| 1. |
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| 2. |
- Coy, R., et al.
(författare)
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Combining in silico and in vitro models to inform cell seeding strategies in tissue engineering
- 2020
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Ingår i: Journal of the Royal Society Interface. - : The Royal Society Publishing. - 1742-5689 .- 1742-5662. ; 17:164
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Tidskriftsartikel (refereegranskat)abstract
- The seeding density of therapeutic cells in engineered tissue impacts both cell survival and vascularization. Excessively high seeded cell densities can result in increased death and thus waste of valuable cells, whereas lower seeded cell densities may not provide sufficient support for the tissue in vivo, reducing efficacy. Additionally, the production of growth factors by therapeutic cells in low oxygen environments offers a way of generating growth factor gradients, which are important for vascularization, but hypoxia can also induce unwanted levels of cell death. This is a complex problem that lends itself to a combination of computational modelling and experimentation. Here, we present a spatio-temporal mathematical model parametrized using in vitro data capable of simulating the interactions between a therapeutic cell population, oxygen concentrations and vascular endothelial growth factor (VEGF) concentrations in engineered tissues. Simulations of collagen nerve repair constructs suggest that specific seeded cell densities and non-uniform spatial distributions of seeded cells could enhance cell survival and the generation of VEGF gradients. These predictions can now be tested using targeted experiments.
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| 3. |
- Sun, M, et al.
(författare)
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In vitro and in vivo testing of novel ultrathin PCL and PCL/PLA blend films as peripheral nerve conduit
- 2010
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Ingår i: Journal of Biomedical Materials Research. Part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 93:4, s. 1470-1481
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to obviate the drawbacks of nerve autograft, ultrathin microporous biodegradable PCL and PCL/PLA films were tested for their compatibility with motor neuron-like NG108-15 cells and primary Schwann cells. Data obtained from MTS colorimetric and DNA fluorimetric assays showed that both cell lines readily attached and proliferated on these materials. Images taken using scanning electron microscope and fluorescence microscope confirmed these observations. Enhanced cell-surface interaction was achieved by pretreating the films in NaOH solution. Importantly, NG108-15 cells could be induced into differentiated phenotype with long, un-branched neurites growing across the surface of the materials. The bipolar spindle-shaped phenotype of Schwann cells was also retained on these scaffolds. Positive immunochemical staining using antibodies against neurofilament for NG108-15 cells and S100 for Schwann cells indicated the expression of these marker proteins. In a small-scaled pilot testing, the performance of PCL conduits in bridging up a 10 mm gap in rat sciatic nerve model was assessed. Immunohistochemical staining showed that regenerated nerve tissue and penetrated Schwann cells have the potential to span the whole length of the conduit in 2 weeks.
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| 4. |
- Armstrong, Stephanie J., et al.
(författare)
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ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth
- 2007
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Ingår i: Tissue engineering. - : Mary Ann Liebert. - 1076-3279 .- 1557-8690. ; 13:12, s. 2863-2870
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Tidskriftsartikel (refereegranskat)abstract
- Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.
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| 5. |
- Armstrong, Stephanie J, et al.
(författare)
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Laminin activates NF-kappaB in Schwann cells to enhance neurite outgrowth.
- 2008
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Ingår i: Neuroscience Letters. - : Elsevier BV. - 0304-3940 .- 1872-7972. ; 439:1, s. 42-6
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular matrix (ECM) molecules and Schwann cells (SCs) are important components of peripheral nerve regeneration. In this study, the role of the transcription factor nuclear factor kappa B (NF-kappaB) in SC activation in response to laminin and the subsequent effect on in vitro neurite outgrowth was investigated. Immunocytochemistry and Western blot analysis showed that compared with poly-d-lysine (PDL), laminin enhanced the phosphorylation of IkappaB and p65 NF-kappaB signalling proteins in SCs. Phospho NF-kappaB-p65 was localised to the nucleus indicating activation of NF-kappaB. To assess the functional effect of NF-kappaB activation, SCs plated on PDL or laminin were pre-treated with NF-kappaB inhibitors, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) or Z-leu-leu-leu-CHO (MG-132) before NG108-15 neuronal cells were seeded on the SC monolayer. After 24h co-culture in the absence of inhibitors, SCs seeded on laminin enhanced the mean number and length of neurites extended by NG108-15 cells (1.87+/-0.13 neurites; 238.74+/-8.53microm) compared with those cultured in the presence of SCs and PDL (1.26+/-0.07 neurites; 157.57+/-9.80microm). At 72h, neurite length had further increased to 321.83+/-6.60microm in the presence of SCs and laminin. Inhibition of NF-kappaB completely abolished the effect of laminin on SC evoked neurite outgrowth at 24h and reduced the enhancement of neurite length by over 60% at 72h. SC proliferation was unaffected by NF-kappaB inhibition suggesting that the NF-kappaB signalling pathway plays a discrete role in the activation of SCs and their neurotrophic potential.
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| 6. |
- Bojmar, Linda, et al.
(författare)
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Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer
- 2024
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Ingår i: Nature Medicine. - : NATURE PORTFOLIO. - 1078-8956 .- 1546-170X.
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Tidskriftsartikel (refereegranskat)abstract
- Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B(+) natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B(+) NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.<br />
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| 7. |
- Caddick, Jenny, et al.
(författare)
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Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage.
- 2006
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Ingår i: Glia. - : Wiley. - 0894-1491 .- 1098-1136. ; 54:8, s. 840-849
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.
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| 8. |
- El-Habta, Roine, et al.
(författare)
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Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling
- 2018
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Ingår i: Muscle and Nerve. - : WILEY. - 0148-639X .- 1097-4598. ; 57:2, s. 305-311
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: In this study we investigated the interaction between adipose tissue-derived stem cells (ASCs) and myoblasts in co-culture experiments. Methods: Specific inductive media were used to differentiate ASCs in vitro into a Schwann cell-like phenotype (differentiated adipose tissuederived stem cells, or dASCs) and, subsequently, the expression of acetylcholine (ACh)-related machinery was determined. In addition, the expression of muscarinic ACh receptors was examined in denervated rat gastrocnemius muscles. Results: In contrast to undifferentiated ASCs, dASCs expressed more choline acetyltransferase and vesicular acetylcholine transporter. When co-cultured with myoblasts, dASCs enhanced the proliferation rate, as did ACh administration alone. Western blotting and pharmacological inhibitor studies showed that phosphorylated extracellular signal-regulated kinase 1/2 signaling mediated these effects. In addition, denervated muscle showed higher expression of muscarinic ACh receptors than control muscle. Discussion: Our findings suggest that dASCs promote proliferation of myoblasts through paracrine secretion of ACh, which could explain some of their regenerative capacity in vivo.
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| 9. |
- El-Habta, Roine, et al.
(författare)
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Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle
- 2021
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Ingår i: Stem Cell Research & Therapy. - : BioMed Central (BMC). - 1757-6512. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recovery of muscle function after peripheral nerve injury is often poor, and this can be attributed to muscle fiber atrophy and cell death. In the current study, we have investigated the effects of stromal vascular fraction (SVF) on muscle cell apoptosis and its potential to preserve muscle tissue following denervation.Methods: Rat gastrocnemius muscle was denervated by sciatic nerve transection. At 2 and 4 weeks after injury, muscles were examined histologically and apoptosis was measured using TUNEL assay and PCR array for a range of apoptotic genes. Additionally, an in vitro TNF-α apoptosis model was established using SVF cells co-cultured indirectly with primary rat myoblasts. Annexin V and TUNEL were used together with Western blotting to investigate the signaling pathways.Results: Denervated muscles showed significantly higher TUNEL reactivity at 2 and 4 weeks following nerve injury, and an increased expression of caspase family genes, mitochondria-related apoptotic genes, and tumor necrosis factor family genes. In cultured rat primary myoblasts, Annexin V labeling was significantly increased at 12 h after TNF-α treatment, and this was followed by a significant increase in TUNEL reactivity at 48 h. Western blotting showed that caspase-7 was activated/cleaved as well as the downstream substrate, poly (ADP-ribose) polymerase (PARP). Co-culture of myoblasts with SVF significantly reduced all these measures of apoptosis. Bax and Bcl-2 levels were not changed suggesting that the TNF-α-induced apoptosis occurred via mitochondria-independent pathways. The protective effect of SVF was also shown in vivo; injections of SVF cells into denervated muscle significantly improved the mean fiber area and diameter, as well as reduced the levels of TUNEL reactivity.Conclusions: This study provides new insights into how adipose tissue-derived cells might provide therapeutic benefits by preserving muscle tissue.
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| 10. |
- El-Habta, Roine, 1988-
(författare)
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Cell therapy for denervated tissue
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Peripheral nerve injury results in denervation of tendons and muscles. The biology of denervated muscle has been well studied but little is known about the associated tendons. Denervation of muscle leads to atrophy which includes muscle fiber shrinkage and cell death, a process that is influenced by the lack of acetylcholine (ACh) signaling to the muscle cells. Recovery of long-term denervated muscle function is often poor. This thesis describes how a cell therapy approach using adipose tissue-derived stromal vascular fraction (SVF) may be used to protect and regenerate denervated muscle. Previous studies have shown how adipose tissue-dervied stem cells (ASCs), commonly expanded from the SVF, have pro-regenerative effects on the injured peripheral nervous system, and how ASCs differentiated towards a “Schwann cell-like phenotype” (dASCs) reduce muscle atrophy. In this thesis work, we studied the possible mechanisms underlying the regenerative potential of both SVF and culture expanded dASCs.Hypotheses: We hypothesized that: 1) denervated tendon displays morphological and biochemical properties that resemble the chronic degenerative tendon condition known as tendinosis; 2) denervated muscle up-regulates expression of muscarinic acetylcholine (ACh) receptors and apoptosis-associated signaling mechanisms; 3) dASCs enhance the proliferation of myoblasts in vitro through secretion of ACh; 4) SVF influences the proliferation, differentiation, and survival of myoblasts in vitro via secretion of growth factors; and 5) SVF can preserve denervated muscle tissue. To test our hypotheses, two model systems were used: an in vitro model based on indirect co-culture, and an in vivo rat sciatic nerve transection model.Results: Denervated tendon displayed morphological changes similar to tendinosis, including hypercellularity, disfigurement of cells, and disorganized collagen architecture, along with an increased expression of type I and type III collagen. In addition, levels of neurokinin 1 receptor (NK-1R) were upregulated in the tendon cells. In denervated muscle, there was an increased expression of muscarinic ACh receptors, as well as of genes associated with apoptosis, such as caspases, cytokines (e.g., tumor necrosis factor-alpha; TNF-a), and death domain receptors. We subsequently used TNF-aas an inducer of apoptosis in an in vitrorat primary myoblast culture model. TNF-aactivated/cleaved caspase 7 and increased poly ADP-ribose polymerase (PARP) levels. Moreover, Annexin V and TUNEL were increased after TNF-atreatment. Indirect co-culture with SVF significantly reduced all these measures of apoptosis. Proliferation studies showed that both dASCs and SVF enhanced growth of myoblasts in vitro. With dASCs, the effect was partially explained by secretion of ACh, and for SVF by released growth factors, such as hepatocyte growth factor (HGF). In both cases, the signal was mediated via phosphorylation of ERK1/2 (MAPK). HGF also had an inhibitory effect on the differentiation of myoblasts into myotubes. Finally, the protective effects of SVF were confirmed in vivo: injections of SVF into denervated muscle significantly increased the mean fiber area and diameter, as well as reduced the expression of apoptotic genes and TUNEL reactivity.Conclusions: Denervated tendons undergo severe degenerative changes similar to tendinosis. Furthermore, SVF has the ability to reduce muscle atrophy in vivo. Using in vitro systems, we showed that this might occur through secretion of growth factors which activate MAPK signaling and anti-apoptotic pathways. In conclusion, SVF offers a promising approach for future clinical application in the treatment of denervated muscle.
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