Local Immunomodulation Using an Adhesive Hydrogel Loaded with miRNA‐Laden Nanoparticles Promotes Wound Healing

Chronic wounds are characterized by impaired healing and uncontrolled inflammation, which compromise the protective role of the immune system and may lead to bacterial infection. Upregulation of miR‐223 microRNAs (miRNAs) shows driving of the polarization of macrophages toward the anti‐inflammatory (M2) phenotype, which could aid in the acceleration of wound healing. However, local‐targeted delivery of microRNAs is still challenging, due to their low stability. Here, adhesive hydrogels containing miR‐223 5p mimic (miR‐223*) loaded hyaluronic acid nanoparticles are developed to control tissue macrophages polarization during wound healing processes. In vitro upregulation of miR‐223* in J774A.1 macrophages demonstrates increased expression of the anti‐inflammatory gene Arg‐1 and a decrease in proinflammatory markers, including TNF‐α, IL‐1β, and IL‐6. The therapeutic potential of miR‐223* loaded adhesive hydrogels is also evaluated in vivo. The adhesive hydrogels could adhere to and cover the wounds during the healing process in an acute excisional wound model. Histological evaluation and quantitative polymerase chain reaction (qPCR) analysis show that local delivery of miR‐223* efficiently promotes the formation of uniform vascularized skin at the wound site, which is mainly due to the polarization of macrophages to the M2 phenotype. Overall, this study demonstrates the potential of nanoparticle‐laden hydrogels conveying miRNA‐223* to accelerate wound healing.     

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.     

Activatable Nanoreporters for Real-Time Tracking of Macrophage Phenotypic States Associated with Disease Progression

Diagnosis of inflammatory diseases is characterized by identifying symptoms, biomarkers, and imaging. However, conventional techniques lack the sensitivities and specificities to detect disease early. Here, it is demonstrated that the detection of macrophage phenotypes, from inflammatory M1 to alternatively activated M2 macrophages, corresponding to the disease state can be used to predict the prognosis of various diseases. Activatable nanoreporters that can longitudinally detect the presence of the enzyme Arginase 1, a hallmark of M2 macrophages, and nitric oxide, a hallmark of M1 macrophages are engineered, in real-time. Specifically, an M2 nanoreporter enables the early imaging of the progression of breast cancer as predicted by selectively detecting M2 macrophages in tumors. The M1 nanoreporter enables real-time imaging of the subcutaneous inflammatory response that rises from a local lipopolysccharide (LPS) administration. Finally, the M1-M2 dual nanoreporter is evaluated in a muscle injury model, where an initial inflammatory response is monitored by imaging M1 macrophages at the site of inflammation, followed by a resolution phase monitored by the imaging of infiltrated M2 macrophages involved in matrix regeneration and wound healing. It is anticipated that this set of macrophage nanoreporters may be utilized for early diagnosis and longitudinal monitoring of inflammatory responses in various disease models.     

Inflammatory response to a porcine membrane composed of fibrous collagen and elastin as dermal substitute

The inflammatory response to a collagen/elastin membrane was studied by measuring the expression of cytokines and function associated antigens in human macrophages. Additionally the angiogenic and inflammatory activity in the chorioallantoic membrane of the chick embryo (CAM-assay) was investigated. Macrophages cultured on the membrane expressed IL-1 mRNA as early as after 4 hours. During prolonged culturing IL-1 mRNA levels decreased. Messenger RNA for IL-8 was detectable over the whole culture period. The anti-inflammatory cytokine IL-10 was expressed up to one day only. Phenotypic analysis revealed a decrease in the number of chronic inflammatory 25F9 positive macrophages not migrating into the membrane but a presence of these cells together with the acute inflammatory 27E10 macrophages within the membrane whereas the anti-inflammatory subtype RM3/1 was absent. In the CAM-assay the membrane stimulated angiogenesis and induced the formation of granulation tissue. Histological analysis showed that the membrane was infiltrated with macrophages, fibroblasts and endothelial cells and locally with granulocytes. These data show that the collagen/elastin membrane causes activation of macrophages, angiogenesis and the formation of inflammatory tissue. Although these processes are essential for wound healing the type of inflammation points to a chronic process which might counteract an efficient scar formation. © 2001 Kluwer Academic Publishers     

Highly Conductive,Stretchable, and Cell‐Adhesive Hydrogel by Nanoclay Doping

Electrically conductive materials that mimic physical and biological properties of tissues are urgently required for seamless brain–machine interfaces. Here, a multinetwork hydrogel combining electrical conductivity of 26 S m^?1, stretchability of 800%, and tissue‐like elastic modulus of 15 kPa with mimicry of the extracellular matrix is reported. Engineering this unique set of properties is enabled by a novel in‐scaffold polymerization approach. Colloidal hydrogels of the nanoclay Laponite are employed as supports for the assembly of secondary polymer networks. Laponite dramatically increases the conductivity of in‐scaffold polymerized poly(ethylene‐3,4‐diethoxy thiophene) in the absence of other dopants, while preserving excellent stretchability. The scaffold is coated with a layer containing adhesive peptide and polysaccharide dextran sulfate supporting the attachment, proliferation, and neuronal differentiation of human induced pluripotent stem cells directly on the surface of conductive hydrogels. Due to its compatibility with simple extrusion printing, this material promises to enable tissue‐mimetic neurostimulating electrodes.     

Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.     

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

Abstract

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.     

Disease‐Triggered Drug Release Effectively Prevents Acute Inflammatory Flare‐Ups,Achieving Reduced Dosing

For conditions with inflammatory flare‐ups, fast drug‐release from a depot is crucial to reduce cell infiltration and prevent long‐term tissue destruction. While this concept has been explored for chronic diseases, preventing acute inflammatory flares has not been explored. To address this issue, a preventative inflammation‐sensitive system is developed and applied to acute gout, a condition where millions of inflammatory cells are recruited rapidly, causing excruciating and debilitating pain. Rapid drug release is first demonstrated from a pH‐responsive acetalated dextran particle loaded with dexamethasone (AcDex‐DXM), reducing proinflammatory cytokines in vitro as efficiently as free drug. Then, using the air pouch model of gout, mice are pretreated 24 h before inducing inflammation. AcDex‐DXM reduces overall cell infiltration with decreased neutrophils, increases monocytes, and diminishes cytokines and chemokines. In a more extended prophylaxis model, murine joints are pretreated eight days before initiating inflammation. After quantifying cell infiltration, only AcDex‐DXM reduces the overall joint inflammation, where neither free drug nor a conventional drug‐depot achieves adequate anti‐inflammatory effects. Here, the superior efficacy of disease‐triggered drug‐delivery to prevent acute inflammation is demonstrated over free drug and slow‐release depots. This approach and results promise exciting treatment opportunities for multiple inflammatory conditions suffering from acute flares.     

Anti‐inflammatory Properties of Cerium Oxide Nanoparticles

The valence and oxygen defect properties of cerium oxide nanoparticles (nanoceria) suggest that they may act as auto‐regenerative free radical scavengers. Overproduction of the free radical nitric oxide (NO) by the enzyme inducible nitric oxide synthase (iNOS) has been implicated as a critical mediator of inflammation. NO is correlated with disease activity and contributes to tissue destruction. The ability of nanoceria to scavenge free radicals, or reactive oxygen species (ROS), and inhibit inflammatory mediator production in J774A.1 murine macrophages is investigated. Cells internalize nanoceria, the treatment is nontoxic, and oxidative stress and pro‐inflammatory iNOS protein expression are abated with stimulation. In vivo studies show nanoceria deposition in mouse tissues with no pathogenicity. Taken together, it is suggested that cerium oxide nanoparticles are well tolerated in mice and are incorporated into cellular tissues. Furthermore, nanoceria may have the potential to reduce ROS production in states of inflammation and therefore serve as a novel therapy for chronic inflammation.     

Immunized Microspheres Engineered Hydrogel Membrane for Reprogramming Macrophage and Mucosal Repair

The “double-edged sword” effect of macrophages under the influence of different microenvironments determines the outcome and prognosis of tissue injury. Accurate and stable reprogramming macrophages (Mφ) are the key to rapid wound healing. In this study, an immunized microsphere-engineered GelMA hydrogel membrane is constructed for oral mucosa treatment. The nanoporous poly(lactide-co-glycolide) (PLGA) microsphere drug delivery system combined with the photo-cross-linkable hydrogel is used to release the soybean lecithin (SL)and IL-4 complexes (SL/IL-4) sustainedly. In this way, it is realized effective wound fit, improvement of drug encapsulation, and stable triphasic release of interleukin-4 (IL-4). In both in vivo and in vitro experiments, it is demonstrated that the hydrogel membrane can reprogram macrophages in the microenvironment into M2Mφ anti-inflammatory types, thereby inhibiting the local excessive inflammatory response. Meanwhile, high levels of platelet-derived growth factor (PDGF) secreted by M2Mφ macrophages enhanced neovascular maturation by 5.7-fold, which assisted in achieving rapid healing of oral mucosa. These findings suggest that the immuno-engineered hydrogel membrane system can re-modulating the biological effects of Mφ, and potentiating the maturation of neovascularization, ultimately achieving the rapid repair of mucosal tissue. This new strategy is expected to be a safe and promising immunomodulatory biomimetic material for clinical translation.     

Liquid Crystalline Networks toward Regenerative Medicine and Tissue Repair

The communication reports the use of liquid crystalline networks (LCNs) for engineering tissue cultures with human cells. Their ability as cell scaffolds for different cell lines is demonstrated. Preliminary assessments of the material biocompatibility are performed on human dermal fibroblasts and murine muscle cells (C2C12), demonstrating that coatings or other treatments are not needed to use the acrylate‐based materials as support. Moreover, it is found that adherent C2C12 cells undergo differentiation, forming multinucleated myotubes, which show the typical elongated shape, and contain bundles of stress fibers. Once biocompatibility is demonstrated, the same LCN films are used as a substrate for culturing human induced pluripotent stem cell‐derived cardiomyocites (hiPSC‐CMs) proving that LCNs are capable to develop adult‐like dimensions and a more mature cell function in a short period of culture in respect to standard supports. The demonstrated biocompatibility together with the extraordinary features of LCNs opens to preparation of complex cell scaffolds, both patterned and stimulated, for dynamic cell culturing. The ability of these materials to improve cell maturation and differentiation will be developed toward engineered heart and skeletal muscular tissues exploring regenerative medicine toward bioartificial muscles for injured sites replacement.     

Personalized Hydrogels for Engineering Diverse Fully Autologous Tissue Implants

Despite incremental improvements in the field of tissue engineering, no technology is currently available for producing completely autologous implants where both the cells and the scaffolding material are generated from the patient, and thus do not provoke an immune response that may lead to implant rejection. Here, a new approach is introduced to efficiently engineer any tissue type, which its differentiation cues are known, from one small tissue biopsy. Pieces of omental tissues are extracted from patients and, while the cells are reprogrammed to become induced pluripotent stem cells, the extracellular matrix is processed into an immunologically matching, thermoresponsive hydrogel. Efficient cell differentiation within a large 3D hydrogel is reported, and, as a proof of concept, the generation of functional cardiac, cortical, spinal cord, and adipogenic tissue implants is demonstrated. This versatile bioengineering approach may assist to regenerate any tissue and organ with a minimal risk for immune rejection.     

Hydroxyapatite coating of cellulose sponges attracts bone-marrow-derived stem cells in rat subcutaneous tissue

The presence of bone-marrow-derived stem cells was investigated in a wound-healing model where subcutaneously implanted cellulose sponges were used to induce granulation tissue formation. When cellulose was coated with hydroxyapatite (HA), the sponges attracted circulating haemopoietic and mesenchymal progenitor cells more efficiently than uncoated cellulose. We hypothesized that the giant cells/macrophages of HA-coated sponges recognize HA as foreign material, phagocyte or hydrolyse it and release calcium ions, which are recognized by the calcium-sensing receptors (CaRs) expressed on many cells including haemopoietic progenitors. Our results showed, indeed, that the HA-coated sponges contained more CaR-positive cells than untreated sponges. The stem cells are, most probably, responsible for the richly vascularized granulation tissue formed in HA-coated sponges. This cell-guiding property of HA-coated cellulose might be useful in clinical situations involving impaired wound repair.     

Comparative Effects of Graphene and Molybdenum Disulfide on Human Macrophage Toxicity

Graphene and other 2D materials, such as molybdenum disulfide, have been increasingly used in electronics, composites, and biomedicine. In particular, MoS₂ and graphene hybrids have attracted a great interest for applications in the biomedical research, therefore stimulating a pertinent investigation on their safety in immune cells like macrophages, which commonly engulf these materials. In this study, M1 and M2 macrophage viability and activation are mainly found to be unaffected by few‐layer graphene (FLG) and MoS₂ at doses up to 50 µg mL^?1. The uptake of both materials is confirmed by transmission electron microscopy, inductively coupled plasma mass spectrometry, and inductively coupled plasma atomic emission spectroscopy. Notably, both 2D materials increase the secretion of inflammatory cytokines in M1 macrophages. At the highest dose, FLG decreases CD206 expression while MoS₂ decreases CD80 expression. CathB and CathL gene expressions are dose‐dependently increased by both materials. Despite a minimal impact on the autophagic pathway, FLG is found to increase the expression of Atg5 and autophagic flux, as observed by Western blotting of LC3‐II, in M1 macrophages. Overall, FLG and MoS₂ are of little toxicity in human macrophages even though they are found to trigger cell stress and inflammatory responses.     

Patterning as a signature of human epidermal stem cell regulation

Understanding how stem cells are regulated in adult tissues is a major challenge in cell biology. In the basal layer of human epidermis, clusters of almost quiescent stem cells are interspersed with proliferating and differentiating cells. Previous studies have shown that the proliferating cells follow a pattern of balanced stochastic cell fate. This behaviour enables them to maintain homeostasis, while stem cells remain confined to their quiescent clusters. Intriguingly, these clusters reappear spontaneously in culture, suggesting that they may play a functional role in stem cell auto-regulation. We propose a model of pattern formation that explains how clustering could regulate stem cell activity in homeostatic tissue through contact inhibition and stem cell aggregation.