Minocycline enhances the mesenchymal stromal/stem cell pro-healing phenotype in triple antimicrobial-loaded hydrogels.

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. STATEMENT OF SIGNIFICANCE: 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.

Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

BACKGROUND: 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. METHODS: 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. RESULTS: Minocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB 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. CONCLUSIONS: Minocycline modulates the NFκB 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.

The Anti-Tumor Effects of M1 Macrophage-Loaded Poly (ethylene glycol) and Gelatin-Based Hydrogels on Hepatocellular Carcinoma.

Background and Aims: Recently we reported that direct injection of M1 macrophages significantly caused tumor regression in vivo. Despite the promising result, a major limitation in translating this approach is the induction of acute inflammatory response. To improve the strategy, a biocompatible scaffold for cell presentation and support is essential to control cell fate. Here, we aimed to elucidate the anti-tumor effects of a poly(ethylene glycol) diacrylate (PEGdA) and thiolated gelatin poly(ethylene glycol) (Gel-PEG-Cys) cross-linked hydrogels capsulated with M1 macrophages in both in vitro and in vivo disease models. Methods: Hydrogels were made at 0.5% (w/v) Iragcure 2959 photoinitiator, 10% (w/v) PEGdA, and 10% (w/v) Gel-PEG-Cys. Monocytic THP-1 cells were loaded into hydrogels and differentiated into M1 macrophages with lipopolysaccharide (LPS) and interferon gamma (IFN-γ). The M1 hydrogels were then cocultivated with HCC cell-lines Hep3B and MHCC97L to investigate the anti-tumor capacities and the associated molecular profiles in vitro. A nude mice ectopic liver cancer model with dorsal window chamber (DWC) and a subcutaneous tumor model were both performed to validate the in vivo application of M1 hydrogels. Results: M1 hydrogels significantly decreased the viability of HCC cells (MHCC97L: -46%; Hep3B: -56.9%; P<0.05) compared to the control in vitro. In response to HCC cells, the hydrogel embedded M1 macrophages up-regulated nitrite and tumor necrosis factor alpha (TNF-α) activating caspase-3 induced apoptosis in the tumor cells. Increased tumor necrosis was observed in DWC filled with M1 hydrogels. In addition, mice treated with M1 hydrogels exhibited a significant 2.4-fold decrease in signal intensity of subcutaneous HCC tumor compared to control (P=0.036). Conclusion: M1 hydrogels induced apoptosis in HCC cells and tumor regression in vivo. Continuous development of the scaffold-based cancer immunotherapy may provide an alternative and innovative strategy against HCC.

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes.

Mesenchymal stromal/stem cells (MSCs) have demonstrated favorable wound healing properties in addition to their differentiation capacity. MSCs encapsulated in biomaterials such as gelatin and polyethylene glycol (PEG) composite hydrogels have displayed an immunophenotype change that leads to the release of cytokines and growth factors to accelerate wound healing. However, therapeutic potential of implanted MSC-loaded hydrogels may be limited by non-specific protein adsorption that facilitates adhesion of bacterial pathogens such as planktonic Staphylococcus aureus (SA) to the surface with subsequent biofilm formation resistant to immune cell recognition and antibiotic activity. In this study, we demonstrate that blood-derived primary leukocytes and bone marrow-derived MSCs cannot inhibit colony-forming abilities of planktonic or biofilm-associated SA. However, we show that hydrogels loaded with MSCs and minocycline significantly inhibit colony-forming abilities of planktonic SA while maintaining MSC viability and multipotency. Our results suggest that minocycline and MSC-loaded hydrogels may decrease the bioburden of SA at implant sites in wounds, and may improve the wound healing capabilities of MSC-loaded hydrogels.