The Management of Posttraumatic Nasal Deformities.

Nasal bone fractures are the most common fractures of the facial skeleton and are often accompanied by bony, cartilaginous, and soft tissue injuries. These injuries are often complex, and when untreated or inadequately treated, can lead to posttraumatic nasal deformity. The most common deformities are the crooked nose and the saddle nose. Both deformities may result in significant cosmetic and functional concerns. The treatment of these deformities can be complex, requiring careful evaluation of the nose and thoughtful planning to correct the cosmetic deformity and restore functional integrity. The rhinoplasty surgeon will benefit from having a large repertoire of techniques to achieve these repairs. In this article, we discuss the options and concepts for the management of nasal bone fractures as well as complicated posttraumatic nasal deformity. Level of evidence is not available.

Evidence of Biofilm and Persister Cell Formation in Revision Rhinoplasty.

Background: Postoperative rhinoplasty infection can lead to serious cosmetic deformity, loss of structural integrity to the nose, and functional deficiencies. Understanding the factors contributing to postoperative infection is important. Microbial biofilms and persister cells play an important role in health care-associated infections. The objective of this study is to identify microbial biofilm and persister cells in the nasal soft tissue of patients undergoing revision rhinoplasty. Methods: Fourteen patients undergoing rhinoplasty were recruited for this study. Nasal soft tissue was removed during rhinoplasty and preserved in 2% paraformaldehyde/2.5% glutaraldehyde. High-resolution images were then obtained from these nasal soft tissue samples. Results: Three samples were positive for the presence of microbial persister cells or biofilms. All samples came from patients undergoing revision rhinoplasty. These patients had between one to six previous rhinoplasty procedures and one patient had previous injectable nasal filler. Conclusions: Biofilms and persister cells are able to form in nasal soft tissue of revision rhinoplasty patients in the absence of an implant and may contribute to increased postoperative infection risk.

Navigating the Collagen Jungle: The Biomedical Potential of Fiber Organization in Cancer.

Recent research has highlighted the importance of key tumor microenvironment features, notably the collagen-rich extracellular matrix (ECM) in characterizing tumor invasion and progression. This led to great interest from both basic researchers and clinicians, including pathologists, to include collagen fiber evaluation as part of the investigation of cancer development and progression. Fibrillar collagen is the most abundant in the normal extracellular matrix, and was revealed to be upregulated in many cancers. Recent studies suggested an emerging theme across multiple cancer types in which specific collagen fiber organization patterns differ between benign and malignant tissue and also appear to be associated with disease stage, prognosis, treatment response, and other clinical features. There is great potential for developing image-based collagen fiber biomarkers for clinical applications, but its adoption in standard clinical practice is dependent on further translational and clinical evaluations. Here, we offer a comprehensive review of the current literature of fibrillar collagen structure and organization as a candidate cancer biomarker, and new perspectives on the challenges and next steps for researchers and clinicians seeking to exploit this information in biomedical research and clinical workflows.

Hierarchical assembly of hyaluronan coated albumin nanoparticles for pancreatic cancer chemoimmunotherapy.

Pancreatic cancer is a highly malignant carcinoma with limited effective treatment options, resulting in a poor patient survival rate of less than 5%. In this study, cationic albumin nanoparticles were assembled with negatively charged hyaluronic acid (HA) to achieve a hierarchical nanostructure and efficient delivery of small molecule drugs to the tumor site in the pancreas. A combination of chemotherapy with indoleamine-2,3-dioxygenase (IDO) inhibition was explored to enhance the chemotherapeutic efficacy in vivo. Hydrophobic celastrol (CLT) and hydrophilic 1-methyltryptophan (MT) were concurrently loaded in HA coated cationic albumin nanoparticles (HNPs) with an average size of ∼300 nm. The size of HNPs was reduced in the presence of hyaluronidase to facilitate penetration into deep tumor tissues. Also, the biodistribution study in the C57BL/6 mice xenograft model showed enhanced tumor accumulation and prolonged circulation of HNPs. Compared with CLT solution, the combination of CLT with MT showed significantly enhanced tumor inhibition in both xenograft and orthotopic pancreatic cancer mice models via downregulating the immunosuppressive tumor microenvironment. Taken together, the combination of CLT with MT administered via HNPs represents a highly promising strategy for targeted pancreatic cancer therapy.

[Digital applicator by 3D printing in contact brachytherapy]. / Applicateur numérique par impression tridimensionnelle en curiethérapie de contact.

Brachytherapy of skin tumours uses custom applicators that are manufactured manually. The integration of 3D printing customization of applicators during hidh dose rate brachytherapy planning could allow a better skin conformation and a better reproducibility of the positioning and treatment. We present the technical implementation of this method for our first two patients. A provisional planning scanner was carried out to create a digital applicator. The creation of the digital applicator used successively several software programs. The first, commercial, was RhinocerosR 3D used via Grasshopper, an integrated open source plug-in. The 3D applicator was then exported to the commercial software Simplify3DR. A g-code format file was generated for the printer. A second scanner was made with a 3D applicator in place to plan the final treatment. The treatment was planned by reverse optimization. The applicator could be designed within 15 days. For patient A, it was noted that 95 % of the clinical target volume received at least 35.4Gy (63Gy EQD2). For patient B, 95 % of the clinical target volume received at least 36Gy (64.8Gy EQD2). The forecast and actual planimetry met the coverage criteria of D95. Contact brachytherapy with 3D bioimpression is feasible, after software training, for complex treatment lesions. This technique could be extended to other indications.

Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis.

OBJECTIVE: Extracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. METHODS: Whole body ablation of Lumican (Lum-/-) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. RESULTS: Lumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. CONCLUSIONS: These data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Site-specific characterization and quantitation of N-glycopeptides in PKM2 knockout breast cancer cells using DiLeu isobaric tags enabled by electron-transfer/higher-energy collision dissociation (EThcD).

The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.

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.

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.

WITHDRAWN: Corrigendum to "Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds" [Acta Biomater. 9(11) (2013) 8802-8814].

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.actbio.2017.06.005. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

The P Value Problem in Otolaryngology: Shifting to Effect Sizes and Confidence Intervals.

There is a lack of reporting effect sizes and confidence intervals in the current biomedical literature. The objective of this article is to present a discussion of the recent paradigm shift encouraging the use of reporting effect sizes and confidence intervals. Although P values help to inform us about whether an effect exists due to chance, effect sizes inform us about the magnitude of the effect (clinical significance), and confidence intervals inform us about the range of plausible estimates for the general population mean (precision). Reporting effect sizes and confidence intervals is a necessary addition to the biomedical literature, and these concepts are reviewed in this article.

Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

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.

Human pancreatic stellate cells modulate 3D collagen alignment to promote the migration of pancreatic ductal adenocarcinoma cells.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the ability for cancer cells to aggressively infiltrate and navigate through a dense stroma during the metastatic process. Key features of the PDAC stroma include an abundant population of activated pancreatic stellate cells (PSCs) and highly aligned collagen fibers; however, important questions remain regarding how collagen becomes aligned and what the biological manifestations are. To better understand how PSCs, aligned collagen, and PDAC cells might cooperate during the transition to invasion, we utilized a microchannel-based in vitro tumor model and advanced imaging technologies to recreate and examine in vivo-like heterotypic interactions. We found that PSCs participate in a collaborative process with cancer cells by orchestrating the alignment of collagen fibers that, in turn, are permissive to enhanced cell migration. Additionally, direct contact between PSCs, collagen, and PDAC cells is critical to invasion and co-migration of both cell types. This suggests PSCs may accompany and assist in navigating PDAC cells through the stromal terrain. Together, our data provides a new role for PSCs in stimulating the metastatic process and underscores the importance of collagen alignment in cancer progression.

Highly aligned stromal collagen is a negative prognostic factor following pancreatic ductal adenocarcinoma resection.

Risk factors for pancreatic ductal adenocarcinoma (PDAC) progression after surgery are unclear, and additional prognostic factors are needed to inform treatment regimens and therapeutic targets. PDAC is characterized by advanced sclerosis of the extracellular matrix, and interactions between cancer cells, fibrillar collagen, and other stromal components play an integral role in progression. Changes in stromal collagen alignment have been shown to modulate cancer cell behavior and have important clinical value in other cancer types, but little is known about its role in PDAC and prognostic value. We hypothesized that the alignment of collagen is associated with PDAC patient survival. To address this, pathology-confirmed tissues from 114 PDAC patients that underwent curative-intent surgery were retrospectively imaged with Second Harmonic Generation (SHG) microscopy, quantified with fiber segmentation algorithms, and correlated to patient survival. The same tissue regions were analyzed for epithelial-to-mesenchymal (EMT), α-SMA, and syndecan-1 using complimentary immunohistostaining and visualization techniques. Significant inter-tumoral variation in collagen alignment was found, and notably high collagen alignment was observed in 12% of the patient cohort. Stratification of patients according to collagen alignment revealed that high alignment is an independent negative factor following PDAC resection (p = 0.0153, multivariate). We also found that epithelial expression of EMT and the stromal expression of α-SMA and syndecan-1 were positively correlated with collagen alignment. In summary, stromal collagen alignment may provide additional, clinically-relevant information about PDAC tumors and underscores the importance of stroma-cancer interactions.

Comparison of Picrosirius Red Staining With Second Harmonic Generation Imaging for the Quantification of Clinically Relevant Collagen Fiber Features in Histopathology Samples.

Stromal collagen alignment has been shown to have clinical significance in a variety of cancers and in other diseases accompanied by fibrosis. While much of the biological and clinical importance of collagen changes has been demonstrated using second harmonic generation (SHG) imaging in experimental settings, implementation into routine clinical pathology practice is currently prohibitive. To translate the assessment of collagen organization into routine pathology workflow, a surrogate visualization method needs to be examined. The objective of the present study was to quantitatively compare collagen metrics generated from SHG microscopy and commonly available picrosirius red stain with standard polarization microscopy (PSR-POL). Each technique was quantitatively compared with established image segmentation and fiber tracking algorithms using human pancreatic cancer as a model, which is characterized by a pronounced stroma with reorganized collagen fibers. Importantly, PSR-POL produced similar quantitative trends for most collagen metrics in benign and cancerous tissues as measured by SHG. We found it notable that PSR-POL detects higher fiber counts, alignment, length, straightness, and width compared with SHG imaging but still correlates well with SHG results. PSR-POL may provide sufficient and additional information in a conventional clinical pathology laboratory for certain types of collagen quantification.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-ß treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.