Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds.
ABSTRACT: Mesenchymal stromal/stem cells (MSCs) are considered promising cellular therapeutics in the fields of tissue engineering and regenerative medicine. MSCs secrete high concentrations of immunomodulatory cytokines and growth factors, which exert paracrine effects on infiltrating immune and resident cells in the wound microenvironment that could favorably promote healing after acute injury. However, better spatial delivery and improved retention at the site of injury are two factors that could improve the clinical application of MSCs. In this study, we utilized thiol-ene Michael-type addition for rapid encapsulation of MSCs within a gelatin/poly(ethylene glycol) biomatrix. This biomatrix was also applied as a provisional dressing to full thickness wounds in Sprague-Dawley rats. The three-way interaction of MSCs, gelatin/poly(ethylene glycol) biomatrices, and host immune cells and adjacent resident cells in the wound microenvironment favorably modulated wound progression and host response. In this model we observed attenuated immune cell infiltration, lack of foreign giant cell (FBGC) formation, accelerated wound closure and re-epithelialization, as well as enhanced neovascularization and granulation tissue formation by 7 days. The MSC entrapped in the gelatin/poly(ethylene glycol) biomatrix localized cell presentation adjacent to the wound microenvironment and thus mediated the early resolution of inflammatory events and facilitated the proliferative phases in wound healing.
Project description:Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties including an anti-inflammatory cytokine profile and the promotion of angiogenesis via expression of growth factors in pre-clinical models. MSCs encapsulated in poly(ethylene glycol) diacrylate (PEGdA) and thiolated gelatin poly(ethylene glycol) (Gel-PEG-Cys) crosslinked hydrogels have led to controlled cellular presentation at wound sites with favorable wound healing outcomes. However, the therapeutic potential of MSC-loaded hydrogels may be limited by non-specific protein adsorption on the delivery matrix that could facilitate the initial adhesion of microorganisms and subsequent virulent biofilm formation. Antimicrobials loaded concurrently in the hydrogels with MSCs could reduce microbial bioburden and promote healing, but the antimicrobial effect on the MSC wound healing capacity and the antibacterial efficacy of the hydrogels is unknown. We demonstrate that minocycline specifically induces a favorable change in MSC migration capacity, proliferation, gene expression, extracellular matrix (ECM) attachment, and adhesion molecule and growth factor release with subsequent increased angiogenesis. We then demonstrate that hydrogels loaded with MSCs, minocycline, vancomycin, and linezolid can significantly decrease bacterial bioburden. Our study suggests that minocycline can serve as a dual mechanism for the regenerative capacity of MSCs and the reduction of bioburden in triple antimicrobial-loaded hydrogels.Wound healing is a complex biological process that can be hindered by bacterial infection, excessive inflammation, and inadequate microvasculature. In this study, we develop a new formulation of poly(ethylene glycol) diacrylate and thiolated gelatin poly(ethylene glycol) crosslinked hydrogels loaded with minocycline, vancomycin, linezolid, and mesenchymal stromal/stem cells that induces a favorable wound healing phenotype in mesenchymal stromal/stem cells and prevents bacterial bioburden on the hydrogel. This combinatorial approach to biomaterial development has the potential to impact wound healing for contaminated full thickness cutaneous wounds.
Project description:Although various cell encapsulation materials are available commercially for a wide range of potential therapeutic cells, their combined clinical impact remains inconsistent. Synthetic materials such as poly(ethylene glycol) (PEG) hydrogels are mechanically robust and have been extensively explored but lack natural biofunctionality. Naturally derived materials including collagen, fibrin and alginate-chitosan are often labile and mechanically weak. In this paper we report the development of a hybrid biomatrix based on the thiol-ene reaction of PEG diacrylate (PEGdA) and cysteine/PEG-modified gelatin (gel-PEG-Cys). We hypothesized that covalent crosslinking decreases gelatin dissolution thus increasing gelatin resident time within the matrix and the duration of its biofunctionality; at the same time the relative ratio of PEGdA to gel-PEG-Cys in the matrix formulation directly affects hydrogel bulk and local microenvironment properties. Bulk viscoelastic properties were highly dependent on PEGdA concentration and total water content, while gel-PEG-Cys concentration was more critical to swelling profiles. Microviscoelastic properties were related to polymer concentration. The covalently crosslinked gel-PEG-Cys with PEGdA decreased gelatin dissolution out of the matrix and collagenase-mediated degradation. Fibroblasts and keratinocyte increased adhesion density and formed intercellular connections on stiffer hydrogel surfaces, while cells exhibited more cytoplasmic spreading and proliferation when entrapped within softer hydrogels. Hence, this material system contains multiparametric factors that can easily be controlled to modulate the chemical, physical and biological properties of the biomatrix for soft tissue scaffolding and cell presentation to reconstruct lost tissue architecture and physical functionality.
Project description:Conventional surgical techniques to seal and repair defects in highly stressed elastic tissues are insufficient. Therefore, this study aimed to engineer an inexpensive, highly adhesive, biocompatible, and biodegradable sealant based on a modified and naturally derived biopolymer, gelatin methacryloyl (GelMA). We tuned the degree of gelatin modification, prepolymer concentration, photoinitiator concentration, and crosslinking conditions to optimize the physical properties and adhesion of the photocrosslinked GelMA sealants. Following ASTM standard tests that target wound closure strength, shear resistance, and burst pressure, GelMA sealant was shown to exhibit adhesive properties that were superior to clinically used fibrin- and poly(ethylene glycol)-based glues. Chronic in vivo experiments in small as well as translational large animal models proved GelMA to effectively seal large lung leakages without the need for sutures or staples, presenting improved performance as compared to fibrin glue, poly(ethylene glycol) glue and sutures only. Furthermore, high biocompatibility of GelMA sealant was observed, as evidenced by a low inflammatory host response and fast in vivo degradation while allowing for adequate wound healing at the same time. Combining these results with the low costs, ease of synthesis and application of the material, GelMA sealant is envisioned to be commercialized not only as a sealant to stop air leakages, but also as a biocompatible and biodegradable hydrogel to support lung tissue regeneration.
Project description:The capacity of a biomaterial to innately modulate cell behavior while meeting the mechanical property requirements of the implant is a much sought-after goal within bioengineering. Here we covalently incorporate soluble elastin into a gelatin-poly (ethylene glycol) (PEG) hydrogel for three-dimensional (3D) cell encapsulation to achieve these properties. The inclusion of elastin into a previously optimized gelatin-PEG hydrogel was then evaluated for effects on entrapped fibroblasts, with the aim to assess the hydrogel as an extracellular matrix (ECM)-mimicking 3D microenvironment for cellular guidance. Soluble elastin was incorporated both physically and covalently into novel gelatin/elastin hybrid PEG hydrogels with the aim to harness the cellular interactivity and mechanical tunability of both elastin and gelatin. This design allowed us to assess the benefits of elastin-containing hydrogels in guiding fibroblast activity for evaluation as a potential dermal replacement. It was found that a gelatin-PEG hydrogel with covalently conjugated elastin, supported neonatal fibroblast viability, promoted their proliferation from 7.3% to 13.5% and guided their behavior. The expression of collagen alpha-1(COL1A1) and elastin in gelatin/elastin hybrid gels increased 16-fold and 6-fold compared to control sample at day 9, respectively. Moreover, cells can be loaded into the hydrogel precursor solution, deposited, and the matrix cross-linked without affecting the incorporated cells adversely, thus enabling a potential injectable system for dermal wound healing.
Project description:The osteochondral microenvironment involves a complex milieu of cues that facilitate proper tissue development, homeostasis, and repair. This environment is disrupted in disease states such as osteoarthritis. Mesenchymal stem cells (MSCs) are under clinical investigation for the treatment of osteoarthritis given their capacity to differentiate into chondrocytes as well as to secrete a wide array of biologically active factors that support cell proliferation and tissue formation. In fact, the therapeutic action of these cells in many clinical applications is now thought to be at least partially dependent on their secretory capacity. Previous work demonstrated that MSCs were capable of stimulating chondrocyte growth and tissue production, whereas tissue-derived osteoblasts were not stimulatory. This study investigated the stimulatory capacity of MSCs during osteogenesis and the impact of MSC phenotype on cartilage stimulation. Cell interactions were examined in 3 coculture systems to confirm that trends were not dependent on material: traditional cell culture insert coculture, bilayered poly(ethylene glycol) gels, and a scaffold comprised of a layer of poly(ethylene glycol) polymerized onto a poly(lactic-co-glycolic) acid-based scaffold. Results demonstrated that MSCs predifferentiated toward an osteogenic phenotype for 3 days exhibited enhanced stimulation of chondrocyte extracellular matrix production, whereas longer periods of predifferentiation decreased the magnitude of observed stimulation. Further, tissue formation by the MSCs themselves showed greater dependence on the coculture system than the presence of other cells or length of predifferentiation.
Project description:Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds.Modulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague-Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student's t test was used to determine p values for all assays with multiple or two conditions, respectively.Minocycline leads to the phosphorylation of transcriptional nuclear factor-?B (NF?B), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NF?B activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization.Minocycline modulates the NF?B pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.
Project description:The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system <i>in vitro</i> We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels.
Project description:The intracellular delivery of growth factors increases opportunities for controlling cell behavior and maintaining tissue homeostasis. Recently, VEGFA was reported to enhance osteogenic differentiation of mesenchymal stem cells (MSCs) through an intracrine mechanism, suggesting a new strategy to promote bone tissue formation in osteoporotic patients. The goal of this study was to design and fabricate ligand-conjugated alginate-graft-poly(ethylene glycol) microspheres for intracellular delivery and release of VEGFA in primary human MSCs to enhance osteogenic differentiation as a potential therapeutic. Three types of microspheres were synthesized and characterized by scanning electron microscopy, in vitro drug release kinetics, MSC uptake and internalization: alginate alone (Alg), alginate-graft-poly(ethylene glycol) (Alg-g-PEG) and alginate-graft-poly(ethylene glycol)-S-S-arginine-glycine-aspartic acid (Alg-g-RGD). Each of the different microsphere formulations successfully transported bioactive VEGFA into primary human MSCs within 48h of culture, and significantly enhanced osteogenic differentiation compared to control treatments with empty microspheres (intracellular control) or non-encapsulated VEGFA (extracellular control). Adipogenic differentiation was not affected by the presence of VEGFA intracellularly or extracellularly. These results demonstrating the internalization of alginate-based microspheres and intracellular delivery of VEGFA support the efficacy of using this drug delivery and intracrine mechanism to control the fate of human MSCs and enhance osteogenic differentiation.
Project description:In order to prevent hemorrhage during surgical procedures, a wide range of hemostatic agents have been developed. However, their efficacy is variable; hemostatic devices that use bioactive components to accelerate coagulation are dependent on natural sources, which limits reproducibility. Hybrid devices in which chain-end reactive poly(ethylene glycol) is employed as active component sometimes suffer from irregular cross-linking and dissolution of the polar PEG when blood flow is substantial. Herein, we describe a synthetic, nonbioactive hemostatic product by coating N-hydroxysuccinimide ester (NHS)-functional poly(2-oxazoline)s (POx-NHS) onto gelatin patches, which acts by formation of covalent cross-links between polymer, host blood proteins, gelatin and tissue to seal the wound site and prevent hemorrhage during surgery. We studied different process parameters (including polymer, carrier, and coating technique) in direct comparison with clinical products (Hemopatch and Tachosil) to obtain deeper understanding of this class of hemostatic products. In this work, we successfully prove the hemostatic efficacy of POx-NHS as polymer powders and coated patches both in vitro and in vivo against Hemopatch and Tachosil, demonstrating that POx-NHS are excellent candidate polymers for the development of next generation hemostatic patches.
Project description:We discovered a transient adhesion property in poly(ethylene glycol) dimethacrylate (PEG-DMA) hydrogels and employed it to develop a novel "stem cell bandage" model of cellular delivery. First, we cultured human mesenchymal stromal cells (MSCs) on the surface of PEG-DMA hydrogels with high amounts of arginine-glycine-aspartic acid (RGD) adhesive peptides (RGD++) or without RGD (RGD-). On day 1, MSCs underwent an initial adhesion to RGD- hydrogels that was not significantly different over 13 days (n = 6). In addition, cells appeared to be well spread by day 3. Significantly fewer cells were present on RGD- hydrogels on day 15 compared to day 9, suggesting that RGD- hydrogels allow for an initial cellular adhesion that is stable for multiple days, but transient over longer periods with a decrease by day 15. This initial adhesion is especially surprising considering that PEG-DMA does not contain any biological adhesion motifs and is almost chemically identical to poly(ethylene glycol) diacrylate (PEG-DA), which has been shown to be non-adhesive without RGD. We hypothesized that MSCs could be cultured on RGD- PEG-DMA hydrogels and then applied to a wound site to deliver cells in a novel approach that we refer to as a "stem cell bandage". RGD- donor hydrogels were successfully able to deliver MSCs to PEG-DMA acceptor hydrogels with high RGD content (RGD++) or low amounts of RGD (RGD+). Our novel "bandage" approach promoted cell delivery to these model surfaces while preventing cells from diffusing away. This stem cell delivery strategy may provide advantages over more common stem cell delivery approaches such as direct injections or encapsulation and thus may be valuable as an alternative tissue engineering approach.