| 1. |
- Bermejo, Daniel, 1985-, et al.
(författare)
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First Aldol-Crosslinked Hyaluronic Acid Hydrogel: Fast and Hydrolytically Stable Gel with Tissue Adhesive Properties
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Ingår i: Chemical Sciences Journal. - 2150-3494.
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Tidskriftsartikel (refereegranskat)abstract
- Currently, there are limited approaches to tailor 3D scaffolds crosslinked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-crosslinking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely; an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior crosslinking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of crosslinking at different pH is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogels demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product leads to adhesion to tissue as demonstrated by bonding two bone tissues. The aldehyde functionality also permits facile post-synthetic modifications with nucleophilic reagents such as Alexa FluorTM 488. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3–6 days of study.
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| 2. |
- Bermejo-Velasco, Daniel, 1985-, et al.
(författare)
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First Aldol Cross-Linked Hyaluronic Acid Hydrogel : Fast and Hydrolytically Stable Hydrogel with Tissue Adhesive Properties
- 2019
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Ingår i: ACS Applied Materials and Interfaces. - : American Chemical Society (ACS). - 1944-8244 .- 1944-8252. ; 11:41, s. 38232-38239
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Tidskriftsartikel (refereegranskat)abstract
- Currently, there are limited approaches to tailor 3D scaffolds cross-linked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-cross-linking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely, an enolizable HA-aldehyde (HA-EaI) and a non-enolizable HA-aldehyde (HA-NaI). Hydrogels formed using HA-EaI demonstrate inferior cross linking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-EaI and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of cross-linking at different pH values is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Me). The novel HA cross-aldol hydrogel demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase-mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product rendered the tissue adhesive properties by bonding two bone tissues. The aldehyde functionality also facilitated facile post-synthetic modifications with nucleophilic reagents. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3-6 days of study.
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| 3. |
- Beusch, Christian M., et al.
(författare)
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Analysis of local extracellular matrix identifies different aetiologies behind bicuspid and tricuspid aortic valve degeneration and suggests therapies
- 2023
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Nature. - 1420-682X .- 1420-9071. ; 80:9
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Tidskriftsartikel (refereegranskat)abstract
- Aortic valve degeneration (AVD) is a life-threatening condition that has no medical treatment and lacks individual therapies. Although extensively studied with standard approaches, aetiologies behind AVD are unclear. We compared abundances of extracellular matrix (ECM) proteins from excised valve tissues of 88 patients with isolated AVD of normal tricuspid (TAV) and congenital bicuspid aortic valves (BAV), quantified more than 1400 proteins per ECM sample by mass spectrometry, and demonstrated that local ECM preserves molecular cues of the pathophysiological processes. The BAV ECM showed enrichment with fibrosis markers, namely Tenascin C, Osteoprotegerin, and Thrombospondin-2. The abnormal physical stress on BAV may cause a mechanical injury leading to a continuous Tenascin C-driven presence of myofibroblasts and persistent fibrosis. The TAV ECM exhibited enrichment with Annexin A3 (p = 1.1 x 10(-16) and the fold change 6.5) and a significant deficit in proteins involved in high-density lipid metabolism. These results were validated by orthogonal methods. The difference in the ECM landscape suggests distinct aetiologies between AVD of BAV and TAV; warrants different treatments of the patients with BAV and TAV; elucidates the molecular basis of AVD; and implies possible new therapeutic approaches. Our publicly available database (human_avd_ecm.surgsci. uu.se) is a rich source for medical doctors and researchers who are interested in AVD or heart ECM in general. Systematic proteomic analysis of local ECM using the methods described here may facilitate future studies of various tissues and organs in development and disease.
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| 4. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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PDMS leaching and its implications for on-chip studies focusing on bone regeneration applications
- 2020
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Ingår i: Organs-on-a-Chip. - : Elsevier. - 2666-1020. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite itsmultiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMSon the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMSon cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS,namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chipswere prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel whenkept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concen-tration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spec-trometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment,were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and humanmesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional tothe tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnightinstead of 2 h, regardless of the curing temperature (65 and 80 C), a large reduction in silicon concentration wasfound, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligo-mers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as nochanges in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates theimportance of optimization steps when using PDMS devices for biological studies, in particular PDMS curingconditions and extensive washing steps prior to an experiment.
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| 5. |
- Kadekar, Sandeep, et al.
(författare)
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Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures
- 2021
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Ingår i: Cellular and Molecular Bioengineering. - : Springer Nature. - 1865-5025 .- 1865-5033. ; 14, s. 121-130
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments.Methods: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 mu M) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPAR gamma (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated.Results: Daily delivering 5-AzaC caused a higher upregulation of PPAR gamma, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPAR gamma (tenfold-change) could indicate priming of hMSCs towards adipocytes.Conclusions: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.
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| 6. |
- Kadekar, Sandeep
(författare)
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Gender differences in chemical carcinogenesis
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gender differences in cancer incidence and mortality is a regular finding in epidemiological studies. In addition to reproductive organs this pattern is also seen in non-reproductive organs, with men being the most affected gender for the majority of cancer-sites. The underlying reasons for the observed disparity are not known, but can partly be explained by differences in exposures, lifestyle factors and biological factors such as hormones and metabolism. Exposure to carcinogenic chemicals is one of the risk factors for cancer, however little is known about gender-specific sensitivity to carcinogens. The overall aim of this thesis was to investigate gender differences in susceptibility to chemical carcinogens and the underlying mechanisms. In the National Toxicology Program (NTP) database detailed technical reports from 2-year bioassays on male and female rats exposed to the same concentration of chemical in well-controlled environments is publicly available. In the first paper, 477 chemicals tested on rats were evaluated for possible gender differences in the carcinogenic effect. The analysis of NTP bioassays showed that male rats were more affected than females. In a total of 278 carcinogens, 201 showed statistically significant gender differences in at least one non-reproductive organ. 69 carcinogens induced male-specific tumors and 19 induced female-specific tumors. Male-specific tumors included for example mesothelioma, kidney-, skin- and pancreas tumors, while female-specific tumors included neoplasms in pituitary, bone marrow and lymphoid tissues, lung and urinary bladder. The study further showed that genotoxicity was more common among male-specific carcinogens, compared to female-specific carcinogens. Based on the results from the NTP study eight male-specific pancreatic carcinogens were studied in more detail in the second study. To find common mechanisms that could clarify the male-specific effect of these carcinogens, the published literature on the eight chemicals was analyzed using a text-mining tool, CRAB. This analysis proposed inflammation as a common mechanism for these carcinogens. In in vitro studies it was found that all eight carcinogens increased the levels of the inflammatory protein Autotaxin (ATX), in parallel with increased invasiveness. Testosterone further increased ATX levels, alone and in combination with carcinogens. These data suggests that ATX may be a target for carcinogens that promote pancreatic tumor development. In the third study, the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Polychlorinated biphenyl (PCB) and estradiol on benzo(a)pyrene (BaP)-induced apoptosis and p53 signaling was investigated. The results showed that BaP induced apoptosis increased nuclear p53 and phosphorylation of FOXO3a. The apoptotic effect of BaP was attenuated by pretreatment of TCDD, PCB or estradiol, leading to a further increase in nuclear p53 and decreased levels of phosphorylated FOXO3a. FOXO3a dephosphorylation was further showed to be essential for the attenuated apoptosis and nuclear trapping of p53, which resulted in restoration of BaP-induced apoptosis. The data suggests an interaction between p53 and FOXO3a, which leads to an attenuated BaP-induced apoptosis in cells co-exposed to TCDD, PCB153 or estradiol. This study also reflects the effect of estradiol as a modulator of the toxic response caused by carcinogens. In conclusion, the results of this thesis show that male rats are more sensitive to chemical carcinogens compared to female rats. The data further suggests interactions between hormones and carcinogens that could be important for the cellular response to carcinogens.
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| 7. |
- Kadekar, Sandeep, et al.
(författare)
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Redox responsive Pluronic micelle mediated delivery of functional siRNA : a modular nano-assembly for targeted delivery
- 2021
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Ingår i: Biomaterials Science. - : Royal Society of Chemistry. - 2047-4830 .- 2047-4849. ; 9:11, s. 3939-3944
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Tidskriftsartikel (refereegranskat)abstract
- There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of similar to 291 nm in DLS and similar to 200-250 nm in SEM at 37 degrees C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in similar to 65% silencing of STAT3 mRNA in BT-13 gliospheres, while only similar to 20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.
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| 8. |
- Kadekar, Sandeep, et al.
(författare)
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Synthetic design of asymmetric miRNA with engineered 3′-overhang to improve strand selection
- 2019
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier. - 2162-2531. ; 16, s. 597-604
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′-end by adding extra nucleotides. Addition of seven nucleotides at the 3′-ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two-ends, which resulted in selective RISC recruitment as demonstrated by the stem-loop quantitative PCR experiment. Such selective recruitment was also corroborated at the protein level by Western blot analysis. In order to investigate the functional effect due to selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that the recruitment of miR strand is responsible for inducing apoptosis as well as to inhibit invasiveness of cancer cells. Recruitment of miR* strand, on the other hand, showed opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNA to enhance their therapeutic potential.
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| 9. |
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| 10. |
- Nawale, Ganesh N., et al.
(författare)
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4 '-Guanidinium-modified siRNA : a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading
- 2019
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Ingår i: Chemical Communications. - : ROYAL SOC CHEMISTRY. - 1359-7345 .- 1364-548X. ; 55:62, s. 9112-9115
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Tidskriftsartikel (refereegranskat)abstract
- We designed novel 4 '-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in approximate to 4.9-fold more efficient knockdown than unmodified siRNA.
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