Janus liposozyme for the modulation of redox and immune homeostasis in infected diabetic wounds.

Diabetic foot ulcers often become infected, leading to treatment complications and increased risk of loss of limb. Therapeutics to manage infection and simultaneously promote healing are needed. Here we report on the development of a Janus liposozyme that treats infections and promotes wound closure and re-epithelialization. The Janus liposozyme consists of liposome-like selenoenzymes for reactive oxygen species (ROS) scavenging to restore tissue redox and immune homeostasis. The liposozymes are used to encapsulate photosensitizers for photodynamic therapy of infections. We demonstrate application in methicillin-resistant Staphylococcus aureus-infected diabetic wounds showing high ROS levels for antibacterial function from the photosensitizer and nanozyme ROS scavenging from the liposozyme to restore redox and immune homeostasis. We demonstrate that the liposozyme can directly regulate macrophage polarization and induce a pro-regenerative response. By employing single-cell RNA sequencing, T cell-deficient Rag1-/- mice and skin-infiltrated immune cell analysis, we further reveal that IL-17-producing γδ T cells are critical for mediating M1/M2 macrophage transition. Manipulating the local immune homeostasis using the liposozyme is shown to be effective for skin wound repair and tissue regeneration in mice and mini pigs.

Decellularized Scaffolds with Double-Layer Aligned Microchannels Induce the Oriented Growth of Bladder Smooth Muscle Cells: Toward Urethral and Ureteral Reconstruction.

There is a great clinical need for regenerating urinary tissue. Native urethras and ureters have bidirectional aligned smooth muscle cells (SMCs) layers, which plays a pivotal role in micturition and transporting urine and inhibiting reflux. Thus far, urinary scaffolds have not been designed to induce the native-mimicking aligned arrangement of SMCs. In this study, a tubular decellularized extracellular matrix (dECM) with an intact internal layer and bidirectional aligned microchannels in the tubular wall, which is realized by the subcutaneous implantation of a template, followed by the removal of the template, and decellularization, is engineered. The dense and intact internal layer effectively increases the leakage pressure of the tubular dECM scaffolds. Rat-derived dECM scaffolds with three different sizes of microchannels are fabricated by tailoring the fiber diameter of the templates. The rat-derived dECM scaffolds exhibiting microchannels of ≈65 µm show suitable mechanical properties, good ability to induce the bidirectional alignment and growth of human bladder SMCs, and elevated higher functional protein expression in vitro. These data indicate that rat-derived tubular dECM scaffolds manifesting double-layer aligned microchannels may be promising candidates to induce the native-mimicking regeneration of SMCs in urethra and ureter reconstruction.

The loading of C-type natriuretic peptides improved hemocompatibility and vascular regeneration of electrospun poly(ε-caprolactone) grafts.

As a result of thrombosis or intimal hyperplasia, synthetic artificial vascular grafts had a low success rate when they were used to replace small-diameter arteries (inner diameter < 6 mm). C-type natriuretic peptides (CNP) have anti-thrombotic effects, and can promote endothelial cell (EC) proliferation and inhibit vascular smooth muscle cell (SMC) over-growth. In this study, poly(ε-caprolactone) (PCL) vascular grafts loaded with CNP (PCL-CNP) were constructed by electrospinning. The PCL-CNP grafts were able to continuously release CNP at least 25 days in vitro. The results of scanning electron microscopy (SEM) and mechanical testing showed that the loading of CNP did not change the microstructure and mechanical properties of the PCL grafts. In vitro blood compatibility analysis displayed that PCL-CNP grafts could inhibit thrombin activity and reduce platelet adhesion and activation. In vitro cell experiments demonstrated that PCL-CNP grafts activated ERK1/2 and Akt signaling in human umbilical vein endothelial cells (HUVECs), as well as increased cyclin D1 expression, enhanced proliferation and migration, and increased vascular endothelial growth factor (VEGF) secretion and nitric oxide (NO) production. The rabbit arteriovenous (AV)-shunt ex vitro indicated that CNP loading significantly improved the antithrombogenicity of PCL grafts. The assessment of vascular grafts in rat abdominal aorta implantation model displayed that PCL-CNP grafts promoted the regeneration of ECs and contractile SMCs, modulated macrophage polarization toward M2 phenotype, and enhanced extracellular matrix remodeling. These findings confirmed for the first time that loading CNP is an effective approach to improve the hemocompatibility and vascular regeneration of synthetic vascular grafts. STATEMENT OF SIGNIFICANCE: Small-diameter (< 6 mm) vascular grafts (SDVGs) have not been made clinically available due to their prevalence of thrombosis, limited endothelial regeneration and intimal hyperplasia. The incorporation of bioactive molecules into SDVGs serves as an effective solution to improve hemocompatibility and endothelialization. In this study, for the first time, we loaded C-type natriuretic peptides (CNP) into PCL grafts by electrospunning and confirmed the effectiveness of loading CNP on improving the hemocompatibility and vascular regeneration of artificial vascular grafts. Regenerative advantages included enhancement of endothelialization, modulation of macrophage polarization toward M2 phenotypes, and improved contractile smooth muscle cell regeneration. Our investigation brings attention to CNP as a valuable bioactive molecule for modifying cardiovascular biomaterial.

Mechanically reinforced biotubes for arterial replacement and arteriovenous grafting inspired by architectural engineering.

There is a lack in clinically-suitable vascular grafts. Biotubes, prepared using in vivo tissue engineering, show potential for vascular regeneration. However, their mechanical strength is typically poor. Inspired by architectural design of steel fiber reinforcement of concrete for tunnel construction, poly(ε-caprolactone) (PCL) fiber skeletons (PSs) were fabricated by melt-spinning and heat treatment. The PSs were subcutaneously embedded to induce the assembly of host cells and extracellular matrix to obtain PS-reinforced biotubes (PBs). Heat-treated medium-fiber-angle PB (hMPB) demonstrated superior performance when evaluated by in vitro mechanical testing and following implantation in rat abdominal artery replacement models. hMPBs were further evaluated in canine peripheral arterial replacement and sheep arteriovenous graft models. Overall, hMPB demonstrated appropriate mechanics, puncture resistance, rapid hemostasis, vascular regeneration, and long-term patency, without incidence of luminal expansion or intimal hyperplasia. These optimized hMPB properties show promise as an alternatives to autologous vessels in clinical applications.

The effect of hypoxia-mimicking responses on improving the regeneration of artificial vascular grafts.

Cellular transition to hypoxia following tissue injury, has been shown to improve angiogenesis and regeneration in multiple tissues. To take advantage of this, many hypoxia-mimicking scaffolds have been prepared, yet the oxygen access state of implanted artificial small-diameter vascular grafts (SDVGs) has not been investigated. Therefore, the oxygen access state of electrospun PCL grafts implanted into rat abdominal arteries was assessed. The regions proximal to the lumen and abluminal surfaces of the graft walls were normoxic and only the interior of the graft walls was hypoxic. In light of this differential oxygen access state of the implanted grafts and the critical role of vascular regeneration on SDVG implantation success, we investigated whether modification of SDVGs with HIF-1α stabilizer dimethyloxalylglycine (DMOG) could achieve hypoxia-mimicking responses resulting in improving vascular regeneration throughout the entirety of the graft wall. Therefore, DMOG-loaded PCL grafts were fabricated by electrospinning, to support the sustained release of DMOG over two weeks. In vitro experiments indicated that DMOG-loaded PCL mats had significant biological advantages, including: promotion of human umbilical vein endothelial cells (HUVECs) proliferation, migration and production of pro-angiogenic factors; and the stimulation of M2 macrophage polarization, which in-turn promoted macrophage regulation of HUVECs migration and smooth muscle cells (SMCs) contractile phenotype. These beneficial effects were downstream of HIF-1α stabilization in HUVECs and macrophages in normoxic conditions. Our results indicated that DMOG-loaded PCL grafts improved endothelialization, contractile SMCs regeneration, vascularization and modulated the inflammatory reaction of grafts in abdominal artery replacement models, thus promoting vascular regeneration.

Targeted Repair of Vascular Injury by Adipose-Derived Stem Cells Modified with P-Selectin Binding Peptide.

Percutaneous coronary intervention for coronary artery disease treatment often results in pathological vascular injury, characterized by P-selectin overexpression. Adipose-derived stem cells (ADSCs) therapeutic efficacy remains elusive due to poor ADSCs targeting and retention in injured vessels. Here, conjugated P-selectin binding peptide (PBP) to polyethylene glycol-conjugated phospholipid derivative (DMPE-PEG) linkers (DMPE-PEG-PBP; DPP) are used to facilitate the modification of PBP onto ADSCs cell surfaces via hydrophobic interactions between DMPE-PEG and the phospholipid bilayer. DPP modification neither has influence on ADSCs proliferation nor apoptosis/paracrine factor gene expression. A total of 5 × 10-6 m DPP-modified ADSCs (DPP-ADSCs) strongly binds to P-selectin-displaying activated platelets and endothelial cells (ECs) in vitro and to wire-injured rat femoral arteries when administered by intra-arterial injection. Targeted binding of ADSCs shields injury sites from platelet and leukocyte adhesion, thereby decreasing inflammation at injury sites. Furthermore, targeted binding of ADSCs recovers injured ECs functionality and reduces platelet-initiated vascular smooth muscle cells (VSMCs) chemotactic migration. Targeted binding of DPP-human ADSCs to balloon-injured human femoral arteries is also demonstrated in ex vivo experiments. Overall, DPP-ADSCs promote vascular repair, inhibit neointimal hyperplasia, increase endothelium functionality, and maintain normal VSMCs alignment, supporting preclinical noninvasive utilization of DPP-ADSCs for vascular injury.

Construction of vascular graft with circumferentially oriented microchannels for improving artery regeneration.

Design and fabrication of scaffolds with three-dimensional (3D) topological cues inducing regeneration of the neo-tissue comparable to native one remains a major challenge in both scientific and clinical fields. Here, we developed a well-designed vascular graft with 3D highly interconnected and circumferentially oriented microchannels by using the sacrificial sugar microfiber leaching method. The microchannels structure was capable of promoting the migration, oriented arrangement, elongation, and the contractile phenotype expression of vascular smooth muscle cells (VSMCs) in vitro. After implantation into the rat aorta defect model, the microchannels in vascular grafts simultaneously improved the infiltration and aligned arrangement of VSMCs and the oriented deposition of extracellular matrix (ECM), as well as the recruitment and polarization of macrophages. These positive results also provided protection and support for ECs growth, and ultimately accelerated the endothelialization. Our research provides a new strategy for the fabrication of grafts with the capability of inducing arterial regeneration, which could be further extended to apply in preparing other kinds of oriented scaffolds aiming to guide oriented tissue in situ regeneration.

Local Delivery of Dual MicroRNAs in Trilayered Electrospun Grafts for Vascular Regeneration.

Globally growing problems related to cardiovascular diseases lead to a considerable need for synthetic vascular grafts. For small-caliber vascular prosthesis, it remains essential to fulfill rapid endothelialization, inhibit intimal hyperplasia, and prevent calcification for keeping patency. To modulate vascular regeneration, herein, we developed a bioactive trilayered tissue-engineered vascular graft encapsulating both microRNA-126 and microRNA-145 in the fibrous inner and middle layers, respectively. In vitro cell activities demonstrated that the trilayered electrospun membranes had significant biological advantages in enhanced growth and intracellular nitric oxide production of vascular endothelial cells, modulation of phenotypes of vascular smooth muscle cells (SMCs), and restraint of calcium deposition through fast-releasing microRNA-126 and slow-releasing microRNA-145. Histological and immunofluorescent analyses of in vivo implantation in a rat abdominal aorta interposition model suggested that the dual-microRNA-loading trilayered electrospun graft exerted a positive effect on accelerating endothelialization, improving contractile SMC regeneration, and promoting normal extracellular matrix formation. Meanwhile, the local bioactivity of microRNA-126 and microRNA-145 in the trilayered vascular graft could regulate inflammation and depress calcification possibly by facilitating transformation of macrophages into the anti-inflammatory M2 phenotype. These findings indicated that the trilayered electrospun graft by local delivery of dual microRNAs could be possibly used as a bioactive substitute for replacement of artificial small-caliber blood vessels.

Assessment of combustion and emission behavior of corn straw biochar briquette fuels under different temperatures.

The 350 °C and 700 °C corn straw biochars were used to produce solid fuel briquettes. NovoGro (NG), an industrial by-product, were selected as a binder in the briquetting process. The ratios of the raw material to NG was assumed as 100:1 and 50:1 (denoted as 350NB1, 350NB2, 700NB1 and 700NB2, respectively). The physicochemical and morphological properties, combustion characteristics and gas emissions of the four briquettes were investigated. The results revealed that the biochars and the NG binder performed a good combination. The low temperature biochar briquettes, especially 350NB2, had excellent combustion characteristics, including low H/C and O/C ratios (0.17 and 0.82), low gas emissions (104.06 mg/m3 of CO, 157.25 mg/m3 of NOx and 18.92 mg/m3 of SO2), optimal resistance to mechanical shock (~90%) and high calorific values (21.48 MJ/kg). Thus, NG is a good binder for the briquetting of biochar. The low temperature biochar was a good feedstock for solid fuel production in the improvement of the combustion and emission quality.

Supramolecular Nanofibers with Superior Bioactivity to Insulin-Like Growth Factor-I.

Bioactive peptides derived from proteins generally need to be folded into secondary structures to activate downstream signaling pathways. However, synthetic peptides typically form random-coils, thus losing their bioactivities. Here, we show that by introducing a self-assembling peptide motif and using different preparation pathways, a peptide from insulin-like growth factor-I (IGF-1) can be folded into an α-helix and ß-sheet. The ß-sheet one exhibits a low dissociation constant to the IGF-1 receptor (IGF-1R, 11.5 nM), which is only about 3 times higher than that of IGF-1 (4.3 nM). However, the α-helical one and the peptide without self-assembling motif show weak affinities to IGF-1R ( KD = 179.1 and 321.6 nM, respectively). At 10 nM, the ß-sheet one efficiently activates the IGF-1 downstream pathway, significantly enhancing HUVEC proliferation and preventing cell apoptosis. The ß-sheet peptide shows superior performance to IGF-1 in vivo, and it improves ischemic hind-limb salvage by significantly reducing muscle degradation and enhancing limb vascularization. Our study provides a useful strategy to constrain peptides into different conformations, which may lead to the development of supramolecular nanomaterials mimicking biofunctional proteins.

Cobalt-mediated multi-functional dressings promote bacteria-infected wound healing.

Wound dressings with multiple functions are required to meet the complexity of the wound healing process. The multifunctionality often leads to an increase in the complexity and difficulty in dressing preparation. To surmount this problem, we used a facile preparation and fabrication process to fabricate a multi-functional dressing by integrating four widely accessible materials: plain gauze, sodium alginate (SA), Ca2+ and Co2+. Firstly, mixed Ca2+/Co2+ ion solutions with different concentration were applied to gauzes. After drying, SA solution was added to ionized gauze and Co2+-Ca2+/Gauze/SA (Ion-GSA) composite dressings were formed easily. In vitro results showed that all Ion-GSA dressings exhibited strong mechanical properties, uniform dispersion and sustained release of Ca2+ and Co2+, and the ability to retain moisture and absorb wound exudate. Besides the above advantages, dressings prepared with 0.25 g/L Co2+ and 4 g/L Ca2+ (Co2+0.25-Ca2+4 GSA composite dressings) exhibited the best overall effect for inducing a hypoxia-like response, and favorable cytocompatibility, hemostatic property and antibacterial activity. In vivo wound healing assays revealed that Co2+0.25-Ca2+4 GSA composite dressings inhibited bacterial growth, increased local Hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1) protein expression, and accelerated full-thickness skin wound healing in mouse bacterial-infected wound model. The quick healing wounds had improved angiogenesis, macrophages regulation, re-epithelialization and dense collagen deposition. Collectively, our results indicated that Co2+0.25-Ca2+4 GSA composite dressings promote wound healing. STATEMENT OF SIGNIFICANCE: Wound dressings with integrated functionalities are required to meet complex clinical requirements. However, there is often a trade-off between reducing preparation complexity and increasing the multifunctionality of the dressing's properties. In this study, we prepared multifunctional composite dressings by a facile preparation process using widely accessible materials. The composite dressings possessed the mechanical strength of gauze, had the effective wound exudate absorption, moisture maintenance and hemostatic property capacity of calcium alginate hydrogels, and had the hypoxia-like induction and the antimicrobial effects of Co2+. These functions all together promote bacteria-infected wound healing. Thus, we believed that the composite dressings can be widely applied in skin wound repair duo to their facile preparation method and good therapeutic effect.

In situ blood vessel regeneration using neuropeptide substance P-conjugated small-diameter vascular grafts.

In situ blood vessel regeneration through host stem/progenitor cell mobilization may hold great promise for vascular reconstruction. Neuropeptide substance P (SP) has been shown to accelerate tissue repair by endogenous cell mobilization and recruitment. This study was aimed to evaluate the vascular regeneration potential of SP and heparin co-tethered vascular grafts. Polycaprolactone (PCL), PCL/SP-conjugated poly(L-lactide-co-ε-caprolactone) (PLCL-SP) (SP), and PCL/PLCL-SP/heparin-conjugated PLCL (Hep/SP) vascular grafts were implanted as rat abdominal aorta substitutes for up to 2 weeks and 4 weeks. Ex vivo results delineate that heparin can improve the hemocompatibility and SP can recruit mesenchymal stem cells. Histological and immunohistochemical staining reveal higher cellular infiltration and homogeneous cell distribution in SP and Hep/SP grafts than that of the control grafts. At 4 weeks, SP and Hep/SP grafts show the presence of cobblestone-like cells on the luminal side, whereas the surface of PCL grafts remains bare. Immunoflourescence staining using von Willibrand factor (vWF) antibody shows improved endothelialization in SP and Hep/SP grafts compared with the PCL grafts. SP and Hep/SP grafts also exhibit more numbers of α-smooth muscle actin-positive cells and laminin+ blood vessels than that of the control group. Evaluation of inflammatory response reveals that three groups did not differ in terms of the numbers of CD68+ macrophages, whereas SP and Hep/SP grafts show higher numbers of CD206+ macrophages. These results indicate that SP can induce endogenous tissue regeneration in cell-free grafts, which may be of great interest for regenerative medicine and tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1669-1683, 2019.

In Situ Blood Vessel Regeneration Using SP (Substance P) and SDF (Stromal Cell-Derived Factor)-1α Peptide Eluting Vascular Grafts.

OBJECTIVE: The objective of this study was to develop small-diameter vascular grafts capable of eluting SDF (stromal cell-derived factor)-1α-derived peptide and SP (substance P) for in situ vascular regeneration. APPROACH AND RESULTS: Polycaprolactone (PCL)/collagen grafts containing SP or SDF-1α-derived peptide were fabricated by electrospinning. SP and SDF-1α peptide-loaded grafts recruited significantly higher numbers of mesenchymal stem cells than that of the control group. The in vivo potential of PCL/collagen, SDF-1, and SP grafts was assessed by implanting them in a rat abdominal aorta for up to 4 weeks. All grafts remained patent as observed using color Doppler and stereomicroscope. Host cells infiltrated into the graft wall and the neointima was formed in peptides-eluting grafts. The lumen of the SP grafts was covered by the endothelial cells with cobblestone-like morphology, which were elongated in the direction of the blood flow, as discerned using scanning electron microscopy. Moreover, SDF-1α and SP grafts led to the formation of a confluent endothelium as evaluated using immunofluorescence staining with von Willebrand factor antibody. SP and SDF-1α grafts also promoted smooth muscle cell regeneration, endogenous stem cell recruitment, and blood vessel formation, which was the most prominent in the SP grafts. Evaluation of inflammatory response showed that 3 groups did not significantly differ in terms of the numbers of proinflammatory macrophages, whereas SP grafts showed significantly higher numbers of proremodeling macrophages than that of the control and SDF-1α grafts. CONCLUSIONS: SDF-1α and SP grafts can be potential candidates for in situ vascular regeneration and are worthy for future investigations.

Functional Modification of Electrospun Poly(ε-caprolactone) Vascular Grafts with the Fusion Protein VEGF-HGFI Enhanced Vascular Regeneration.

Synthetic artificial vascular grafts have exhibited low patency rate and severe neointimal hyperplasia in replacing small-caliber arteries (<6 mm) because of their failure to generate a functional endothelium. In this study, small-caliber (2.0 mm) electrospun poly(ε-caprolactone) (PCL) vascular grafts were modified with a fusion protein VEGF-HGFI which consists of the class I hydrophobin (HGFI) and vascular endothelial growth factor (VEGF), via hydrophobic interactions. Immunofluorescence staining with the anti-VEGF antibody showed that VEGF-HGFI formed a protein layer on the surface of fibers in the grafts. Scanning electron microscopy (SEM) and mechanical measurements showed that VEGF-HGFI modification had no effect on the structure and mechanical properties of PCL grafts. Blood compatibility tests demonstrated a lower level of fibrinogen (FGN) absorption, platelet activation, and aggregation on the VEGF-HGFI-modified PCL mats than that on the bare PCL mats. The hemolysis rate was comparable in both the modified and bare PCL mats. In vitro culture of human umbilical vein endothelial cells (HUVECs) demonstrated that VEGF-HGFI modification could remarkably enhance nitric oxide (NO) production, prostacyclin2 (PGI2) release, and the uptake of acetylated low-density lipoprotein (Ac-LDL) by HUVECs. The healing characteristics of the modified grafts were examined in the replacement of rat abdominal aorta for up to 1 month. Immunofluorescence staining revealed that endothelialization, vascularization, and smooth muscle cell (SMC) regeneration were markedly improved in the VEGF-HGFI-modified PCL grafts. These results suggest that modification with fusion protein VEGF-HGFI is an effective method to improve the regeneration capacity of synthetic vascular grafts.