| 1. |
- Yan, Hongji, et al.
(författare)
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Synthetic design of growth factor sequestering extracellular matrix mimetic hydrogel for promoting in vivo bone formation
- 2018
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 161, s. 190-202
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Tidskriftsartikel (refereegranskat)abstract
- Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor.
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| 2. |
- Bermejo-Velasco, Daniel, 1985-, et al.
(författare)
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Modulating thiol pKa promotes disulfide formation at physiological pH : An elegant strategy to design disulfide cross-linked hyaluronic acid hydrogels
- 2019
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 20:3, s. 1412-1420
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Tidskriftsartikel (refereegranskat)abstract
- The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the β-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as L-cysteine indicated disulfide formation at a 2.8-fold higher (k1 = 5.04 x 10-4 min-1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid (k1 = 1.80 x 10-4 min-1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-L-cysteine substrate (k1 = 0.51 x 10-4 min-1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the pKa value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the β-position reduced the pKa of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys) and 8.1 for HA-thiol (HA-SH) derivatives respectively. These experiments further confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 minutes or 10 hours, respectively at pH 7.4. After completion of crosslinking reaction both gels demonstrated a storage modulus G’ ≈3300–3500 Pa, indicating comparable levels of crosslinking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.
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| 3. |
- Ferreira, Silvia A, et al.
(författare)
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Bi-directional cell-pericellular matrix interactions direct stem cell fate
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells' 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells' reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC's interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.
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| 4. |
- Roshanbinfar, Kaveh, et al.
(författare)
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Injectable and thermoresponsive pericardial matrix derived conductive scaffold for cardiac tissue engineering
- 2017
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Ingår i: RSC Advances. - 2046-2069. ; 7:51, s. 31980-31988
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Tidskriftsartikel (refereegranskat)abstract
- Scaffolds derived from decellularized cardiac tissue offer an enormous advantage for cardiac applications as they recapitulate biophysical and cardiac specific cues. However, poor electrical conductivity and mechanical properties severely compromise the therapeutic potential of these matrices. Dispersion of multiwall carbon nanotubes (MWCTs) in these scaffolds could improve their mechanical and electrical properties. However, the inherent hydrophobicity and poor dispersibility of these materials under aqueous conditions limit their outcome. We have developed a modified MWCNT functionalized with carbodihydrazide (CDH) residues that significantly improved their dispersibility and suppressed cytotoxicity in HL-1 cardiomyocytes. We found that the doping of CDH functionalized MWCNT (CDH-MWCNT) as low as 0.5 wt% to the pericardial matrix hydrogel (PMNT) induced the necessary electrical conductivity and significantly improved the mechanical properties of the hydrogel. Cardiomyocytes cultured on a PMNT scaffold triggered proliferation and significantly increased the expression of cardiac-specific gap junction protein, namely connexin 43. Such a phenomenon was not observed when cardiomyocytes were cultured on the pericardial matrix derived gels without MWCNT or on gelatin-fibronectin coated 2D cultures. The PMNT gels displayed excellent biophysical characteristics resulting in the clustering of cardiomyocytes with synchronous contraction, which is crucial for the successful integration to the host tissue.
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| 5. |
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| 6. |
- 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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| 7. |
- Bermejo-Velasco, Daniel, et al.
(författare)
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Thiazolidine chemistry revisited : a fast, efficient and stable click-type reaction at physiological pH
- 2018
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 54:88, s. 12507-12510
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Tidskriftsartikel (refereegranskat)abstract
- We describe the fast reaction kinetics between 1,2-aminothiols and aldehydes. Under physiological conditions such a click-type reaction afforded a thiazolidine product that remains stable and did not require any catalyst. This type of bioorthogonal reaction offers enormous potential for the coupling of biomolecules in an efficient and biocompatible manner.
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| 8. |
- 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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| 9. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
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An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier : The First Biomimetic, Anionic, Non-Viral Transfection Method
- 2019
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Ingår i: Angewandte Chemie International Edition. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 58:9, s. 2815-2819
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Tidskriftsartikel (refereegranskat)abstract
- Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size- and concentration-dependent gene silencing in a CD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNA transfection represents the first report on an anionic, non-viral delivery method that resulted in approximately 60% gene knockdown in both cell types tested, which correlated with a reduction in translation levels.
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| 10. |
- Rangasami, Vignesh K., et al.
(författare)
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Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function
- 2023
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Ingår i: Biomaterials Advances. - : Elsevier. - 2772-9516 .- 2772-9508. ; 147
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and miti-gated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and dif-ferentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.
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| 11. |
- Samal, Jay R. K., et al.
(författare)
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Discrepancies on the Role of Oxygen Gradient and Culture Condition on Mesenchymal Stem Cell Fate
- 2021
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Ingår i: Advanced Healthcare Materials. - : John Wiley & Sons. - 2192-2640 .- 2192-2659. ; 10:6
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Forskningsöversikt (refereegranskat)abstract
- Over the past few years, mesenchymal stem (or stromal) cells (MSCs) have garnered enormous interest due to their therapeutic value especially for their multilineage differentiation potential leading to regenerative medicine applications. MSCs undergo physiological changes upon in vitro expansion resulting in expression of different receptors, thereby inducing high variabilities in therapeutic efficacy. Therefore, understanding the biochemical cues that influence the native local signals on differentiation or proliferation of these cells is very important. There have been several reports that in vitro culture of MSCs in low oxygen gradient (or hypoxic conditions) upregulates the stemness markers and promotes cell proliferation in an undifferentiated state, as hypoxia mimics the conditions the progenitor cells experience within the tissue. However, different studies report different oxygen gradients and culture conditions causing ambiguity in their interpretation of the results. In this progress report, it is aimed to summarize recent studies in the field with specific focus on conflicting results reported during the application of hypoxic conditions for improving the proliferation or differentiation of MSCs. Further, it is tried to decipher the factors that can affect characteristics of MSC under hypoxia and suggest a few techniques that could be combined with hypoxic cell culture to better recapitulate the MSC tissue niche.
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| 12. |
- Samanta, Sumanta, et al.
(författare)
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Heparin-Derived Theranostic Nanoprobes Overcome the Blood-Brain Barrier and Target Glioma in Murine Model
- 2022
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Ingår i: Advanced Therapeutics. - : John Wiley & Sons. - 2366-3987. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- The poor permeability of theranostic agents across the blood-brain barrier (BBB) significantly hampers the development of new treatment modalities for neurological diseases. A new biomimetic nanocarrier is discovered using heparin (HP) that effectively passes the BBB and targets glioblastoma. Specifically, HP-coated gold nanoparticles (HP-AuNPs) are designed that are labeled with three different imaging modalities namely, fluorescein (FITC-HP-AuNP), radioisotope (68)Gallium (Ga-68-HP-AuNPs), and MRI active gadolinium (Gd-HP-AuNPs). The systemic infusion of FITC-HP-AuNPs in three different mouse strains (C57BL/6JRj, FVB, and NMRI-nude) displays excellent penetration and reveals uniform distribution of fluorescent particles in the brain parenchyma (69-86%) with some accumulation in neurons (8-18%) and microglia (4-10%). Tail-vein administration of radiolabeled Ga-68-HP-AuNPs in healthy rats also show Ga-68-HP-AuNP inside the brain parenchyma and in areas containing cerebrospinal fluid, such as the lateral ventricles, the cerebellum, and brain stem. Finally, tail-vein administration of Gd-HP-AuNPs (that displays approximate to threefold higher relaxivity than that of commercial Gd-DTPA) in an orthotopic glioblastoma (U87MG xenograft) model in nude mice demonstrates enrichment of T1-contrast at the intracranial tumor with a gradual increase in the contrast in the tumor region between 1 and 3 h. It is believed, the finding offers the untapped potential of HP-derived-NPs to deliver cargo molecules for treating neurological disorders.
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| 13. |
- Samanta, Sumanta, et al.
(författare)
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Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype
- 2022
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Ingår i: Acta Biomaterialia. - : Elsevier. - 1742-7061 .- 1878-7568. ; 142, s. 36-48
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Tidskriftsartikel (refereegranskat)abstract
- Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-β, and TGF-βR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1β, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-β. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions.Statement of significanceHost immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.
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| 14. |
- Tavakoli, Shima, et al.
(författare)
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Fine-tuning Dynamic Cross-linking for Enhanced 3D Bioprinting of Hyaluronic Acid Hydrogels
- 2024
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Ingår i: Advanced Functional Materials. - : Wiley-VCH Verlagsgesellschaft. - 1616-301X .- 1616-3028. ; 34:4
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Tidskriftsartikel (refereegranskat)abstract
- 3D bioprinting of stem cells shows promise for medical applications, but the development of an efficient bioink remains a challenge. Recently, the emergence of dynamically cross-linked hydrogels has advanced this field to obtain self-healing materials. However, more advanced bioinks are needed that display optimum gelling kinetics, viscoelasticity, shear-thinning property, structural fidelity, and hold the printed structures sufficiently long enough that allow maturation of the new tissue. Here, a novel extracellular matrix-based bioink for human mesenchymal stem cells (hMSCs) is presented. Hyaluronic acid (HA) is modified with cysteine and aldehyde functional groups, creating hydrogels with dual cross-linking of disulfide and thiazolidine products. The investigation demonstrates that this cross-linking significantly improves hydrogel stability and biological properties. The bioink exhibits fast gelation kinetics, shear-thinning, shape-maintaining properties, high cell survival after printing with >2-fold increase in stemness marker (OCT3/4 and NANOG), and supports cell proliferation and migration. Disulfide cross-linking contributes to self-healing and cell migration, while thiazolidine cross-linking reduces gelation time, enhances long-term stability, and supports cell proliferation. Overall, the HA-based bioink fulfills the requirements for successful 3D printing of stem cells, providing a promising solution for cell therapy and regenerative medicine.
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| 15. |
- Tavakoli, Shima, et al.
(författare)
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Unveiling extracellular matrix assembly : Insights and approaches through bioorthogonal chemistry
- 2023
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Ingår i: MATERIALS TODAY BIO. - : Elsevier BV. - 2590-0064. ; 22
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Tidskriftsartikel (refereegranskat)abstract
- Visualizing cells, tissues, and their components specifically without interference with cellular functions, such as biochemical reactions, and cellular viability remains important for biomedical researchers worldwide. For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or in vitro differentiation of stem cells.
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| 16. |
- Wang, Shujiang, et al.
(författare)
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Dynamic covalent crosslinked hyaluronic acid hydrogels and nanomaterials for biomedical applications
- 2022
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Ingår i: Biomaterials Science. - : Royal Society of Chemistry (RSC). - 2047-4830 .- 2047-4849. ; 10:22, s. 6399-6412
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Forskningsöversikt (refereegranskat)abstract
- Hyaluronic acid (HA), one of the main components of the extracellular matrix (ECM), is extensively used in the design of hydrogels and nanoparticles for different biomedical applications due to its critical role in vivo, degradability by endogenous enzymes, and absence of immunogenicity. HA-based hydrogels and nanoparticles have been developed by utilizing different crosslinking chemistries. The development of such crosslinking chemistries indicates that even subtle differences in the structure of reactive groups or the procedure of crosslinking may have a profound impact on the intended mechanical, physical and biological outcomes. There are widespread examples of modified HA polymers that can form either covalently or physically crosslinked biomaterials. More recently, studies have been focused on dynamic covalent crosslinked HA-based biomaterials since these types of crosslinking allow the preparation of dynamic structures with the ability to form in situ, be injectable, and have self-healing properties. In this review, HA-based hydrogels and nanomaterials that are crosslinked by dynamic-covalent coupling (DCC) chemistry have been critically assessed.
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| 17. |
- Wang, Shujiang, et al.
(författare)
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Influence of ions to modulate hydrazone and oxime reaction kinetics to obtain dynamically cross-linked hyaluronic acid hydrogels
- 2019
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Ingår i: Polymer Chemistry. - : ROYAL SOC CHEMISTRY. - 1759-9954 .- 1759-9962. ; 10:31, s. 4322-4327
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic covalent chemistry forming hydrazone and oxime linkages is attractive due to its simplicity, selectivity and compatibility under aqueous conditions. However, the low reaction rate at physiological pH hampers its use in biomedical applications. Herein, we present different monovalent and bivalent aqueous salt solutions as bio-friendly, non-toxic catalysts which can drive the hydrazone and oxime reactions with excellent efficacy at physiological pH. Direct comparison of hydrazone and oxime reactions using a small molecule model, without any salt catalysis, indicated that oxime formation is 6-times faster than hydrazone formation. Addition of different salts (NaCl, NaBr, KCl, LiCl, LiClO4, Na2SO4, MgCl2 and CaCl2) accelerated the pseudo-first-order reaction kinetics by similar to 1.2-4.9-fold for acylhydrazone formation and by similar to 1.5-6.9-fold for oxime formation, in a concentration-dependent manner. We further explored the potential of such catalysts to develop acylhydrazone and oxime cross-linked hyaluronic acid (HA) hydrogels with different physicochemical properties without changing the degree of chemical modification. Analogous to the small molecule model system, the addition of monovalent and divalent salts as catalysts significantly reduced the gelling time. The gelling time for the acylhydrazone cross-linked HA-hydrogel (1.6 wt%) could be reduced from 300 min to 1.2 min by adding 100 mM CaCl2, while that for the oxime cross-linked HA-hydrogel (1.2 wt%) could be reduced from 68 min to 1.1 min by adding 50 mM CaCl2. This difference in the gelling time also resulted in hydrogels with differential swelling properties as measured after 24 h. Our results are the first to demonstrate the use of salts, for catalyzing hydrogel formation under physiologically relevant conditions.
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| 18. |
- Wang, Shujiang, et al.
(författare)
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Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.
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| 19. |
- Wang, Shujiang, et al.
(författare)
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Saline catalyse oxime reaction at physiological pH : overcoming a major limitation of bioorthogonal reaction
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Tidskriftsartikel (refereegranskat)abstract
- We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most bio-friendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.
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| 20. |
- 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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| 21. |
- 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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| 22. |
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| 23. |
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| 24. |
- Podiyan, Oommen, 1977-, et al.
(författare)
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Smart design of stable hydrazone crosslinked extracellular matrix mimetic hydrogel for tissue engineering application
- 2012
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Ingår i: Journal of Tissue Engineering and Regenerative Medicine. - : Hindawi Limited. - 1932-6254. ; 6:suppl 1, s. 192-192
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Injectable hydrogels are important biomaterials with enormous applications. They are used for various biomedical applications such as diagnostics, 3D cell culture matrix, drug reservoir, encapsulation of bioactive compounds and growth factors, scaffold for tissue engineering etc. We here present our recent development in our efforts to develop hydrogel scaffolds with enhanced rigidity, stability, swelling characteristics. Hydrazone crosslinked gels are attractive due to its simplicity and versatility which could be formed by mixing appropriate aldehyde and hydrazide functionalized hyaluronan. By fine-tuning the electronic character around the hydrazone linkage, we succeeded in developing extremely stable hydrazone bond and utilized it for developing hyaluronan (HA) based synthetic extracellular matrix (ECM) hydrogel. Among the different hydrazides tested, we identified carbonyldihydrazide (CDH) as the best candidate to deliver stable hydrazone linkage. This stability is presumably due to extensive delocalization of the positive charge across neighboring amino groups of CDH. The hydrolytic stability imparted by this group was found to be several folds under acidic, basic and physiological pH when compared to other hydrazones. This tailored hydrogel with CDH also exhibited superior swelling and mechanical properties and enzymatic stability which makes it ideal for tissue engineering application.
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| 25. |
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| 26. |
- Mittapelli, Lavanya L., et al.
(författare)
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A turn-on fluorescent GFP chromophore analog for highly selective and efficient detection of H2S in aqueous and in living cells
- 2019
-
Ingår i: Sensors and actuators. B, Chemical. - : ELSEVIER SCIENCE SA. - 0925-4005 .- 1873-3077. ; 298
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen sulphide is a gaseous neurotransmitter responsible for neuronal function and controls vast range of physiological functions. Herein, we report the synthesis and evaluation of novel Green Fluorescent Protein (GFP) chromophore analog, acryloyl-4-(p-hydroxybenzylidene)-5-imidazolidinone (AHBI) for turn-on fluorescent detection of H2S over wide range of anions and various biologically important competitive thiols. AHBI probe exhibited high selectivity and sensitivity, high fluorescence stability, large stokes shift and lower detection limit (15.85 ppb) for H2S in complete water medium. Cell imaging studies in human colon cancer cells (HCT116) and normal human dermal fibroblasts (HDF) confirmed the compatibility and versatility of AHBI probe at micromolar level. Overall, we believe the AHBI, as an optical probe will be useful to investigate the role of H2S in various physiological processes, regulation of cancer cell growth, and in pathogenic events.
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| 27. |
- Bajic, Andrej, et al.
(författare)
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Physically and Chemically Crosslinked Hyaluronic Acid-Based Hydrogels Differentially Promote Axonal Outgrowth from Neural Tissue Cultures
- 2024
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Ingår i: Biomimetics. - : MDPI. - 2313-7673. ; 9:3
-
Tidskriftsartikel (refereegranskat)abstract
- Our aim was to investigate axonal outgrowth from different tissue models on soft biomaterials based on hyaluronic acid (HA). We hypothesized that HA-based hydrogels differentially promote axonal outgrowth from different neural tissues. Spinal cord sliced cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) were maintained on a collagen gel, a physically crosslinked HA-based hydrogel (Healon 5®) and a novel chemically crosslinked HA-based hydrogel, with or without the presence of neurotrophic factors (NF). Time-lapse microscopy was performed after two, five and eight days, where axonal outgrowth was assessed by automated image analysis. Neuroprotection was investigated by PCR. Outgrowth was observed in all groups; however, in the collagen group, it was scarce. At the middle timepoint, outgrowth from SCSCs was superior in both HA-based groups compared to collagen, regardless of the presence of NF. In DRGCs, the outgrowth in Healon 5® with NF was significantly higher compared to the rest of the groups. PCR revealed upregulation of NeuN gene expression in the HA-based groups compared to controls after excitotoxic injury. The differences in neurite outgrowth from the two different tissue models suggest that axons differentially respond to the two types of biomaterials.
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| 28. |
- Bermejo-Velasco, Daniel, 1985-
(författare)
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Insights into Covalent Chemistry for the Development of Biomaterials
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Covalent cross-linking chemistry is currently exploited in the preparation of biomaterial for biomedical applications. Choice of these chemistries for the preparation of biomaterials and bioconjugates strongly influences the biological output of these materials. Therefore, this thesis aims to develop novel bioconjugation strategies understanding their advantages and drawbacks. Our results provide new insight to adapt these chemical transformations for a specific application.The first part of this thesis points out the relevance of tuning different properties of biomaterials with specific emphasis on the development of hyaluronic acid (HA) hydrogels. The second part of the thesis describes how different chemical transformations including hydrazone formation (Paper I), thiazolidine formation (Paper II), cross-aldol addition reaction (Paper III) and disulfide formation (Paper IV) dictate material properties.This thesis explores both basic organic reaction mechanism and application of these reactions to influence material characteristics. The detailed study of the reaction conditions, kinetics, and stability of the products will help to understand the mechanical properties, hydrolytic stability, and degradability of the materials described here.Additionally, we performed degradation studies of gadolinium labeled HA hydrogels using magnetic resonance imaging. Furthermore, we also explored post-synthetic modification of hydrogels to link model fluorescent moieties as well as explored the tissue adhesive properties using Schiff-base formation.In summary, this thesis presents a selection of different covalent chemistries for the design of advanced biomaterials. The advantages and disadvantages of these chemistries are rigorously investigated. We believe, such an investigation provides a better understanding of the bioconjugation strategies for the preparation of biomaterials with potential clinical translation.
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| 29. |
- 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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| 30. |
- Han, Yuanyuan, et al.
(författare)
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MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification
- 2019
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 127, s. 188-193
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Tidskriftsartikel (refereegranskat)abstract
- DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.
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| 31. |
- Hilborn, Jöns, 1956-, et al.
(författare)
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Keynote: Safe and efficient in-vivo gene gene transfer and silencing technologies using natural pathways
- 2012
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Ingår i: Journal of Tissue Engineering and Regenerative Medicine. - : Hindawi Limited. - 1932-6254. ; 6:suppl 1, s. 190-190
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: The advance in nucleic acids therapeutic had been outstanding in recent years, which have opened new possibilities in regenerative medicine to tackle many serious diseases. To make nucleic acids based therapy a reality, the fundamental problem of tissue specific cellular delivery has to be accomplished. Here we present a new approach to develop the next generation of delivery vectors.Materials and methods: Hyaluronic acid were modified to complex DNA plasmids for luciferase transfection. Transfection: HCT 116 cells and mice where tranfected by adding HA-DNA complex. The transfection experiment with commercially available reagent polyethyleneimine (PEI) and plasmid alone were used as controls.Results and discussion: Efficient tranfection was shown using the new non toxic and safe non viral vector. Using ECM components (such as hyaluronic acid or HA) that allow both in vitroandin vivo transfection of plasmid DNA in CD44 positive cells. The chemically modified ECM components (non-toxic) binds to nucleic acids and are taken in by cells by natural receptor mediated endocytosis. The amount transported via these routes naturally is around 10 gram per day to give a potential capacity far exceeding the need for transfection if it is targeted. By molecular association strategies (joining the bandwagon) it is now possible for nucleic acids to follow these pathways that utilise the highly efficient receptor mediated endocytosis- door opener into cells.
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| 32. |
- 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
-
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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| 33. |
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| 34. |
- Laskar, Partha, et al.
(författare)
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Advances in Intracellular and On-Surface Polymerization in Living Cells : Implications for Nanobiomedicines
- 2023
-
Ingår i: Advanced NanoBiomed Research. - : Wiley-VCH Verlagsgesellschaft. - 2699-9307. ; 3:8
-
Forskningsöversikt (refereegranskat)abstract
- The cellular environment offers some unique features to carry out polymerizations under controlled conditions. Polymerization of monomers in cellular compartments and on the surface of living organisms holds much promise in the engineering of biofunctional synthetic polymers for sensing and probing cell behavior and of late has received significant interest. This effort lies at the interface of synthetic biology and polymer chemistry and can pave the way for innovative solutions to many existing challenges in healthcare, environment, energy, and the study of the "origin of life". Herein, recent advances in controlled polymerization strategies for intracellular and surface of living cells are presented with a particular emphasis on nanobiomedicines. Furthermore, polymerization strategies, cytocompatible monomer structures, compatible cell lines and microorganisms, nature of stimulus, catalysts, along with specific polymerization conditions to produce non-natural biofunctional polymers that can undergo polymerization-induced self-assembly within and onto the living cells are presented in detail. Furthermore, the review offers a window into the future of such novel emerging synthetic bionano systems in biomedical sciences.
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| 35. |
- 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
-
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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| 36. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
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Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2
- 2019
-
Ingår i: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422.
-
Tidskriftsartikel (refereegranskat)abstract
- Designing strategies to deliver functional proteins at physiologically relevant concentrations using chemically cross-linked biocompatible hydrogels is a major field of research. However, the impact of cross-linking chemistry on the encapsulated protein bioactivity is rarely studied. Here we examine the two well-known cross-linking reactions namely; hydrazone cross-linking chemistry and thiol-Michael addition reaction to form hyaluronic acid (HA) hydrogels. As a therapeutic protein, we employed recombinant human bone morphogenetic protein-2 (rhBMP-2) for this study. Incubation of rhBMP-2 with HA functionalized with a thiol diminished phosphorylation of Smad 1/5/8, a signal transducer for osteogenic differntiation, whereas an aldehyde functionalized HA had no effect. This indicates that thiol functionalized polymers indeed has an impact on protein function. To validate this result in an in vivo setting we performed BMP-2 induced bone formation in a rat ectopic model. These experiments revealed that the hydrazone-cross-linked HA-hydrogel induced significantly higher bone formation (18.90 ± 4.25 mm3) as compared to the HA-thiol-Michael hydrogels (1.25 ± 0.52 mm3) after 8 weeks as determined by micro-computed tomography. The histological examination of the neo-bone indicated that hydrazone-hydrogels promoted a better quality of bone formation with improved mineralization and collagen formation as compared to the thiol-Michael hydrogels. We believe such a direct comparison of two cross-linking chemistries will provide new insight for developing biomaterials for protein delivery for in vivo applications.
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| 37. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
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Insights into siRNA Transfection in Suspension : Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel
- 2019
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 20:3, s. 1317-1324
-
Tidskriftsartikel (refereegranskat)abstract
- Small interfering RNAs (siRNAs) are powerful toolsfor post-transcriptional gene silencing, which offers enormousopportunities for tissue engineering applications. However, poorserum stability, inefficient intracellular delivery, and inevitabletoxicity of transfection reagents are the key barriers for their clinicaltranslation. Thus, innovative strategies that allow safe and efficientintracellular delivery of the nucleic acid drugs at the desired site isurgently needed for a smooth clinical translation of therapeuticallyappealing siRNA-based technology. In this regard, we havedeveloped an innovative siRNA transfection protocol that employsa short incubation time of just 5 min. This allows easy transfection insuspension followed by transplantation of the cells in a hyaluronicacid (HA) hydrogel system. We also report here the unique ability ofsiRNA to bind HA that was quantified by siRNA release andrheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA insuspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to beessential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with orwithout removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removalof excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improvedknockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) asdetermined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the proteinlevels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allowsminimal manipulation of cells that are to be transplanted and reduce toxicity.
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| 38. |
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| 39. |
- Paidikondala, Maruthibabu, 1985-
(författare)
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Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bone is a tissue that heals by itself, unless the defect is too large (critical size). Today, novel regenerative medicine approaches have emerged as an alternative to treat such defects. This thesis explores alternative therapeutic strategies for bone tissue engineering which are biocompatible and clinically translatable. Many types of scaffolds that can act as reservoirs for growth factors such as rh-BMP-2 have been developed for bone tissue engineering in the past. However, the role of cross-linking chemistries that are employed to make hydrogels on the integrity and function of the loaded growth factors is not well understood. In this thesis, we have explored the influence of cross-linking chemistry on rh-BMP-2 integrity and bioactivity both in-vitro and in-vivo. These studies have demonstrated that thiol-Michael addition cross-linking chemistry greatly affects the integrity and bio-functionality of the loaded protein BMP-2 and leads to poor bone formation in an in-vivo rat model. On the other hand, hydrogels employing hydrazone chemistry did not significantly affect the integrity and bioactivity of BMP-2, which lead to a superior bone formation in-vivo. Since the high dose of rh-BMP-2 is known to confer many side effects, alternative ex-vivo strategies involving transient transfection of BMP-2 expressing plasmid DNA and silencing of anti-osteogenic genes using siRNA are developed. Our optimized method involves rapid transfection of hMSCs in suspension (5 minutes) with plasmid DNA followed by centrifugation and encapsulation in a hydrogel not only reduced cytotoxicity but also lead to efficient osteoblast differentiation of stem cells. Furthermore, this thesis presents the role of ECM-derived polymer HA in interacting with siRNA and trafficking across the plasma membrane, presumably through CD44 receptors and successfully silencing the target gene in-vitro. We explored the potential of such a non-cationic transfection method to deliver functional siRNA (anti-Pleckho-1 siRNA) in MSCs and compared it with commercially available cationic lipid LipofectamineTMRNAiMAX, using our optimized suspension transfection method. Our novel ex-vivo strategy employing HA hydrogels enabled efficient silencing of BMP-2 signaling pathway antagonist Pleckho-1 while avoiding the cytotoxicity issues in 3D, which further qualifies them for potential clinical application for cell-based therapies.
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| 40. |
- Ranamalla, Saketh Reddy, et al.
(författare)
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A quality by design approach to optimise disulfide-linked hyaluronic acid hydrogels
- 2024
-
Ingår i: Carbohydrate Polymers. - : Elsevier. - 0144-8617 .- 1879-1344. ; 339
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Tidskriftsartikel (refereegranskat)abstract
- n this study, the disulfide-linked hyaluronic acid (HA) hydrogels were optimised for potential application as a scaffold in tissue engineering through the Quality by Design (QbD) approach. For this purpose, HA was first modified by incorporating the cysteine moiety into the HA backbone, which promoted the formation of disulfide cross-linked HA hydrogel at physiological pH. Utilising a Design of Experiments (DoE) methodology, the critical factors to achieve stable biomaterials, i.e. the degree of HA substitution, HA molecular weight, and coupling agent ratio, were explored. To establish a design space, the DoE was performed with 65 kDa, 138 kDa and 200 kDa HA and variable concentrations of coupling agent to optimise conditions to obtain HA hydrogel with improved rheological properties. Thus, HA hydrogel with a 12 % degree of modification, storage modulus of ≈2321 Pa and loss modulus of ≈15 Pa, was achieved with the optimum ratio of coupling agent. Furthermore, biocompatibility assessments in C28/I2 chondrocyte cells demonstrated the non-toxic nature of the hydrogel, underscoring its potential for tissue regeneration. Our findings highlight the efficacy of the QbD approach in designing HA hydrogels with tailored properties for biomedical applications.
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| 41. |
- Todeschi, Maria R., et al.
(författare)
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Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment
- 2017
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Ingår i: Regenerative Medicine. - : FUTURE MEDICINE LTD. - 1746-0751 .- 1746-076X. ; 12:5, s. 525-539
-
Tidskriftsartikel (refereegranskat)abstract
- Aim: This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Materials & methods: Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Results & conclusion: Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.
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| 42. |
- Varghese, Oommen P., 1977-
(författare)
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Conformationally Constrained Nucleosides : Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG‡ = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring.The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.
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