Automated Extraction of Skin Wound Healing Biomarkers From In Vivo Label-Free Multiphoton Microscopy Using Convolutional Neural Networks.

BACKGROUND AND OBJECTIVES: Histological analysis is a gold standard technique for studying impaired skin wound healing. Label-free multiphoton microscopy (MPM) can provide natural image contrast similar to histological sections and quantitative metabolic information using NADH and FAD autofluorescence. However, MPM analysis requires time-intensive manual segmentation of specific wound tissue regions limiting the practicality and usage of the technology for monitoring wounds. The goal of this study was to train a series of convolutional neural networks (CNNs) to segment MPM images of skin wounds to automate image processing and quantification of wound geometry and metabolism. STUDY DESIGN/MATERIALS AND METHODS: Two CNNs with a 4-layer U-Net architecture were trained to segment unstained skin wound tissue sections and in vivo z-stacks of the wound edge. The wound section CNN used 380 distinct MPM images while the in vivo CNN used 5,848 with both image sets being randomly distributed to training, validation, and test sets following a 70%, 20%, and 10% split. The accuracy of each network was evaluated on the test set of images, and the effectiveness of automated measurement of wound geometry and optical redox ratio were compared with hand traced outputs of six unstained wound sections and 69 wound edge z-stacks from eight mice. RESULTS: The MPM wound section CNN had an overall accuracy of 92.83%. Measurements of epidermal/dermal thickness, wound depth, wound width, and % re-epithelialization were within 10% error when evaluated on six full wound sections from days 3, 5, and 10 post-wounding that were not included in the training set. The in vivo wound z-stack CNN had an overall accuracy of 89.66% and was able to isolate the wound edge epithelium in z-stacks from eight mice across post-wound time points to quantify the optical redox ratio within 5% of what was recorded by manual segmentations. CONCLUSION: The CNNs trained and presented in this study can accurately segment MPM imaged wound sections and in vivo z-stacks to enable automated and rapid calculation of wound geometry and metabolism. Although MPM is a noninvasive imaging modality well suited to imaging living wound tissue, its use has been limited by time-intensive user segmentation. The use of CNNs for automated image segmentation demonstrate that it is possible for MPM to deliver near real-time quantitative readouts of tissue structure and function. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.

Reducing Chlorothalonil Use in Fungicide Spray Programs for Powdery Mildew, Anthracnose, and Gummy Stem Blight in Melons.

Fungicides are applied to nearly 80% of U.S. melon acreage to manage the numerous foliar and fruit diseases that threaten yield. Chlorothalonil is the most widely used fungicide but has been associated with negative effects on human and bee health. We designed alternative fungicide programs to examine the impact of reducing chlorothalonil use (Bravo Weather Stik) on watermelon, cantaloupe, and honeydew melon in 2016, 2017, and 2018 in Maryland. Chlorothalonil was replaced in the tank mix of weekly sprays of targeted fungicides with either polyoxin D zinc salt (Oso) or an extract of Reynoutria sachalinensis (Regalia). Powdery mildew (PM; Podosphaera xanthii), gummy stem blight (GSB; Stagonosporopsis spp.), and anthracnose (Colletotrichum orbiculare) were the most prevalent diseases to occur in the 3 years. Replacing chlorothalonil with the biopesticides as the tank-mix component of the fungicide spray program was successful in reducing GSB and PM severity in cantaloupe, honeydew melon, and watermelon compared with the untreated control, with the exception of GSB in 2017 in cantaloupe, and similar to the program including chlorothalonil in all cases, except anthracnose in watermelon. Anthracnose disease severity was not significantly reduced compared with the untreated control when chlorothalonil was replaced with the biopesticides and yields were not improved over the chlorothalonil-alone treatment in any of the trials. Therefore, replacement of chlorothalonil may not fully address its loss as a fungicide resistance management tool but efficacy can be maintained when polyoxin D is alternated with R. sachalinensis as a tank mix with targeted fungicides to manage PM and GSB.

Characterizing differences in the collagen fiber organization of skin wounds using quantitative polarized light imaging.

Collagen fiber organization requires characterization in many biomedical applications, but it is difficult to objectively quantify in standard histology tissue sections. Quantitative polarized light imaging is a low-cost technique that allows for rapid measurement of collagen fiber orientation and thickness. In this study, we utilize a quantitative polarized light imaging system to characterize fiber orientation and thickness from wound sections. Full thickness skin wound sections that were previously stained with hematoxylin and eosin were used to assess collagen fiber content and organization at different points during the wound healing process. Overall, wounds exhibited a measurable increase in collagen fiber thickness and a nonlinear change in fiber reorganization within the wound. Our study demonstrates that quantitative polarized light imaging is an inexpensive alternative or supplement to standard histology protocols, requiring no additional stains or dyes, and yields repeatable quantitative assessments of collagen organization.

Quantification of age-related changes in the structure and mechanical function of skin with multiscale imaging.

The mechanical properties of skin change during aging but the relationships between structure and mechanical function remain poorly understood. Previous work has shown that young skin exhibits a substantial decrease in tissue volume, a large macro-scale Poisson's ratio, and an increase in micro-scale collagen fiber alignment during mechanical stretch. In this study, label-free multiphoton microscopy was used to quantify how the microstructure and fiber kinematics of aged mouse skin affect its mechanical function. In an unloaded state, aged skin was found to have less collagen alignment and more non-enzymatic collagen fiber crosslinks. Skin samples were then loaded in uniaxial tension and aged skin exhibited a lower mechanical stiffness compared to young skin. Aged tissue also demonstrated less volume reduction and a lower macro-scale Poisson's ratio at 10% uniaxial strain, but not at 20% strain. The magnitude of 3D fiber realignment in the direction of loading was not different between age groups, and the amount of realignment in young and aged skin was less than expected based on theoretical fiber kinematics affine to the local deformation. These findings provide key insights on how the collagen fiber microstructure changes with age, and how those changes affect the mechanical function of skin, findings which may help guide wound healing or anti-aging treatments.

A phase 3, multicenter, randomized, double-blind, active-comparator-controlled study to evaluate the safety, tolerability, and immunogenicity of a 4-dose regimen of V114, a 15-valent pneumococcal conjugate vaccine, in healthy infants (PNEU-PED).

BACKGROUND: Pneumococcal disease (PD) remains a major health concern with considerable morbidity and mortality in children. Currently licensed pneumococcal conjugate vaccines (PCVs) confer protection against PD caused by most vaccine serotypes, but non-vaccine serotypes contribute to residual disease. V114 is a 15-valent PCV containing all 13 serotypes in Prevnar 13™ (PCV13) and additional serotypes 22F and 33F. This pivotal phase 3 study compared safety and immunogenicity of V114 and PCV13. METHODS: 1720 healthy infants were randomized 1:1 to receive a 4-dose regimen of V114 or PCV13 concomitantly with other routine pediatric vaccines. Safety was evaluated after each dose as proportion of participants with adverse events (AEs). Serotype-specific anti-pneumococcal immunoglobulin G (IgG) was measured at 1-month post-dose 3 (PD3), pre-dose 4, and 1-month post-dose 4 (PD4). IgG response rates, geometric mean concentrations (GMCs), and opsonophagocytic activity (OPA) were compared between vaccination groups. RESULTS: The proportion, maximum intensity, and duration of injection-site, systemic, and serious AEs were generally comparable between V114 and PCV13 groups. In comparison to PCV13, V114 met non-inferiority criteria for all 15 serotypes based on IgG response rates at PD3. V114 met non-inferiority criteria by IgG GMCs for all serotypes at PD3 and PD4, except for serotype 6A at PD3. V114-induced antibodies had bactericidal activity as assessed by OPA. Further, V114 met superiority criteria for shared serotype 3 and unique serotypes 22F and 33F compared to PCV13 by serotype-specific IgG GMCs at both PD3 and PD4. Immunogenicity of concomitantly administered routine pediatric vaccines was comparable in V114 and PCV13 groups. CONCLUSIONS: In healthy infants, V114 displays acceptable safety and tolerability profiles and generates comparable immune responses to PCV13. V114 also met superiority criteria for serotypes 3, 22F, and 33F. These results support use of V114 for prevention of PD as part of routine infant vaccination schedules. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03893448; EudraCT: 2018-004109-21.

Three-Dimensional Quantification of Collagen Microstructure During Tensile Mechanical Loading of Skin.

Skin is a heterogeneous tissue that can undergo substantial structural and functional changes with age, disease, or following injury. Understanding how these changes impact the mechanical properties of skin requires three-dimensional (3D) quantification of the tissue microstructure and its kinematics. The goal of this study was to quantify these structure-function relationships via second harmonic generation (SHG) microscopy of mouse skin under tensile mechanical loading. Tissue deformation at the macro- and micro-scale was quantified, and a substantial decrease in tissue volume and a large Poisson's ratio was detected with stretch, indicating the skin differs substantially from the hyperelastic material models historically used to explain its behavior. Additionally, the relative amount of measured strain did not significantly change between length scales, suggesting that the collagen fiber network is uniformly distributing applied strains. Analysis of undeformed collagen fiber organization and volume fraction revealed a length scale dependency for both metrics. 3D analysis of SHG volumes also showed that collagen fiber alignment increased in the direction of stretch, but fiber volume fraction did not change. Interestingly, 3D fiber kinematics was found to have a non-affine relationship with tissue deformation, and an affine transformation of the micro-scale fiber network overestimates the amount of fiber realignment. This result, along with the other outcomes, highlights the importance of accurate, scale-matched 3D experimental measurements when developing multi-scale models of skin mechanical function.

Quantifying Age-Related Changes in Skin Wound Metabolism Using In Vivo Multiphoton Microscopy.

Objective: The elderly are at high risk for developing chronic skin wounds, but the effects of intrinsic aging on skin healing are difficult to isolate due to common comorbidities like diabetes. Our objective is to use multiphoton microscopy (MPM) to find endogenous, noninvasive biomarkers to differentiate changes in skin wound healing metabolism between young and aged mice in vivo. Approach: We utilized MPM to monitor skin metabolism at the edge of full-thickness, excisional wounds in 24- and 4-month-old mice of both sexes for 10 days. MPM can assess quantitative biomarkers of cellular metabolism in vivo by utilizing autofluorescence from the cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Results: An optical redox ratio of FAD/(NADH+FAD) autofluorescence and NADH fluorescence lifetime imaging revealed dynamic changes in keratinocyte function during healing. Aged female mice demonstrated an attenuation of keratinocyte proliferation during wound healing detectable optically through a higher redox ratio and longer NADH fluorescence lifetime. By measuring the correlation between NADH lifetime and the optical redox ratio at each day, we also demonstrate sensitivity to the proliferative phase of wound healing. Innovation: Label-free MPM was used to longitudinally monitor individual wounds in vivo, which revealed age-dependent differences in wound metabolism. Conclusion: These results indicate in vivo MPM can provide quantitative biomarkers of age-related delays in healing, which can be used in the future to provide patient-specific wound care.

Skin Structure-Function Relationships and the Wound Healing Response to Intrinsic Aging.

Significance: Chronic wounds, such as diabetic foot ulcers, venous stasis ulcers, and pressure ulcers affect millions of Americans each year, and disproportionately afflict our increasingly older population. Older individuals are predisposed to wound infection, repeated trauma, and the development of chronic wounds. However, a complete understanding of how the attributes of aging skin affect the wound healing process has remained elusive. Recent Advances: A variety of studies have demonstrated that the dermal matrix becomes thinner, increasingly crosslinked, and fragmented with advanced age. These structural changes, as well as an increase in cell senescence, result in altered collagen fiber remodeling and increased stiffness. Studies combining mechanical testing with advanced imaging techniques are providing new insights into the relationships between these age-related changes. Emerging research into the mechanobiology of aging and the wound healing process indicate that the altered mechanical environment of aged skin may have a significant effect on age-related delays in healing. Critical Issues: The interpretation and synthesis of clinical studies is confounded by the effects of common comorbidities that also contribute to the development of chronic wounds. A lack of quantitative biomarkers of wound healing and age-related changes makes understanding structure-function relationships during the wound healing process challenging. Future Directions: Additional work is needed to establish quantitative and mechanistic relationships among age-related changes in the skin microstructure, mechanical function, and the cellular responses to wound healing.

In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing.

Chronic wounds are difficult to diagnose and characterize due to a lack of quantitative biomarkers. Label-free multiphoton microscopy has emerged as a useful imaging modality capable of quantifying changes in cellular metabolism using an optical redox ratio of FAD/(NADH+FAD) autofluorescence. However, the utility of an optical redox ratio for long-term in vivo monitoring of tissue metabolism has not been robustly evaluated. In this study, we demonstrate how multiphoton microscopy can be used to monitor changes in the metabolism of individual full-thickness skin wounds in vivo. 3D optical redox ratio maps and NADH fluorescence lifetime images identify differences between diabetic and control mice during the re-epithelialization of wounds. These metabolic changes are associated with a transient increase in keratinocyte proliferation at the wound edge. Our study demonstrates that high-resolution, non-invasive autofluorescence imaging can be performed in vivo and that optical redox ratios can serve as quantitative optical biomarkers of impaired wound healing.

Rejuvenation of aged rat skin with pulsed electric fields.

The demand for skin rejuvenation procedures has progressively increased in the past decade. Additionally, clinical trials have shown that current therapies might cause downtime and side effects in patients including prolonged erythema, scarring, and dyspigmentation. The goal of this study was to explore the effect of partial irreversible electroporation (pIRE) with pulsed electric fields in aged skin rejuvenation as a novel, non-invasive skin resurfacing technique. In this study, we used an experimental model of aged rats. We showed that treatment with pIRE promoted keratinocyte proliferation and blood flow in aged rat skin. We also found significant evidence indicating that pIRE reformed the dermal extracellular matrix (ECM). Both the collagen protein and fibre density in aged skin increased after pIRE administration. Furthermore, using an image-processing algorithm, we found that the collagen fibre orientation in the histological sections did not change, indicating a lack of scar formation in the treated areas. The results showed that pIRE approach could effectively stimulate keratinocyte proliferation, ECM synthesis, and angiogenesis in an aged rat model.

Cell derived extracellular matrix fibers synthesized using sacrificial hollow fiber membranes.

The therapeutic potential of biological scaffolds as adjuncts to synthetic polymers motivates the engineering of fibers formed using the extracellular matrix (ECM) secreted by cells. To capture the ECM secreted by cells during in vitro culture, a solvent degradable hollow fiber membrane (HFM) was created and utilized as a cell culture platform. NIH/3T3 fibroblasts were injected into the narrow (0.986 ± 0.042 mm) lumina of mesoporous polysulfone HFMs and maintained in culture for up to 3 weeks. Following cell culture, HFMs were dissolved using N-methyl-2-pyrrolidone and the accumulated ECM was collected. The ECM retained the filamentous dimensions of the HFM lumen. The process yielded up to 0.89 ± 0.20 mg of ECM for every mm of HFM dissolved. Immunofluorescence, second-harmonic generation microscopy, and tandem mass spectrometry indicated the presence of an array of ECM constituents, including collagen, fibronectin, and proteoglycans, while FTIR spectra suggested thorough HFM material dissolution. Isolated ECM fibers, although fragile, were amenable to handling and exhibited an average elastic modulus of 34.6 ± 15.3 kPa, ultimate tensile strength of 5.2 ± 2.2 kPa, and elongation-at-break of 29% ± 18%. ECM fibers consisted of an interconnected yet porous (32.7% ± 5.8% open space) network which supported the attachment and in vitro proliferation of mammalian cells. ECM fibers were similarly synthesized using muscle and astrocyte cells, suggesting process robustness across different cell types. Ultimately, these ECM fibers could be utilized as an alternative to synthetics for the manufacture of woven meshes targeting wound healing or regenerative medicine applications.

Codelivery of Infusion Decellularized Skeletal Muscle with Minced Muscle Autografts Improved Recovery from Volumetric Muscle Loss Injury in a Rat Model.

Skeletal muscle is capable of robust self-repair following mild trauma, yet in cases of traumatic volumetric muscle loss (VML), where more than 20% of a muscle's mass is lost, this capacity is overwhelmed. Current autogenic whole muscle transfer techniques are imperfect, which has motivated the exploration of implantable scaffolding strategies. In this study, the use of an allogeneic decellularized skeletal muscle (DSM) scaffold with and without the addition of minced muscle (MM) autograft tissue was explored as a repair strategy using a lower-limb VML injury model (n = 8/sample group). We found that the repair of VML injuries using DSM + MM scaffolds significantly increased recovery of peak contractile force (81 ± 3% of normal contralateral muscle) compared to unrepaired VML controls (62 ± 4%). Similar significant improvements were measured for restoration of muscle mass (88 ± 3%) in response to DSM + MM repair compared to unrepaired VML controls (79 ± 3%). Histological findings revealed a marked decrease in collagen dense repair tissue formation both at and away from the implant site for DSM + MM repaired muscles. The addition of MM to DSM significantly increased MyoD expression, compared to isolated DSM treatment (21-fold increase) and unrepaired VML (37-fold) controls. These findings support the further exploration of both DSM and MM as promising strategies for the repair of VML injury.