Quantifying innervation facilitated by deep learning in wound healing.

The peripheral nerves (PNs) innervate the dermis and epidermis, and are suggested to play an important role in wound healing. Several methods to quantify skin innervation during wound healing have been reported. Those usually require multiple observers, are complex and labor-intensive, and the noise/background associated with the immunohistochemistry (IHC) images could cause quantification errors/user bias. In this study, we employed the state-of-the-art deep neural network, Denoising Convolutional Neural Network (DnCNN), to perform pre-processing and effectively reduce the noise in the IHC images. Additionally, we utilized an automated image analysis tool, assisted by Matlab, to accurately determine the extent of skin innervation during various stages of wound healing. The 8 mm wound is generated using a circular biopsy punch in the wild-type mouse. Skin samples were collected on days 3, 7, 10 and 15, and sections from paraffin-embedded tissues were stained against pan-neuronal marker- protein-gene-product 9.5 (PGP 9.5) antibody. On day 3 and day 7, negligible nerve fibers were present throughout the wound with few only on the lateral boundaries of the wound. On day 10, a slight increase in nerve fiber density appeared, which significantly increased on day 15. Importantly, we found a positive correlation (R2 = 0.926) between nerve fiber density and re-epithelization, suggesting an association between re-innervation and re-epithelization. These results established a quantitative time course of re-innervation in wound healing, and the automated image analysis method offers a novel and useful tool to facilitate the quantification of innervation in the skin and other tissues.

A system for bioelectronic delivery of treatment directed toward wound healing.

The development of wearable bioelectronic systems is a promising approach for optimal delivery of therapeutic treatments. These systems can provide continuous delivery of ions, charged biomolecules, and an electric field for various medical applications. However, rapid prototyping of wearable bioelectronic systems for controlled delivery of specific treatments with a scalable fabrication process is challenging. We present a wearable bioelectronic system comprised of a polydimethylsiloxane (PDMS) device cast in customizable 3D printed molds and a printed circuit board (PCB), which employs commercially available engineering components and tools throughout design and fabrication. The system, featuring solution-filled reservoirs, embedded electrodes, and hydrogel-filled capillary tubing, is assembled modularly. The PDMS and PCB both contain matching through-holes designed to hold metallic contact posts coated with silver epoxy, allowing for mechanical and electrical integration. This assembly scheme allows us to interchange subsystem components, such as various PCB designs and reservoir solutions. We present three PCB designs: a wired version and two battery-powered versions with and without onboard memory. The wired design uses an external voltage controller for device actuation. The battery-powered PCB design uses a microcontroller unit to enable pre-programmed applied voltages and deep sleep mode to prolong battery run time. Finally, the battery-powered PCB with onboard memory is developed to record delivered currents, which enables us to verify treatment dose delivered. To demonstrate the functionality of the platform, the devices are used to deliver H[Formula: see text] in vivo using mouse models and fluoxetine ex vivo using a simulated wound environment. Immunohistochemistry staining shows an improvement of 35.86% in the M1/M2 ratio of H[Formula: see text]-treated wounds compared with control wounds, indicating the potential of the platform to improve wound healing.

Quantifying innervation facilitated by deep learning in wound healing.

The peripheral nerves (PNs) innervate the dermis and epidermis, which have been suggested to play an important role in wound healing. Several methods to quantify skin innervation during wound healing have been reported. Those usually require multiple observers, are complex and labor-intensive, and noise/background associated with the Immunohistochemistry (IHC) images could cause quantification errors/user bias. In this study, we employed the state-of-the-art deep neural network, DnCNN, to perform pre-processing and effectively reduce the noise in the IHC images. Additionally, we utilized an automated image analysis tool, assisted by Matlab, to accurately determine the extent of skin innervation during various stages of wound healing. The 8mm wound is generated using a circular biopsy punch in the wild-type mouse. Skin samples were collected on days 3,7,10 and 15, and sections from paraffin-embedded tissues were stained against pan-neuronal marker- protein-gene-product 9.5 (PGP 9.5) antibody. On day 3 and day 7, negligible nerve fibers were present throughout the wound with few only on the lateral boundaries of the wound. On day 10, a slight increase in nerve fiber density appeared, which significantly increased on day 15. Importantly we found a positive correlation (R- 2 = 0.933) between nerve fiber density and re-epithelization, suggesting an association between re-innervation and re-epithelization. These results established a quantitative time course of re-innervation in wound healing, and the automated image analysis method offers a novel and useful tool to facilitate the quantification of innervation in the skin and other tissues.

Consistent application of compression: An under-considered variable in the prevention of venous leg ulcers.

Venous leg ulcers, the most common leg ulcer, occur in patients with chronic venous insufficiency due to venous hypertension. Evidence supports the conservative treatment with lower extremity compression, ideally between 30-40 mm Hg. Pressures in this range provide enough force to partially collapse lower extremity veins without restricting arterial flow in patients without peripheral arterial disease. There are many options for applying such compression, and those who apply these devices have varying levels of training and backgrounds. In this quality improvement project, a single observer utilised a reusable pressure monitor to compare pressures applied using different devices by individuals in wound clinics with diverse training from specialties of dermatology, podiatry, and general surgery. Average compression was higher in the dermatology wound clinic (n = 153) compared to the general surgery clinic (n = 53) (35.7 ± 13.3 and 27.2 ± 8.0 mm Hg, respectively, p < 0.0001), and wraps applied by clinic staff (n = 194) were nearly twice as likely as a self-applied wrap (n = 71) to have pressures greater than 40 mm Hg (relative risk: 2.2, 95% confidence interval: 1.136-4.423, p = 0.02). Pressures were also dependent upon the specific compression device used, with CircAid®s (35.5 mm Hg, SD: 12.0 mm Hg, n = 159) providing higher average pressures than Sigvaris Compreflex (29.5 mm Hg, SD: 7.7 mm Hg, n = 53, p = 0.009) and Sigvaris Coolflex (25.2 mm Hg, SD: 8.0 mm Hg, n = 32, p < 0.0001). These results indicate that the device-provided pressure may be dependent on both the compression device and the background and training of the applicator. We propose that standardisation in the training of compression application and increased use of a point-of-care pressure monitor may improve the consistency of applied compression, thus improving adherence to treatment and outcomes in patients with chronic venous insufficiency.

Drawn-on-Skin Sensors from Fully Biocompatible Inks toward High-Quality Electrophysiology.

The need to develop wearable devices for personal health monitoring, diagnostics, and therapy has inspired the production of innovative on-demand, customizable technologies. Several of these technologies enable printing of raw electronic materials directly onto biological organs and tissues. However, few of them have been thoroughly investigated for biocompatibility of the raw materials on the cellular, tissue, and organ levels or with different cell types. In addition, highly accurate multiday in vivo monitoring using such on-demand, in situ fabricated devices has yet to be done. Presented herein is the first fully biocompatible, on-skin fabricated electronics for multiple cell types and tissues that can capture electrophysiological signals with high fidelity. While also demonstrating improved mechanical and electrical properties, the drawn-on-skin ink retains its properties under various writing conditions, which minimizes the variation in electrical performance. Furthermore, the drawn-on-skin ink shows excellent biocompatibility with cardiomyocytes, neurons, mice skin tissue, and human skin. The high signal-to-noise ratios of the electrophysiological signals recorded with the DoS sensor over multiple days demonstrate its potential for personalized, long-term, and accurate electrophysiological health monitoring.

Chronic wounds.

Chronic wounds are characterized by their inability to heal within an expected time frame and have emerged as an increasingly important clinical problem over the past several decades, owing to their increasing incidence and greater recognition of associated morbidity and socio-economic burden. Even up to a few years ago, the management of chronic wounds relied on standards of care that were outdated. However, the approach to these chronic conditions has improved, with better prevention, diagnosis and treatment. Such improvements are due to major advances in understanding of cellular and molecular aspects of basic science, in innovative and technological breakthroughs in treatment modalities from biomedical engineering, and in our ability to conduct well-controlled and reliable clinical research. The evidence-based approaches resulting from these advances have become the new standard of care. At the same time, these improvements are tempered by the recognition that persistent gaps exist in scientific knowledge of impaired healing and the ability of clinicians to reduce morbidity, loss of limb and mortality. Therefore, taking stock of what is known and what is needed to improve understanding of chronic wounds and their associated failure to heal is crucial to ensuring better treatments and outcomes.

A machine learning based model accurately predicts cellular response to electric fields in multiple cell types.

Many cell types migrate in response to naturally generated electric fields. Furthermore, it has been suggested that the external application of an electric field may be used to intervene in and optimize natural processes such as wound healing. Precise cell guidance suitable for such optimization may rely on predictive models of cell migration, which do not generalize. Here, we present a machine learning model that can forecast directedness of cell migration given a timeseries of previous directedness and electric field values. This model is trained using time series galvanotaxis data of mammalian cranial neural crest cells obtained through time-lapse microscopy of cells cultured at 37 °C in a galvanotaxis chamber at ambient pressure. Next, we show that our modeling approach can be used for a variety of cell types and experimental conditions with very limited training data using transfer learning methods. We adapt the model to predict cell behavior for keratocytes (room temperature, ~ 18-20 °C) and keratinocytes (37 °C) under similar experimental conditions with a small dataset (~ 2-5 cells). Finally, this model can be used to perform in silico studies by simulating cell migration lines under time-varying and unseen electric fields. We demonstrate this by simulating feedback control on cell migration using a proportional-integral-derivative (PID) controller. This data-driven approach provides predictive models of cell migration that may be suitable for designing electric field based cellular control mechanisms for applications in precision medicine such as wound healing.

Automatic wound detection and size estimation using deep learning algorithms.

Evaluating and tracking wound size is a fundamental metric for the wound assessment process. Good location and size estimates can enable proper diagnosis and effective treatment. Traditionally, laboratory wound healing studies include a collection of images at uniform time intervals exhibiting the wounded area and the healing process in the test animal, often a mouse. These images are then manually observed to determine key metrics -such as wound size progress- relevant to the study. However, this task is a time-consuming and laborious process. In addition, defining the wound edge could be subjective and can vary from one individual to another even among experts. Furthermore, as our understanding of the healing process grows, so does our need to efficiently and accurately track these key factors for high throughput (e.g., over large-scale and long-term experiments). Thus, in this study, we develop a deep learning-based image analysis pipeline that aims to intake non-uniform wound images and extract relevant information such as the location of interest, wound only image crops, and wound periphery size over-time metrics. In particular, our work focuses on images of wounded laboratory mice that are used widely for translationally relevant wound studies and leverages a commonly used ring-shaped splint present in most images to predict wound size. We apply the method to a dataset that was never meant to be quantified and, thus, presents many visual challenges. Additionally, the data set was not meant for training deep learning models and so is relatively small in size with only 256 images. We compare results to that of expert measurements and demonstrate preservation of information relevant to predicting wound closure despite variability from machine-to-expert and even expert-to-expert. The proposed system resulted in high fidelity results on unseen data with minimal human intervention. Furthermore, the pipeline estimates acceptable wound sizes when less than 50% of the images are missing reference objects.

Simultaneous determination of tryptophan, 5-hydroxytryptophan, tryptamine, serotonin, and 5-HIAA in small volumes of mouse serum using UHPLC-ED.

In this paper we report a simple and efficient method for the concurrent analysis of tryptophan, 5-HTP, tryptamine, serotonin, and 5-HIAA in mouse serum using UHPLC-ED after protein precipitation and dilution. These compounds are neuroactive and are of interest in studies of mood and behavior; They are also biomarkers for the presence of neuroendocrine tumors and are used in the diagnosis of these cancers. After a brief series of validation experiments, this method was applied to serum from mouse behaviour experiments.•A convenient UHPLC method with electrochemical detection for concomitant analysis of the serotonin pathway in serum, including, for the first time, tryptamine.•The method met all performance criteria established for use in our lab and was applied in rodent experiments.

Wearable electronics for skin wound monitoring and healing.

Wound healing is one of the most complex processes in the human body, supported by many cellular events that are tightly coordinated to repair the wound efficiently. Chronic wounds have potentially life-threatening consequences. Traditional wound dressings come in direct contact with wounds to help them heal and avoid further complications. However, traditional wound dressings have some limitations. These dressings do not provide real-time information on wound conditions, leading clinicians to miss the best time for adjusting treatment. Moreover, the current diagnosis of wounds is relatively subjective. Wearable electronics have become a unique platform to potentially monitor wound conditions in a continuous manner accurately and even to serve as accelerated healing vehicles. In this review, we briefly discuss the wound status with some objective parameters/biomarkers influencing wound healing, followed by the presentation of various novel wearable devices used for monitoring wounds and accelerating wound healing. We further summarize the associated device working principles. This review concludes by highlighting some major challenges in wearable devices toward wound healing that need to be addressed by the research community.

Transcriptome analysis of human dermal fibroblasts following red light phototherapy.

Fibrosis occurs when collagen deposition and fibroblast proliferation replace healthy tissue. Red light (RL) may improve skin fibrosis via photobiomodulation, the process by which photosensitive chromophores in cells absorb visible or near-infrared light and undergo photophysical reactions. Our previous research demonstrated that high fluence RL reduces fibroblast proliferation, collagen deposition, and migration. Despite the identification of several cellular mechanisms underpinning RL phototherapy, little is known about the transcriptional changes that lead to anti-fibrotic cellular responses. Herein, RNA sequencing was performed on human dermal fibroblasts treated with RL phototherapy. Pathway enrichment and transcription factor analysis revealed regulation of extracellular matrices, proliferation, and cellular responses to oxygen-containing compounds following RL phototherapy. Specifically, RL phototherapy increased the expression of MMP1, which codes for matrix metalloproteinase-1 (MMP-1) and is responsible for remodeling extracellular collagen. Differential regulation of MMP1 was confirmed with RT-qPCR and ELISA. Additionally, RL upregulated PRSS35, which has not been previously associated with skin activity, but has known anti-fibrotic functions. Our results suggest that RL may benefit patients by altering fibrotic gene expression.

Sling Training with Positive Reinforcement to Facilitate Porcine Wound Studies.

Domestic swine have become important large animal models for dermatologic and wound studies owing to the similarity of their skin architecture to that of human skin. To improve on current porcine wound protocols and accomplish postoperational daily wound care or treatment in a welfare-centered, low-stress setting, we developed a unique sling-training program using a commercially available Panepinto-like sling in combination with positive reinforcement of desired behaviors. Training using these methods is initiated during the acclimation period of 7-10 days before the initial surgical manipulation and continued throughout project-specific treatments for the duration of the study. Using this protocol, daily treatments can be administered without additional anesthesia while the animals rest in the sling with the administration of simultaneous nutritional enrichment. This low-stress handling program successfully facilitates the postoperational treatments and wound care without the use of potentially confounding anesthesia or sedation. It has a wide range of potential applications in translational medicine and in data acquisition from a resting state where baseline readouts of unstressed animals can be achieved.

Use of Topical Timolol Maleate as Re-Epithelialization Agent for Treatment of Recalcitrant Wounds of Varying Etiologies.

Background: Chronic wounds remain a challenge for the clinician and healthcare system. It is therefore vital for additional therapies that target steps involved in wound recalcitrance. Recently, topical timolol has shown promising results for use in wound healing. Objective: The goal of this study was to assess timolol's effectiveness in healing wounds of varying etiologies. Methods: This multi-center series took place from 2016¬­2019 at the wound healing centers at the University of Miami Health System and the Veterans Affairs Northern California Healthcare. We identified all wound patients who received treatment with topical timolol maleate 0.5% for at least 4 weeks after failing previous treatments. Timolol drops at a dose of 1 drop per cm2 of wound area were instilled with dressing changes twice a day, once a day, every other day, or continuous application. Once they began the study, they stopped all concurrent therapies aside from standard of care. Healing outcomes were classified into 3 categories: healed, defined as complete re-epithelialization of the wound and closure, improved, defined as decreasing wound size area (WSA), and worsening, defined as increasing WSA. Results: We identified 39 patients, 32 males and 7 females that had a total of 55 chronic wounds of varying etiologies. Thirty-four of the wounds had completely healed, 15 wounds improved in WSA, 4 wounds were unchanged in WSA, and 2 wounds worsened in WSA. Conclusions: In line with our previous experience, we found topical timolol to be a safe, cost-effective, and efficacious treatment for recalcitrant wounds of varying etiologies.

Microbiome-skin-brain axis: A novel paradigm for cutaneous wounds.

Chronic wounds cause a significant burden on society financially, medically, and psychologically. Unfortunately, patients with nonhealing wounds often suffer from comorbidities that further compound their disability. Given the high rate of depressive symptoms experienced by patients with chronic wounds, further studies are needed to investigate the potentially linked pathophysiological changes in wounds and depression in order to improve patient care. The English literature on wound healing, inflammatory and microbial changes in chronic wounds and depression, and antiinflammatory and probiotic therapy was reviewed on PubMed. Chronic wound conditions and depression were demonstrated to share common pathologic features of dysregulated inflammation and altered microbiome, indicating a possible relationship. Furthermore, alternative treatment strategies such as immune-targeted and probiotic therapy showed promising potential by addressing both pathophysiological pathways. However, many existing studies are limited to a small study population, a cross-sectional design that does not establish temporality, or a wide range of confounding variables in the context of a highly complex and multifactorial disease process. Therefore, additional preclinical studies in suitable wound models, as well as larger clinical cohort studies and trials are necessary to elucidate the relationship between wound microbiome, healing, and depression, and ultimately guide the most effective therapeutic and management plan for chronic wound patients.

Absorption and Safety of Topically Applied Timolol for Treatment of Chronic Cutaneous Wounds.

Objective: There are no safety or absorption studies to guide topical timolol therapy for treatment of chronic wounds. This study was undertaken to address this gap. Approach: A prospective, observational, cross-sectional comparative study of timolol plasma levels in patients after topical administration to a chronic wound, compared with levels in patients after timolol ocular administration for the indication of glaucoma. Results: There was no statistically significant difference in the average plasma level of timolol in wound as compared with glaucoma patients. No bradycardia or wheezing was observed after administration. Innovation: We determined the single time point concentration of timolol in plasma 1 h after application of timolol 0.5% gel-forming solution to debrided chronic wounds, providing insight as to the safety of this emerging off-label treatment. Conclusion: The topical application of timolol for chronic wounds shares the same safety profile as the widely used application of ocular administration for glaucoma.

Prolotherapy: Potential for the Treatment of Chronic Wounds?

Significance: Chronic skin ulcers, including venous, diabetic, and pressure ulcers, constitute a major health care burden, affecting 2-6 million people in the United States alone, with projected increases in incidence owing to the aging population and rising epidemic of diabetes. The ulcers are often accompanied by pain. Standard of care fails to heal ∼50% of diabetic foot ulcers and 25% of venous leg ulcers. Even advanced therapies do not heal >60%. Thus there is an unmet need for novel therapies that promote healing and also address the concomitant pain issue. Recent Advances: Prolotherapy involves injection of small amounts of an irritant material to the site of degenerated or painful joints, ligaments, and tendons. Multiple irritants are reported to be efficacious, but the focus here is on dextrose prolotherapy. In vitro and in vivo studies support translation to clinical use. Concentrations as low as 5% dextrose have resulted in production of growth factors that have critical roles in repair. Numerous clinical trials report pro-reparative effects of dextrose prolotherapy in joint diseases, tendon, and ligament damage, and for painful musculoskeletal issues. However, most of the studies have limitations that result in low-quality evidence. Critical Issues: The preclinical data support a role for dextrose prolotherapy in promoting tissue repair that is required for healing chronic wounds and ameliorating the associated pain. Critical issues include provision of evidence of efficacy in human chronic wounds. Another potential obstacle is limitation of reimbursement by third-party payers for a therapy with as yet limited evidence. Future Directions: Preclinical studies in models of chronic wounds would support clinical translation. As dextrose prolotherapy has some mechanistic similarities to already approved honey therapies, it may have a shortened pathway for clinical translation. The gold standard for widespread adoption would be a well-designed clinical trial.

Topical Fluoxetine as a Novel Therapeutic That Improves Wound Healing in Diabetic Mice.

Diabetic foot ulcers represent a significant source of morbidity in the U.S., with rapidly escalating costs to the health care system. Multiple pathophysiological disturbances converge to result in delayed epithelialization and persistent inflammation. Serotonin (5-hydroxytryptamine [5-HT]) and the selective serotonin reuptake inhibitor fluoxetine (FLX) have both been shown to have immunomodulatory effects. Here we extend their utility as a therapeutic alternative for nonhealing diabetic wounds by demonstrating their ability to interact with multiple pathways involved in wound healing. We show that topically applied FLX improves cutaneous wound healing in vivo. Mechanistically, we demonstrate that FLX not only increases keratinocyte migration but also shifts the local immune milieu toward a less inflammatory phenotype in vivo without altering behavior. By targeting the serotonin pathway in wound healing, we demonstrate the potential of repurposing FLX as a safe topical for the challenging clinical problem of diabetic wounds.

Development of a novel ion-pairing UPLC method with cation-exchange solid-phase extraction for determination of free timolol in human plasma.

A novel UPLC-UV method was developed for analysis of timolol in human plasma using a simple, fast, and cost effective ion-exchange SPE procedure, followed by separation on a C18 UPLC column with a mobile phase consisting of acetonitrile, phosphate buffer, and sodium 1-octane sulfonate as an ion pairing agent. The method was fully validated according to US-FDA guidelines, and was found to be sufficiently accurate and precise for analysis of timolol in human plasma for clinical pharmacokinetic studies. The application of ion-exchange SPE cartridges for purification of timolol in plasma produced excellent percent recoveries and good sample clean-up, while the ion-pairing separation described here allowed quantitation of timolol without interference from endogenous sample components. The method lower limit of detection was 1.7 ng/mL and the lower limit of quantitation was 5.0 ng/mL, allowing for analysis of therapeutic concentrations of timolol in plasma.

A Concise Review of the Conflicting Roles of Dopamine-1 versus Dopamine-2 Receptors in Wound Healing.

Catecholamines play an important regulatory role in cutaneous wound healing. The exact role of dopamine in human epidermis has yet to be fully elucidated. Current published evidence describes its differential effects on two separate families of G protein coupled receptors: D1-like and D2-like dopamine receptors. Dopamine may enhance angiogenesis and wound healing through its action on dopamine D1 receptors, while impairing wound healing when activating D2 receptors. This review summarizes the evidence for the role of dopamine in wound healing and describes potential mechanisms behind its action on D1 versus D2-like receptors in the skin.