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Tailoring the surface of biomaterial scaffolds has been a key strategy to modulate the cellular interactions that are helpful for tissue healing process. In particular, nanotopological surfaces have been demonstrated to regulate diverse behaviors of stem cells, such as initial adhesion, spreading and lineage specification. Here, we tailor the surface of biopolymer nanofibers with carbon nanotubes (CNTs) to create a unique bi-modal nanoscale topography (500 nm nanofiber with 25 nm nanotubes) and report the performance in modulating diverse in vivo responses including inflammation, angiogenesis, and bone regeneration. When administered to a rat subcutaneous site, the CNT-coated nanofiber exhibited significantly reduced inflammatory signs (down-regulated pro-inflammatory cytokines and macrophages gathering). Moreover, the CNT-coated nanofibers showed substantially promoted angiogenic responses, with enhanced neoblood vessel formation and angiogenic marker expression. Such stimulated tissue healing events by the CNT interfacing were evidenced in a calvarium bone defect model. The in vivo bone regeneration of the CNT- coated nanofibers was significantly accelerated, with higher bone mineral density and up-regulated osteogenic signs (OPN, OCN, BMP2) of in vivo bone forming cells. The in vitro studies using MSCs could demonstrate accelerated adhesion and osteogenic differentiation and mineralization, supporting the osteo-promoting mechanism behind the in vivo bone forming event. These findings highlight that the CNTs interfacing of biopolymer nanofibers is highly effective in reducing inflammation, promoting angiogenesis, and driving adhesion and osteogenesis of MSCs, which eventually orchestrate to accelerate tissue healing and bone regeneration process. Here we demonstrate that the interfacing of biopolymer nanofibers with carbon nanotubes (CNTs) could modulate multiple interactions of cells and tissues that are ultimately helpful for the tissue healing and bone regeneration process. The CNT-coated scaffolds significantly reduced the pro-inflammatory signals while stimulating the angiogenic marker expressions. Furthermore, the CNT-coated scaffolds increased the bone matrix production of bone forming cells in vivo as well as accelerated the adhesion and osteogenic differentiation of MSCs in vitro. These collective findings highlight that the CNTs coated on the biopolymer nanofibers allow the creation of a promising platform for nanoscale engineering of biomaterial surface that can favor tissue healing and bone regeneration process, through a series of orchestrated events in anti-inflammation, pro-angiogenesis, and stem cell stimulation. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Cell encapsulating hydrogels with tunable mechanical and biological properties are of special importance for cell delivery and tissue engineering. Silk fibroin and collagen, two typical important biological proteins, are considered potential as cell culture hydrogels. However, both have been used individually, with limited properties ( e.g., collagen has poor mechanical properties and cell-mediated shrinkage, and silk fibroin from Bombyx mori (mulberry) lacks cell adhesion motifs). Therefore, the combination of them is considered to achieve improved mechanical and biological properties with respect to individual hydrogels. Here, we show that the cell-encapsulating hydrogels of mulberry silk fibroin / collagen are implementable over a wide range of compositions, enabled simply by combining the different gelation mechanisms. Not only the gelation reaction but also the structural characteristics, consequently, the mechanical properties and cellular behaviors are accelerated significantly by the silk fibroin / collagen hybrid hydrogel approach. Of note, the mechanical and biological properties are tunable to represent the combined merits of individual proteins. The shear storage modulus is tailored to range from 0.1 to 20 kPa along the iso-compositional line, which is considered to cover the matrix stiffness of soft-to-hard tissues. In particular, the silk fibroin / collagen hydrogels are highly elastic, exhibiting excellent resistance to permanent deformation under different modes of stress; without being collapsed or water-squeezed out ( vs. not possible in individual proteins) – which results from the mechanical synergism of interpenetrating networks of both proteins. Furthermore, the role of collagen protein component in the hybrid hydrogels provides adhesive sites to cells, stimulating anchorage and spreading significantly with respect to mulberry silk fibroin gel, which lacks cell adhesion motifs. The silk fibroin / collagen hydrogels can encapsulate cells while preserving the viability and growth over a long 3D culture period. Our findings demonstrate that the silk / collagen hydrogels possess physical and biological properties tunable and significantly improved ( vs. the individual protein gels), implying their potential uses for cell delivery and tissue engineering. Statement of Significance Development of cell encapsulating hydrogels with excellent physical and biological properties is important for the cell delivery and cell-based tissue engineering. Here we communicate for the first time the novel protein composite hydrogels comprised of ‘Silk’ and ‘Collagen’ and report their outstanding physical, mechanical and biological properties that are not readily achievable with individual protein hydrogels. The properties include i) gelation accelerated over a wide range of compositions, ii) stiffness levels covering 0.1 kPa to 20 kPa that mimic those of soft-to-hard tissues, iii) excellent elastic behaviors under various stress modes (bending, twisting, stretching, and compression), iv) high resistance to cell-mediated gel contraction, v) rapid anchorage and spreading of cells, and vi) cell encapsulation ability with a long-term survivability. These results come from the synergism of individual proteins of alpha-helix and beta-sheet structured networks. We consider the current elastic cell-encapsulating hydrogels of silk-collagen can be potentially useful for the cell delivery and tissue engineering in a wide spectrum of soft-to-hard tissues. [ABSTRACT FROM AUTHOR]

Multifunctional nanocarrier-based theranostics is currently considered to solve some key unmet challenges in cancer treatment. Here we report a nanocarrier platform, named carbon dot (CD) created mesoporous hollow organosilica (C-hMOS) nanoparticles, to deliver anticancer drug and to enable optical imaging. The hollow structure was formed by the removal of a nanorod core template, and at the same time, the fluorescent signal was endowed from the heat-treated organosilica network. Thanks to the hollow and mesoporous structure, the C-hMOS effectively loaded doxorubicin (DOX) for cancer chemotherapy. The DOX was released from C-hMOS highly sustainably (over 12 days) and pH-dependently (pH 5.0 > pH 7.4). The DOX-loading C-hMOS internalized cancer cells efficiently (>90%), and induced cellular apoptosis including the expression of caspase-3. The treatment of C-hMOS to cancer cells enabled multi-color visualization in vitro, suggesting the possibility of cell tracing. Moreover, when injected intratumorally in mice, the C-hMOS exhibited strong optical signals in vivo along with a high optical stability (over a week). The injected C-hMOS were distributed only a fraction in liver but not in heart, lung, spleen or kidney and displayed good biocompatibility. The DOX-delivering C-hMOS significantly suppressed the in vivo tumor growth associated with apoptotic functions. Taken together, the developed C-hMOS nanoparticles can be a promising nanoplatform for drug delivery and in vivo imaging in cancer treatment. Statement of Significance Multifunctional nanoparticles that combine chemotherapeutic ability with imaging modality comprise promising platform for cancer theranostics. Here we developed a novel theranostic nanoparticle, i.e., carbon-dot created mesoporous hollow silica nanoparticle, to offer unique merit for this purpose. The in vitro and in vivo findings to support this include: i) carbon dots with 1–2 nm size in situ generated discretely and uniformly within silica network, ii) hollow and mesoporous structure effective for loading of DOX at high content, iii) release behavior of DOX in a sustainable and pH-dependent manner, iv) chemotherapeutic efficacy in killing cancer cells and suppressing tumor growth through DOX delivery, and v) carbon dot induced multi-color fluorescence imaging within cells and tumor tissues. These collective multifaceted properties may facilitate the novel carbon dot nanocarriers to be a potential candidate for delivering anticancer drug and non-invasive imaging in cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract: We prepared chitosan–gelatin (Chi–Gel) composite coatings on Ti via electrophoretic deposition (EPD) method for utilization in tissue repair and drug delivery. Uniform coatings were produced over a wide compositional range (0–75% Gel) with coating gains dependent on the EPD parameters including voltage and time. Coating degradation increased as the Gel content increased, with 16–54% weight losses after 3weeks of immersion in phosphate buffered saline. Ampicillin, used as a model drug, was successfully incorporated within the coatings during the EPD process, and the release was highly sustainable with no burst effect up to a month, proving the potential of these materials as long-term drug eluting coatings. The release rate was dependent on the coating degradation, i.e., the more degradable with increasing Gel content, suggesting a rate-controllable drug release by a compositional change. Preliminary cell tests showed favorable cell adhesion and spreading on the composite coatings, with significant improvement in cell proliferation as Gel content increased. While more in-depth biological assays remain, the Chi–Gel might be useful as a drug eluting electrophoretic coating system on metallic implants for tissue repair. [Copyright &y& Elsevier]

Abstract: Development of biocompatible and multifunctional nanocarriers is important for the therapeutic efficacy of drug molecules in the treatment of disease and tissue repair. A novel nanocarrier of luminescent hollowed mesoporous silica (L-hMS) was explored for the loading and controlled delivery of drugs. For the synthesis of L-hMS, self-activated luminescence hydroxyapatite (LHA) was used as a template. Different thicknesses (∼7–62nm) of mesoporous silica shell were obtained by varying the volume of silica precursor and the subsequent removal of the LHA core, which resulted in hollow-cored (size of ∼40nm×10nm) mesoporous silica nanoreservoirs, L-hMS. While the silica shell provided a highly mesoporous structure, enabling an effective loading of drug molecules, the luminescent property of LHA was also well preserved in both the silica-shelled and the hollow-cored nanocarriers. Doxorubicin (DOX), used as a model drug, was shown to be effectively loaded onto the mesopore structure and within the hollow space of the nanoreservoir. The DOX release was fairly pH-dependent, occurring more rapidly at pH 5.3 than at pH 7.4, and a long-term sustainable delivery over the test period of 2weeks was observed. The nanoreservoir exhibited favorable cell compatibility with low cytotoxicity and excellent cell uptake efficiency (over 90%). Treatment of HeLa cells with DOX-loaded L-hMS elicited a sufficient degree of biological efficacy of DOX, as confirmed in the DOX-induced apoptotic behaviors, including stimulation in caspase-3 expression, and was even more effective than the direct DOX treatment. Overall, the newly developed L-hMS nanoreservoirs may be potentially useful as a multifunctional (luminescent, mesoporous and biocompatible) carrier system to effectively load and sustainably deliver small molecules, including anticancer drugs. [Copyright &y& Elsevier]

Abstract: Three-dimensional (3-D) open-channeled scaffolds of biopolymers are a promising candidate matrix for tissue engineering. When scaffolds have the capacity to deliver bioactive molecules the potential for tissue regeneration should be greatly enhanced. In order to improve drug-delivery capacity, we exploit 3-D poly(lactic acid) (PLA) scaffolds by creating microporosity within the scaffold network. Macroporous channeled PLA with a controlled pore configuration was obtained by a robotic dispensing technique. In particular, a room temperature ionic liquid (RTIL) bearing hydrophilic counter-anions, such as OTf and Cl, was introduced to the biopolymer solution at varying ratios. The RTIL–biopolymer slurry was homogenized by ultrasonication, and then solidified through the robotic dispensing process, during which the biopolymer and RTIL formed a bicontinuous interpenetrating network. After ethanol wash-out treatment the RTIL was completely removed to leave highly microporous open channels throughout the PLA network. The resultant pore size was observed to be a few micrometers (average 2.43μm) and microporosity was determined to be ∼70%. The microporous surface was also shown to favor initial cell adhesion, stimulating cell anchorage on the microporous structure. Furthermore, in vivo tissue responses assessed in rat subcutaneous tissue revealed good tissue compatibility, with minimal inflammatory reactions, while gathering a larger population of fibroblastic cells than the non-microporous scaffolds, and even facilitating invasion of the cells within the microporous structure. The efficacy of the micropore networks generated within the 3-D scaffolds in loading and releasing therapeutic molecules was addressed using antibiotic sodium ampicillin and protein cytochrome C as model drugs. The microporous scaffolds exhibited significantly enhanced drug loading capacity: 4–5 times increase in ampicillin and 9–10 times increase in cytochrome C compared to the non-microporous scaffolds. The release of ampicillin loaded within the microporous scaffolds was initially fast (∼85% for 1week), and was then slowed down, showing a continual release up to a month. On the other hand, cytochrome C was shown to release in a highly sustainable manner over a month, without showing an initial burst release effect. This study provides a novel insight into the generation of 3-D biopolymer scaffolds with high performance in loading and delivery of biomolecules, facilitated by the creation of microporous channels through the scaffold network. The capacity to support tissue cells while in situ delivering drug molecules makes the current scaffolds potentially useful for therapeutic tissue engineering. [Copyright &y& Elsevier]

Long term outcomes of lung transplantation are impacted by the occurrence of chronic lung allograft dysfunction (CLAD). Recent evidence suggests a role for the lung microbiome in the occurrence of CLAD, but the exact mechanisms are not well defined. We hypothesize that the lung microbiome inhibits epithelial autophagic clearance of pro-fibrotic proteins in an IL-33 dependent manner, thereby augmenting fibrogenesis and risk for CLAD. Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast differentiation markers. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of Beclin-1. Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further, PsA exposure increased myofibroblast differentiation and collagen formation. IL-33 blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated myofibroblast activation in a Beclin-1 dependent manner. CLAD is associated with increased airway IL-33 expression and reduced basal autophagy. PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an IL-33 dependent manner. [ABSTRACT FROM AUTHOR]

Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking the microbiome to CLAD are poorly delineated. We investigated a possible mechanism involved in microbial modulation of mucosal response leading to CLAD with the hypothesis that a Proteobacteria dominant lung microbiome would inhibit N-myc-interactor (NMI) expression and induce epithelial to mesenchymal transition (EMT). Explant CLAD, non-CLAD, and healthy nontransplant lung tissue were collected, as well as bronchoalveolar lavage from 14 CLAD and matched non-CLAD subjects, which were followed by 16S rRNA amplicon sequencing and quantitative polymerase chain reaction (PCR) analysis. Pseudomonas aeruginosa (PsA) or PsA-lipopolysaccharide was cocultured with primary human bronchial epithelial cells (PBEC). Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate NMI expression and EMT in explants and in PsA-exposed PBECs. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of EMT regulator NMI. 16S rRNA amplicon analyses revealed that CLAD patients have a higher abundance of phyla Proteobacteria and reduced abundance of the phyla Bacteroidetes. At the genera level, CLAD subjects had an increased abundance of genera Pseudomonas and reduced Prevotella. Human CLAD airway cells showed a downregulation of the N-myc-interactor gene and presence of EMT. Furthermore, exposure of human primary bronchial epithelial cells to PsA resulted in downregulation of NMI and induction of an EMT phenotype while NMI upregulation resulted in attenuation of this PsA-induced EMT response. CLAD is associated with increased bacterial biomass and a Proteobacteria enriched airway microbiome and EMT. Proteobacteria such as PsA induces EMT in human bronchial epithelial cells via NMI, demonstrating a newly uncovered mechanism by which the microbiome induces cellular metaplasia. [ABSTRACT FROM AUTHOR]

Cell reprogramming can satisfy the demands of obtaining specific cell types for applications such as tissue regeneration and disease modeling. Here we report the reprogramming of human fibroblasts to produce chemically-induced osteogenic cells (ciOG), and explore the potential uses of ciOG in bone repair and disease treatment. A chemical cocktail of RepSox, forskolin, and phenamil was used for osteogenic induction of fibroblasts by activation of RUNX2 expression. Following a maturation, the cells differentiated toward an osteoblast phenotype that produced mineralized nodules. Bulk and single-cell RNA sequencing identified a distinct ciOG population. ciOG formed mineralized tissue in an ectopic site of immunodeficiency mice, unlike the original fibroblasts. Osteogenic reprogramming was modulated under engineered culture substrates. When generated on a nanofiber substrate ciOG accelerated bone matrix formation in a calvarial defect, indicating that the engineered biomaterial promotes the osteogenic capacity of ciOG in vivo. Furthermore, the ciOG platform recapitulated the genetic bone diseases Proteus syndrome and osteogenesis imperfecta, allowing candidate drug testing. The reprogramming of human fibroblasts into osteogenic cells with a chemical cocktail thus provides a source of specialized cells for use in bone tissue engineering and disease modeling. [ABSTRACT FROM AUTHOR]

Objective Although polymethyl methacrylate (PMMA) is widely used as a dental material, a major challenge of using this substance is its poor antimicrobial (anti-adhesion) effects, which increase oral infections. Here, graphene-oxide nanosheets (nGO) were incorporated into PMMA to introduce sustained antimicrobial-adhesive effects by increasing the hydrophilicity of PMMA. Methods After characterizing nGO and nGO-incorporated PMMA (up to 2 wt%) in terms of morphology and surface characteristics, 3-point flexural strength and hardness were evaluated. The anti-adhesive effects were determined for 4 different microbial species with experimental specimens and the underlying anti-adhesive mechanism was investigated by a non-thermal oxygen plasma treatment. Sustained antimicrobial-adhesive effects were characterized with incubation in artificial saliva for up to 28 days. Results The typical nanosheet morphology was observed for nGO. Incorporating nGO into PMMA roughened its surface and increased its hydrophilicity without compromising flexural strength or surface hardness. An anti-adhesive effect after 1 h of exposure to microbial species in artificial saliva was observed in nGO-incorporated specimens, which accelerated with increasing levels of nGO without significant cytotoxicity to oral keratinocytes. Plasma treatment of native PMMA demonstrated that the antimicrobial-adhesive effects of nGO incorporation were at least partially due to increased hydrophilicity, not changes in the surface roughness. A sustained antimicrobial-adhesive property against Candida albicans was observed in 2% nGO for up to 28 days. Significance The presence of sustained anti-adhesion properties in nGO-incorporated PMMA without loading any antimicrobial drugs suggests the potential usefulness of this compound as a promising antimicrobial dental material for dentures, orthodontic devices and provisional restorative materials. [ABSTRACT FROM AUTHOR]