Exogenous Signaling Molecules Released from Aptamer-Functionalized Hydrogels Promote the Survival of Mesenchymal Stem Cell Spheroids.
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ABSTRACT:
SUBMITTER: Zhao N
PROVIDER: S-EPMC7883300 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bone regeneration heavily relies on bone marrow mesenchymal stem cells (BMSCs). However, recruiting endogenous BMSCs for in situ bone regeneration remains challenging. In this study, we developed a novel BMSC-aptamer (BMSC-apt) functionalized hydrogel (BMSC-aptgel) and evaluated its functions in recruiting BMSCs and promoting bone regeneration. The functional hydrogels were synthesized between maleimide-terminated 4-arm polyethylene glycols (PEG) and thiol-flanked PEG crosslinker, allowing rapid in situ gel formation. The aldehyde group-modified BMSC-apt was covalently bonded to a thiol-flanked PEG crosslinker to produce high-density aptamer coverage on the hydrogel surface. In vitro and in vivo studies demonstrated that the BMSC-aptgel significantly increased BMSC recruitment, migration, osteogenic differentiation, and biocompatibility. In vivo fluorescence tomography imaging demonstrated that functionalized hydrogels effectively recruited DiR-labeled BMSCs at the fracture site. Consequently, a mouse femur fracture model significantly enhanced new bone formation and mineralization. The aggregated BMSCs stimulated bone regeneration by balancing osteogenic and osteoclastic activities and reduced the local inflammatory response via paracrine effects. This study's findings suggest that the BMSC-aptgel can be a promising and effective strategy for promoting in situ bone regeneration.
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Project description:Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs. STATEMENT OF SIGNIFICANCE:Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu.
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Project description:Encapsulating pancreatic islets in a semipermeable poly(ethylene glycol) (PEG) hydrogel membrane holds potential as an immuno-isolation barrier for the treatment of type 1 diabetes mellitus. The semipermeable PEG hydrogel not only permits free diffusion of nutrients, metabolic waste, and insulin produced from the encapsulated beta-cells, but also provides a size-exclusion effect to prevent direct contact of entrapped islets to host immune cells and antibodies. However, the use of unmodified PEG hydrogels for islet encapsulation is not ideal, as there is no bioactive cue to promote the long-term survival and function of the encapsulated cells. Herein, we report the synthesis and characterization of a bioactive glucagon-like peptide 1 (GLP-1) analog, namely, GLP-1-cysteine or GLP-1C, and the fabrication of functional GLP-1 immobilized PEG hydrogels via a facile thiol-acrylate photopolymerization. The immobilization of bioactive GLP-1C within PEG hydrogels is efficient and does not alter the bulk hydrogel properties. Further, the GLP-1 immobilized PEG hydrogels enhance the survival and insulin secretion of encapsulated islets. Overall, this study demonstrates a strategy to modify PEG hydrogels with bioactive peptide moieties that can significantly enhance the efficacy of islet encapsulation.
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Project description:Mesenchymal stem cells (MSCs) have great therapeutic potential for the repair of nonhealing bone defects, because of their proliferative capacity, multilineage potential, trophic factor secretion and lack of immunogenicity. However, a major challenge to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesize that forming MSCs into more physiologic three-dimensional spheroids, rather than employing dissociated cells from two-dimensional monolayer culture, will enhance their survival when exposed to a harsh microenvironment but maintain their osteogenic potential. MSC spheroids were formed by using the hanging drop method with increasing cell numbers. Compared with larger spheroids, the smallest spheroids, which contained 15,000 cells, exhibited increased metabolic activity, reduced apoptosis and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free medium and 1 % oxygen. Compared with identical numbers of dissociated MSCs in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more vascular endothelial growth factor. Moreover, fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. Thus, MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential while maintaining similar osteogenic potential to dissociated MSCs entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair.
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Project description:A variety of hydrogels have been synthesized for controlling the release of signaling molecules in applications such as drug delivery and regenerative medicine. However, it remains challenging to synthesize hydrogels with the ability to control the release of signaling molecules sequentially or periodically under physiological conditions as living cells do in response to the variation of metabolism. The purpose of this work was to study a novel biomimetic hydrogel system with the ability of recapitulating the procedure of cellular signal transduction and controlling the sequential release of signaling molecules under physiological conditions. In the presence of a small chemical, the signaling molecule is regulated to change from a DNA-bound state to a free state and the freed signaling molecule is able to regulate intracellular signal transduction and cell migration. Moreover, periodic exposure of the hydrogel system to the small chemical leads to sequential protein release. Since signaling molecules are important for every activity of the cell, this hydrogel system holds potential as a metabolism-responsive platform for controlled release of signaling molecules and cell regulation in various applications.
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Project description:Restoring the tooth-supporting tissues lost during periodontitis is a significant clinical challenge, despite advances in both biomaterial and cell-based approaches. This study investigated poly(ethylene glycol) (PEG) hydrogels functionalized with integrin-binding peptides RGD and GFOGER for controlling periodontal ligament cell (PDLC) activity and promoting periodontal tissue regeneration. Dual presentation of RGD and GFOGER within PEG hydrogels potentiated two key PDLC functions, alkaline phosphatase (ALP) activity and matrix mineralization, over either peptide alone and could be tuned to differentially promote each function. Hydrogel matrix mineralization, fostered by high concentrations of GFOGER together with RGD, identified a PDLC phenotype with accelerated matrix adhesion formation and expression of cementoblast and osteoblast genes. In contrast, maximizing ALP activity through high RGD and low GFOGER levels resulted in minimal hydrogel mineralization, in part, through altered PDLC pyrophosphate regulation. Transplantation of PDLCs in hydrogels optimized for either outcome promoted cementum formation in rat periodontal defects; however, only hydrogels optimized for in vitro mineralization improved new bone formation. Overall, these results highlight the utility of engineered hydrogel systems for controlling PDLC functions and their promise for promoting periodontal tissue regeneration.
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Project description:The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.
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Project description:Previously we developed a fibrin hydrogel (FH) decorated with laminin-111 peptides (L1p-FH) and supports three-dimensional (3D) gland microstructures containing polarized acinar cells. Here we expand on these results and show that co-culture of rat parotid Par-C10 cells with mesenchymal stem cells produces migrating branches of gland cells into the L1p-FH and we identify FGF-7 as the principal morphogenetic signal responsible for branching. On the other hand, another FGF family member and gland morphogen, FGF-10 increased proliferation but did not promote migration and therefore, limited the number and length of branched structures grown into the gel. By controlling the mode of growth factor presentation and delivery, we can control the length and cellularity of branches as well as formation of new nodes/clusters within the hydrogel. Such spatial delivery of two or more morphogens may facilitate engineering of anatomically complex tissues/mini organs such as salivary glands that can be used to address developmental questions or as platforms for drug discovery. STATEMENT OF SIGNIFICANCE: Hyposalivation leads to the development of a host of oral diseases. Current treatments only provide temporary relief. Tissue engineering may provide promising permanent solutions. Yet current models are limited to salivary spheroids with no branching networks. Branching structures are vital to an effective functioning gland as they increase the surface area/glandular volume ratio of the tissue, allowing a higher output from the small-sized gland. We describe a strategy that controls branch network formation in salivary glands that is a key in advancing the field of salivary gland tissue engineering.
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Project description:Antimicrobial peptides (AMPs) are seen as a promising replacement to conventional antibiotics for the prevention of skin wound infections. However, due to the short half-life of AMPs in biological environments, such as blood, their use in clinical applications has been limited. The covalent immobilization of AMPs onto suitable substrates is an effective solution to create contact-killing surfaces with increased long-term stability. In this work, an antimicrobial peptide, RRPRPRPRPWWWW-NH2 (RRP9W4N), was covalently attached to amphiphilic and ordered mesoporous Pluronic F127 hydrogels made of cross-linked lyotropic liquid crystals through 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. The AMP-hydrogels showed high antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, methicillin-resistant S. aureus (MRSA), and multidrug-resistant Escherichia coli for up to 24 h. Furthermore, the AMP-hydrogels did not present any toxicity to human fibroblasts. The AMPs retained their antimicrobial activity up to 48 h in human blood serum, which is a significant increase in stability compared to when used in dissolved state. A pilot in vivo rat model showed 10-100× less viable counts of S. aureus on AMP-hydrogels compared with control hydrogels during the first 3 days of infection. Studies performed on human whole blood showed that blood coagulated more readily in the presence of AMP-hydrogels as compared to hydrogels without AMPs, indicating potential hemostatic activity. Overall, the results suggest that the combination of amphiphilic hydrogels with covalently bonded AMPs has potential to be used as antibacterial wound dressing material to reduce infections and promote hemostatic activity as an alternative to antibiotics or other antimicrobial agents, whose use should be restricted.
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