Metallic alloys are normally composed of multiple constituent elements in order to achieve integration of a plurality of properties required in technological applications. However, conventional alloy ...development paradigm, by sequential trial-and-error approach, requires completely unrelated strategies to optimize compositions out of a vast phase space, making alloy development time consuming and labor intensive. Here, we challenge the conventional paradigm by proposing a combinatorial strategy that enables parallel screening of a multitude of alloys. Utilizing a typical metallic glass forming alloy system Zr-Cu-Al-Ag as an example, we demonstrate how glass formation and antibacterial activity, two unrelated properties, can be simultaneously characterized and the optimal composition can be efficiently identified. We found that in the Zr-Cu-Al-Ag alloy system fully glassy phase can be obtained in a wide compositional range by co-sputtering, and antibacterial activity is strongly dependent on alloy compositions. Our results indicate that antibacterial activity is sensitive to Cu and Ag while essentially remains unchanged within a wide range of Zr and Al. The proposed strategy not only facilitates development of high-performing alloys, but also provides a tool to unveil the composition dependence of properties in a highly parallel fashion, which helps the development of new materials by design.
Thrombospondin (TSP) 1 and 2, share the same overall structure and interact with a number of the same cell-surface receptors. In an attempt to elucidate their biological roles more clearly, we ...generated double-TSP1/TSP2-null animals and compared their phenotype to those of TSP1- and TSP2-null mice. Double-null mice exhibited an apparent phenotype that primarily represented the sum of the abnormalities observed in the single-null mice. However, surprisingly, the wound-healing response in double-null mice resembled that in TSP1-null animals and differed from that in TSP2-nulls. Thus, although the excisional wounds of TSP2-null mice are characterized by increased neovascularization and heal at an accelerated rate, TSP1-null and double-null animals demonstrated delayed healing, as indicated by the prolonged persistence of inflammation and delayed scab loss. Immunohistochemical analysis showed that, similar to TSP1-null mice, the granulation tissue of double-null mice was not excessively vascularized. Furthermore as in TSP1-nulls, decreases in macrophage recruitment and in the levels of monocyte chemoattractant protein-1 indicated that the inflammatory phase of the wound-healing response was impaired in double-null mice. Our data demonstrate that the consequences of a lack of TSP1 predominate in the response of double-null mice, and dictate the course of wound healing. These findings reflect distinct temporal and spatial expressions of TSP1 and TSP2 in the healing wound.
Bacterial infection within the synovial joint, commonly known as septic arthritis, remains a clinical challenge as it presents two concurrent therapeutic goals of reducing bacterial burden and ...preservation of articular cartilage from destructive host inflammation. We hypothesized that mitigation of MRSA-induced inflammatory signaling could diminish destruction of articular cartilage in the setting of septic arthritis when used in conjunction with antibiotics. Herein, we provide evidence which supports a new therapeutic notion that concurrent antimicrobial therapy to address the ‘septic’ component of the disease with inflammation mitigation to manage the destructive ‘arthritis’ component. We established a murine model to mimic septic knee arthritis, as well as a variety of other inflammatory joint conditions. This murine septic arthritis model, in conjunction with
in vitro
and ex-vivo models, was utilized to characterize the inflammatory profile seen in active septic arthritis, as well as post-antibiotic treatment,
via
transcriptomic and histologic studies. Finally, we provided the clinical rationale for a novel therapeutic strategy combining enhanced antibiotic treatment with rifampin and adjuvant immunomodulation to inhibit post-infectious, excess chondrolysis and osteolysis. We identified that septic arthritis secondary to MRSA infection in our murine model led to increased articular cartilage damage compared to various types of inflammatory arthritis. The activation of the pERK1/2 signaling pathway, which is implicated with the mounting of an immune response and generation of inflammation, was increased in intracellular MRSA-infected synovial tissue and persisted despite antibiotic treatment. Trametinib, an inhibitor of ERK signaling through suppression of MEK1/2, alleviated the inflammation produced by the addition of intra-articular, heat-killed MRSA. Further, when combined with vancomycin and rifampin, mitigation of inflammation by pERK1/2 targeting improved outcomes for MRSA septic arthritis by conferring chondroprotection to articular cartilage and diminishing inflammatory osteolysis within bone. Our results support a new therapeutic notion that cell/biofilm-penetrating antibiotics alongside adjuvant mitigation of excessive intra-articular inflammation accomplish distinct therapeutic goals: reduction of bacterial burden and preservation of articular cartilage integrity.
The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ...ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization.
Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis.
Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.
Infection is a devastating complication following an open fracture. We investigated whether local rifampin‐loaded hydrogel can combat infection and improve healing in a murine model of ...methicillin‐resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10–12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony‐forming units (CFU) of MRSA was inoculated. A collagen‐based hydrogel containing low‐dose (60 μg) and high‐dose (300 μg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (μCT), immunohistochemistry, and histological analyses. All untreated MRSA‐infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin‐1β stain were associated with poor callus formation. High‐dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low‐dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high‐dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). μCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin‐loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin‐loaded hydrogels is required to correlate these findings with clinical efficacy.
Sustained changes in blood flow modulate the size of conduit arteries through structural alterations of the vessel wall that are dependent on the transient accumulation and activation of perivascular ...macrophages. The leukocytic infiltrate appears to be confined to the adventitia, is responsible for medial remodeling, and resolves once hemodynamic stresses have normalized without obvious intimal changes. We report that inward remodeling of the mouse common carotid artery after ligation of the ipsilateral external carotid artery is dependent on the chemokine receptor CXCR3. Wild-type myeloid cells restored flow-mediated vascular remodeling in CXCR3-deficient recipients, adventitia-infiltrating macrophages of Gr1(low) resident phenotype expressed CXCR3, the perivascular accumulation of macrophages was dependent on CXCR3 signaling, and the CXCR3 ligand IP-10 was sufficient to recruit monocytes to the adventitia. CXCR3 also contributed to selective features of macrophage activation required for extracellular matrix turnover, such as production of the transglutaminase factor XIII A subunit. Human adventitial macrophages displaying a CD14(+)/CD16(+) resident phenotype, but not circulating monocytes, expressed CXCR3, and such cells were more frequent at sites of disturbed flow. Our observations reveal a CXCR3-dependent accumulation and activation of perivascular macrophages as a necessary step in homeostatic arterial remodeling triggered by hemodynamic stress in mice and possibly in humans as well.
Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family ...member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.
Abstract We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We ...synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly( l -lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full-thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds.
Injury- and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces ...angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.
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