| 1. |
- Al-Khalili, Lubna, et al.
(författare)
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Characterization of Human CD133+Cells in Biocompatible Poly(l-lactic acid) Electrospun Nano-Fiber Scaffolds
- 2016
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Ingår i: Journal of Biomaterials and Tissue Engineering. - : American Scientific Publishers. - 2157-9083 .- 2157-9091. ; 6:12, s. 959-966
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Tidskriftsartikel (refereegranskat)abstract
- CD133+ cells are potential myogenic progenitors for skeletal muscle regeneration to treat muscular dystrophies. The proliferation of human CD133+ stem cells was studied for 14 days in 3D biomimetic electrospun poly-L-lactic acid (PLLA) nano-fiber scaffolds. Additionally, the myogenic differentiation of the cells was studied during the last 7 days of the culture period. The cells were homogeneously distributed in the 3D scaffolds while colony formation and myotube formation occurred in 2D. After a lag phase due to lower initial cell attachment and an adaptation period, the cell growth rate in 3D was comparable to 2D after 7 and 14 days of culture. The expression of the stem cell (SC) marker PAX7 was 1.5-fold higher in 3D than 2D while the differentiation markers MyoG, Desmin and MyoD were only slightly changed (or remain unchanged) in 3D but strongly increased in 2D (12.6, 3.9, and 7.9-fold), and the myotube formation observed in 2D was absent in 3D. The marker expression during proliferation and differentiation, together with the absence of myotubes in 3D, indicates a better maintenance of stemness in 3D PLLA and stronger tendency for spontaneous differentiation in 2D culture. This makes 3D PLLA a promising biomaterial for the expansion of functional CD133+ cells.
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| 3. |
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| 4. |
- Johansson, Ulrika, 1974-, et al.
(författare)
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Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.
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| 5. |
- Leino, Mattias, et al.
(författare)
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Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin-521 and E-cadherin-Fc
- 2018
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Ingår i: Journal of Biomedical Materials Research. Part B - Applied biomaterials. - : Wiley. - 1552-4973 .- 1552-4981. ; 106:3, s. 1226-1236
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Tidskriftsartikel (refereegranskat)abstract
- Advances in human pluripotent cell cultivation and differentiation protocols have led to production of stem cell-derived progenitors as a promising cell source for replacement therapy. Three-dimensional (3-D) culture is a better mimic of the natural niche for stem cells and is widely used for disease modeling. Here, we describe a nonaggregate culture system of human embryonic stem cells inside electrospun polycaprolactone (PCL) fiber scaffolds combined with defined extracellular proteins naturally occurring in the stem cell niche. PCL fiber scaffolds coated with recombinant human laminin-521 readily supported initial stem cell attachment and growth from a single-cell suspension. The combination of recombinant E-cadherin-Fc and laminin-521 further improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in E-cadherin-Fc- and laminin-521-coated PCL scaffolds could differentiate into all three germ layers. Importantly, we provided a chemically defined 3-D system in which pluripotent stem cells grown and differentiated avoiding the formation of cell aggregates.
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| 6. |
- Ornithopoulou, Eirini, et al.
(författare)
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Self-assembly of RGD-functionalized recombinant spider silk protein into microspheres in physiological buffer and in the presence of hyaluronic acid
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e. membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk, and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.
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| 7. |
- Ornithopoulou, Eirini, et al.
(författare)
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Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid
- 2023
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Ingår i: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422. ; 6:9, s. 3696-3705
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Tidskriftsartikel (refereegranskat)abstract
- Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.
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| 8. |
- Ravichandran, Ranjithkumar, et al.
(författare)
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Intelligent ECM mimetic injectable scaffolds based on functional collagen building blocks for tissue engineering and biomedical applications
- 2017
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Ingår i: RSC Advances. - : ROYAL SOC CHEMISTRY. - 2046-2069. ; 7:34, s. 21068-21078
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogels comprising natural extracellular matrix (ECM) components are very attractive as scaffolds for regenerative medicine applications due to their inherent biointeractive properties. Responsive materials that adapt to their surrounding environments and regulate transport of ions and bioactive molecules manifest significant advantages for biomedical applications. Although there are many exciting challenges, the opportunity to design, fabricate and engineer stimuli-responsive polymeric systems based on ECM components is particularly attractive for regenerative medicine. Here we describe a one-pot approach to fabricate in situ fast gellable intelligent ECM mimetic scaffolds, based on methacrylated collagen building blocks with mechanical properties that can be modulated in the kPa-MPa range and that are suitable for both soft and hard tissues. Physiochemical characterizations demonstrate their temperature and pH responsiveness, together with the structural and enzymatic resistance that make them suitable scaffolds for long-term use in regenerative medicine and biomedical applications. The multifunctionality of these hydrogels has been demonstrated as an in situ depot-forming delivery platform for the adjustable controlled release of proteins and small drug molecules under physiological conditions and as a structural support for adhesion, proliferation and metabolic activities of human cells. The results presented herein should be useful to the design and fabrication of tailor-made scaffolds with tunable properties that retain and exhibit sustained release of growth factors for promoting tissue regeneration.
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| 9. |
- Åstrand, Carolina, et al.
(författare)
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Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation
- 2020
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Ingår i: Biomaterials Science. - : Royal Society of Chemistry (RSC). - 2047-4830 .- 2047-4849. ; 8:9, s. 2514-2525
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional (3D) neural tissue cultures recapitulate the basic concepts during development and disease better than what can be obtained using conventional two-dimensional cultures. Here, we use a recombinant spider silk protein functionalized with a cell binding motif from fibronectin (FN-silk) in combination with a human recombinant laminin 521 (LN-521) to create a fully defined stem cell niche in 3D. A novel method to assemble silk blended with LN-521 together with human pluripotent stem cells (hPSC) is used to create centimeter-sized foams, which upon cultivation develop into 3D cell constructs supported by a microfibrillar network. After initial cell expansion, neural differentiation was induced to form a homogenous layer of continuous neuroectodermal tissue that allows further differentiation into neuronal subtypes. The silk-supported 3D cell constructs could then be detached from the bottom of the well and cultured as floating entities, where cells appeared in distinctive radial organization resembling early neural tube. This shows that the neural progenitors retain their cellular self-organization ability in the FN-silk/LN-521-supported 3D culture. Calcium imaging demonstrated spontaneous activity, which is important for the formation of neuronal networks. Together, the results show that hPSCs integrated into FN-silk/LN-521 foam develop into neural progenitors and that these stay viable during long-term differentiations. FN-silk/LN-521 also supports morphogenesis mimicking the human brain development and can serve as base for engineering of hPSC-derived neural tissue.
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| 10. |
- Åstrand, Carolina
(författare)
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Chromatin structure and histone modifications in gene regulation
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the living cell, DNA is densely packed into a chromatin structure constituting nucleosomal arrays. One nucleosome core particle includes a disc shaped protein octamer consisting of pairs of histones H2A, H2B, H3 and H4. 146 bp of DNA is wrapped in almost two turns around this protein complex. The N-terminal tails of the histone proteins protrude out from the nucleosome core. These tails are highly flexible and are targets for multiple covalent modifications coupled to gene regulatory events. A linker histone H1 binds at the DNA entry/exit site of the nucleosome and organises the linker DNA and influence higher order chromatin structure. In order to induce gene expression the transcription factors, coactivators and also the transcriptional machinery has to get access to the DNA. This is obtained by modulation of chromatin structure by specific energy-depen dent chromatin remodelling complexes that has the ability to locally unfold the chromatin. In addition, nucleosomes may be positioned to expose specific regulatory DNA segments. The hormone-activated glucocorticoid receptor (GR) driven mouse mammary tumor virus (MMTV) promoter, fused to a thymidine kinase reporter gene was injected into Xenopus laevis oocytes. This was used for studies on chromatin structure coupled to gene regulation. Specific histone acetylations at H3K9, H3K14 and H4K16 were found to correlate with hormone-induced chromatin remodelling but to precede the transcription step. These histone modifications were reversible upon sequential treatment of a hormone antagonist, RU486, which correlated with the disruption of chromatin opening event. Hormone-induction was also found to mediate transient trimethylation of H3K4 during the initiation phase of transcription. Hyperacetylation of histories by Trichostatin A (TSA) mediated complex effects on the chromatin structure. TSA induced a general relaxation of chromatin and a specific rearrangement of nucleosomes in the distal part of the MMTV promoter. This correlated with an enhanced basal transcription. Hormone-activation was impaired by TSA treatment and this correlated with topological changes and an overall increased sensitivity to MNase digestion. The nuclear factor 1 (NF1) was shown to bind to the non-induced MMTV promoter. Upon hormone induction, (NF1) and hormone-activated GR formed an enlarged MNase- resistant structure, that may function as a binding platform for general transcription factors. (NF1) and octamer transcription factor 1 (Oct1) expressed together were found to cooperatively bind and to induce partial nucleosome positioning. This resulted in a faster and stronger hormone response and also correlated with a faster induction of trimethylation of H3K4. Together this serves as a functionally MMTV chromatin presetting. Linker histone H1 was expressed and incorporated into the MMTV chromatin where it dissociated specifically from the active locus upon hormone-induced remodelling. A subsaturated level of specific nucleosome binding of H1 correlated with enhanced binding of hormone-activated GR and also with enhanced transcription. At this specific H1 concentration, a reduction of the general DNA access was seen thus indicating an H1concentration dependent rearrangement of the chromatin fibre. Together this support a mechanism where H1-concentration dependent stimulation of GR binding is achieved through GR-induced disruption of H1 at active locus resulting in preferential HI masking of the nonspecific DNA.
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