipulation.

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Low cost optical particle detection for lab on chip systems based on DVD technology

10.1117/12.759283 ◽

2007 ◽

Cited By ~ 1

Author(s):

Andrew L. Clow ◽

Rainer Künnemeyer ◽

Paul Gaynor ◽

John C. Sharpe

Keyword(s):

Low Cost ◽

Particle Detection ◽

Lab On Chip ◽

On Chip

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Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

Biochemical Journal ◽

10.1042/bj20090542 ◽

2009 ◽

Vol 423 (1) ◽

pp. 53-59 ◽

Cited By ~ 82

Author(s):

Sebastian Kalamajski ◽

Anders Aspberg ◽

Karin Lindblom ◽

Dick Heinegård ◽

Åke Oldberg

Keyword(s):

Matrix Protein ◽

Collagen Type I ◽

Full Length ◽

Extracellular Matrix Protein ◽

Type I ◽

Similar System ◽

Collagen Binding ◽

Osteoblastic Activity ◽

Collagen Mineralization

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10–12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10–12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami™) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).

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Point-of-Care Systems for Rapid DNA Quantification in Oncology

Tumori Journal ◽

10.1177/030089160809400214 ◽

2008 ◽

Vol 94 (2) ◽

pp. 216-225 ◽

Cited By ~ 5

Author(s):

Marco Bianchessi ◽

Sarah Burgarella ◽

Marco Cereda

Keyword(s):

Point Of Care ◽

Low Cost ◽

Semiconductor Industry ◽

Point Of Care Testing ◽

Lab On Chip ◽

Diagnostic Assays ◽

Care Systems ◽

Point Of Care Systems ◽

The Common ◽

On Chip

The development of new powerful applications and the improvement in fabrication techniques are promising an explosive growth in lab-on-chip use in the upcoming future. As the demand reaches significant levels, the semiconductor industry may enter in the field, bringing its capability to produce complex devices in large volumes, high quality and low cost. The lab-on-chip concept, when applied to medicine, leads to the point-of-care concept, where simple, compact and cheap instruments allow diagnostic assays to be performed quickly by untrained personnel directly at the patient's side. In this paper, some practical and economical considerations are made to support the advantages of point-of-care testing. A series of promising technologies developed by STMicroelectronics on lab-on-chips is also presented, mature enough to enter in the common medical practice. The possible use of these techniques for cancer research, diagnosis and treatment are illustrated together with the benefits offered by their implementation in point-of-care testing.

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