124 research outputs found
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The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation
Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P</=0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins
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Slow-release RGD-peptide hydrogel monoliths
We report on the formation of hydrogel monoliths formed by functionalized peptide
Fmoc-RGD (Fmoc: fluorenylmethoxycarbonyl) containing the RGD cell adhesion tripeptide motif.
The monolith is stable in water for nearly 40 days. The gel monoliths present a rigid porous structure
consisting of a network of peptide fibers. The RGD-decorated peptide fibers have a Ī²-sheet secondary
structure. We prove that Fmoc-RGD monoliths can be used to release and encapsulate material,
including model hydrophilic dyes and drug compounds. We provide the first insight into the
correlation between the absorption and release kinetics of this new material and show that both
processes take place over similar time scales
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Transport of cells in hydrogels
The present invention relates to hydrogels which may be used to encapsulate or entrap live cells. The invention further relates to methods for transporting live cells which are encapsulated or entrapped within hydrogels from a first location to a second location. The invention further relates to method of treating a wound, disease or tissue injury, e.g. an ocular injury or a damaged ocular surface in a subject using a hydrogel comprising corneal stem cells. The hydrogels used in such methods may be ones which have been transported from a first location to a second location
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The variation in transparency of amniotic membrane used in ocular surface regeneration
Background/aims: Scant consideration has been given
to the variation in structure of the human amniotic
membrane (AM) at source or to the significance such
differences might have on its clinical transparency.
Therefore, we applied our experience of quantifying
corneal transparency to AM.
Methods: Following elective caesarean, AM from areas
of the fetal sac distal and proximal (ie, adjacent) to the
placenta was compared with freeze-dried AM. The
transmission of light through the AM samples was
quantified spectrophotometrically; also, tissue thickness
was measured by light microscopy and refractive index by
refractometry.
Results: Freeze-dried and freeze-thawed AM samples
distal and proximal to the placenta differed significantly in
thickness, percentage transmission of visible light and
refractive index. The thinnest tissue (freeze-dried AM) had
the highest transmission spectra. The thickest tissue
(freeze-thawed AM proximal to the placenta) had the
highest refractive index. Using the direct summation of
fields method to predict transparency from an equivalent
thickness of corneal tissue, AM was found to be up to
85% as transparent as human cornea.
Conclusion: When preparing AM for ocular surface
reconstruction within the visual field, consideration should
be given to its original location from within the fetal sac
and its method of preservation, as either can influence
corneal transparency
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Influence of substrate on corneal epithelial cell viability within ocular surface models
Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modeling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model
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Tissue engineering a fetal membrane
The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype
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The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair
The human amniotic membrane (AM) is a tissue of fetal origin and has proven to be clinically useful as
a biomaterial in the management of various ocular surface disorders including corneal stem cell
transplantation. However, its success rate displays a degree of clinical unpredictability. We suggest that
the measured variability inAMstiffness offers an explanation for the poor clinical reproducibility when
it is used as a substrate for stem cell expansion and transplantation. Corneal epithelial stem cells were
expanded upon AM samples possessing different mechanical stiffness. To investigate further the
importance of biological substrate stiffness on cell phenotype we replaced AM with type I collagen gels
of known stiffness. Substrate stiffness was measured using shear rheometry and surface topography
was characterized using scanning electron microscopy and atomic force microscopy. The
differentiation status of epithelial cells was examined using RT-PCR, immunohistochemistry and
Western blotting. The level of corneal stem cell differentiation was increased in cells expanded upon
AM with a high dynamic elastic shear modulus and cell expansion on type I collagen gels confirmed
that the level of corneal epithelial stem cell differentiation was related to the substrateās mechanical
properties. In this paper we provide evidence to show that the preparatory method of AM for clinical
use can affect its mechanical properties and that these measured differences can influence the level of
differentiation within expanded corneal epithelial stem cells
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New self-assembling multifunctional templates for the biofabrication and controlled self-release of cultured tissue
The need to source live human tissues for research and clinical applications has been a major driving force for
the development of new biomaterials. Ideally, these should elicit the formation of scaffold-free tissues with
native-like structure and composition. In this study, we describe a biologically interactive coating that combines
the fabrication and subsequent self-release of live purposeful tissues using templateācellāenvironment feedback. This smart coating was formed from a self-assembling peptide amphiphile comprising a proteasecleavable sequence contiguous with a cell attachment and signaling motif. This multifunctional material was subsequently used not only to instruct human corneal or skin fibroblasts to adhere and deposit discreet multiple layers of native extracellular matrix but also to govern their own self-directed release from the template solely through the action of endogenous metalloproteases. Tissues recovered through this physiologically relevant process were carrier-free and structurally and phenotypically equivalent to their natural counterparts. This technology contributes to a new paradigm in regenerative medicine, whereby materials are able to actively direct and respond to cell behavior. The novel application of such materials as a coating capable of directing the formation and detachment of complex tissues solely under physiological conditions can have broad use for fundamental research and in future cell and tissue therapies
Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain
AbstractWith the ever-increasing clinical application of cell-based therapies, it is considered critical to develop systems that facilitate the storage and distribution of cell therapy products (CTPs) between sites of manufacture and the clinic. For such systems to be realized, it is essential that downstream bioprocessing strategies be established that are scalable, reproducible and do not influence the viability or function of the living biologic. To this end, we examined alginate-encapsulation as a method to heighten the preservation of human adipose-derived stem cells (hASCs) during hypothermic storage, and establish a scalable process for high-volume production. A drop-wise method for scalable alginate bead generation, using calcium as the cross-linker, was modified to enable the yield of up to 3500 gelled beads per minute. The effect of alginate concentration on the viscosity of non-gelled sodium alginate and the mechanical properties and internal structure of calcium-crosslinked alginate in response to different alginate and calcium concentrations were investigated. Mechanical strength was chiefly dependent on alginate concentration and 1.2% alginate cross-linked with 100mM calcium chloride could withstand stress in the order of 35kPa. Upon selection of appropriate parameters, we demonstrated the suitability of using this method for immobilizing human stem cells. Encapsulated hASCs demonstrated no loss in cell viability, and had a uniform distribution after high-volume production. Following storage, released cells were able to attach and recover a normal morphology upon return to culture conditions. Thus we present a scalable method for stem cell encapsulation and storage for application within the cell therapy supply chain
Template curvature influences cell alignment to create improved human corneal tissue equivalents
To accurately create corneal stromal equivalents with nativeālike structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with corneaālike shape and composition. Specifically, tissues selfāreleased from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stromaācharacteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo
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