A dose-ranging, parallel group, split-face, single-blind phase II study of light emitting diode-red light (LED-RL) for skin scarring prevention: study protocol for a randomized controlled trial.

BackgroundSkin fibrosis is a significant global health problem that affects over 100 million people annually and has a profoundly negative impact on quality of life. Characterized by excessive fibroblast proliferation and collagen deposition, skin fibrosis underlies a wide spectrum of dermatologic conditions ranging from pathologic scars secondary to injury (e.g., burns, surgery, trauma) to immune-mediated diseases. Effective anti-scarring therapeutics remain an unmet need, underscoring the importance of developing novel approaches to treat and prevent skin fibrosis. Our in vitro data show that light emitting diode-red light (LED-RL) can modulate key cellular and molecular processes involved in skin fibrosis. In two phase I clinical trials (STARS 1 and STARS 2), we demonstrated the safety and tolerability of LED-RL at fluences of 160 J/cm2 up to 480 J/cm2 on normal human skin.Methods/designCURES (Cutaneous Understanding of Red-light Efficacy on Scarring) is a dose-ranging, randomized, parallel group, split-face, single-blind, mock-controlled phase II study to evaluate the efficacy of LED-RL to limit post-surgical skin fibrosis in subjects undergoing elective mini-facelift surgery. Thirty subjects will be randomly allocated to three treatment groups to receive LED-RL phototherapy or temperature-matched mock irradiation (control) to either periauricular incision site at fluences of 160 J/cm2, 320 J/cm2, or 480 J/cm2. Starting one week post-surgery (postoperative days 4-8), treatments will be administered three times weekly for three consecutive weeks, followed by efficacy assessments at 30 days, 3 months, and 6 months. The primary endpoint is the difference in scar pliability between LED-RL-treated and control sites as determined by skin elasticity and induration measurements. Secondary outcomes include clinical and photographic evaluations of scars, 3D skin imaging analysis, histological and molecular analyses, and adverse events.DiscussionLED-RL is a therapeutic modality of increasing importance in dermatology, and has the potential to limit skin fibrosis clinically by decreasing dermal fibroblast activity and collagen production. The administration of LED-RL phototherapy in the early postoperative period may optimize wound healing and prevent excessive scarring. The results from this study may change the current treatment paradigm for fibrotic skin diseases and help to pioneer LED-RL as a safe, non-invasive, cost-effective, portable, at-home therapy for scars.Trial registrationClinicalTrials.gov, NCT03795116 . Registered on 20 December 2018

NONINVASIVE MULTIMODAL DIFFUSE OPTICAL IMAGING OF VULNERABLE TISSUE HEMODYNAMICS

Measurement of tissue hemodynamics provides vital information for the assessment of tissue viability. This thesis reports three noninvasive near-infrared diffuse optical systems for spectroscopic measurements and tomographic imaging of tissue hemodynamics in vulnerable tissues with the goal of disease diagnosis and treatment monitoring. A hybrid near-infrared spectroscopy/diffuse correlation spectroscopy (NIRS/DCS) instrument with a contact fiber-optic probe was developed and utilized for simultaneous and continuous monitoring of blood flow (BF), blood oxygenation, and oxidative metabolism in exercising gastrocnemius. Results measured by the hybrid NIRS/DCS instrument in 37 subjects (mean age: 67 ± 6) indicated that vitamin D supplement plus aerobic training improved muscle metabolic function in older population. To reduce the interference and potential infection risk on vulnerable tissues caused by the contact measurement, a noncontact diffuse correlation spectroscopy/tomography (ncDCS/ncDCT) system was then developed. The ncDCS/ncDCT system employed optical lenses to project limited numbers of sources and detectors on the tissue surface. A motor-driven noncontact probe scanned over a region of interest to collect boundary data for three dimensional (3D) tomographic imaging of blood flow distribution. The ncDCS was tested for BF measurements in mastectomy skin flaps. Nineteen (19) patients underwent mastectomy and implant-based breast reconstruction were measured before and immediately after mastectomy. The BF index after mastectomy in each patient was normalized to its baseline value before surgery to get relative BF (rBF). Since rBF values in the patients with necrosis (n = 4) were significantly lower than those without necrosis (n = 15), rBF levels can be used to predict mastectomy skin flap necrosis. The ncDCT was tested for 3D imaging of BF distributions in chronic wounds of 5 patients. Spatial variations in BF contrasts over the wounded tissues were observed, indicating the capability of ncDCT in detecting tissue hemodynamic heterogeneities. To improve temporal/spatial resolution and avoid motion artifacts due to a long mechanical scanning of ncDCT, an electron-multiplying charge-coupled device based noncontact speckle contrast diffuse correlation tomography (scDCT) was developed. Validation of scDCT was done by imaging both high and low BF contrasts in tissue-like phantoms and human forearms. In a wound imaging study using scDCT, significant lower BF values were observed in the burned areas/volumes compared to surrounding normal tissues in two patients with burn. One limitation in this study was the potential influence of other unknown tissue optical properties such as tissue absorption coefficient (µa) on BF measurements. A new algorithm was then developed to extract both µa and BF using light intensities and speckle contrasts measured by scDCT at multiple source-detector distances. The new algorithm was validated using tissue-like liquid phantoms with varied values of µa and BF index. In-vivo validation and application of the innovative scDCT technique with the new algorithm is the subject of future work

Monitoring Wound Healing with Contactless Measurements and Augmented Reality

Objective: This work presents a device for non-invasive wound parameters assessment, designed to overcome the drawbacks of traditional methods, which are mostly rough, inaccurate, and painful for the patient. The device estimates the morphological parameters of the wound and provides augmented reality (AR) visual feedback on the wound healing status by projecting the wound border acquired during the last examination, thus improving doctor-patient communication. Methods: An accurate 3D model of the wound is created by stereophotogrammetry and refined through self-organizing maps. The 3D model is used to estimate physical parameters for wound healing assessment and integrates AR functionalities based on a miniaturized projector. The physical parameter estimation functionalities are evaluated in terms of precision, accuracy, inter-operator variability, and repeatability, whereas AR wound border projection is evaluated in terms of accuracy on the same phantom. Results: The accuracy and precision of the device are respectively 2% and 1.2% for linear parameters, and 1.7% and 1.3% for area and volume. The AR projection shows an error distance <1 mm. No statistical difference was found between the measurements of different operators. Conclusion: The device has proven to be an objective and non-operator-dependent tool for assessing the morphological parameters of the wound. Comparison with non-contact devices shows improved accuracy, offering reliable and rigorous measurements. Clinical Impact: Chronic wounds represent a significant health problem with high recurrence rates due to the ageing of the population and diseases such as diabetes and obesity. The device presented in this work provides an easy-to-use non-invasive tool to obtain useful information for treatment

Monitoring wound healing of elastic cartilage using multiphoton microscopy

SummaryObjectiveTo demonstrate the ability of multiphoton microscopy (MPM) for monitoring wound healing of elastic cartilage.MethodIn a rabbit ear model, four cartilage specimen groups at 1-day, 1-, 4-, 20-week healing time points as well as a normal elastic cartilage were examined with MPM without using labeling agents. MPM images at wound margins were obtained from specimens at different healing stages, compared with the Hematoxylin and Eosin (H&E) stained images. Image analysis was performed to characterize the collagen morphology for quantifying the wound healing progression of elastic cartilage.ResultsMPM provided high-resolution images of elastic cartilage at varying depths. Comparisons of the images of specimens at different healing stages show obvious cell growth and matrix deposition. The results are consistent with the histological results. Moreover, quantitative analysis results show significant alteration in the collagen cavity size or collagen orientation index during wound healing of elastic cartilage, indicating the possibility to act as indicators for monitoring wound healing.ConclusionOur results suggested that MPM has the ability to monitor the wound healing progression of elastic cartilage, based on the visualization of cell growth and proliferation and quantitative characterization of collagen morphology during wound healing

Imparting 3D representations to artificial intelligence for a full assessment of pressure injuries.

During recent decades, researches have shown great interest to machine learning techniques in order to extract meaningful information from the large amount of data being collected each day. Especially in the medical field, images play a significant role in the detection of several health issues. Hence, medical image analysis remarkably participates in the diagnosis process and it is considered a suitable environment to interact with the technology of intelligent systems. Deep Learning (DL) has recently captured the interest of researchers as it has proven to be efficient in detecting underlying features in the data and outperformed the classical machine learning methods. The main objective of this dissertation is to prove the efficiency of Deep Learning techniques in tackling one of the important health issues we are facing in our society, through medical imaging. Pressure injuries are a dermatology related health issue associated with increased morbidity and health care costs. Managing pressure injuries appropriately is increasingly important for all the professionals in wound care. Using 2D photographs and 3D meshes of these wounds, collected from collaborating hospitals, our mission is to create intelligent systems for a full non-intrusive assessment of these wounds. Five main tasks have been achieved in this study: a literature review of wound imaging methods using machine learning techniques, the classification and segmentation of the tissue types inside the pressure injury, the segmentation of these wounds and the design of an end-to-end system which measures all the necessary quantitative information from 3D meshes for an efficient assessment of PIs, and the integration of the assessment imaging techniques in a web-based application

Quantitative Optical Imaging of Metabolic and Structural Biomarkers in Rodent Injury Models

The assessment of organ metabolic function using optical imaging techniques is an overgrowing field of disease diagnosis. The broad research objective of my PhD thesis is to detect quantitative biomarkers by developing and applying optical imaging and image processing tools to animal models of human diseases. To achieve this goal, I have designed and implemented an optical imaging instrument called in vivo fluorescence imager to study wound healing progress. I have also developed a 3-dimensional (3D) vascular segmentation technique that uses intrinsic fluorescence images of whole organs. Intrinsic fluorophores (autofluorescence signals) provide information about the status of cellular bioenergetics in different tissue types. Reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) are two key Krebs cycle coenzymes in mitochondria, which are autofluorescent. The ratio of these two fluorophores (NADH/FAD) is used as an optical biomarker for mitochondrial redox state of the tissues. The custom-designed optical tools have enabled me to probe the metabolic state of diseases as well as structural information of the organs at different regimes (in vivo, at cryogenic temperature, and in vitro). Here are the main projects that I have conducted and significantly contributed to: 1) Fluorescent metabolic imaging. I have designed and implemented an in vivo fluorescence imaging device to study diabetic wounds in small animals. This device can monitor the dynamics of the metabolism of the skin by capturing the images of the surface fluorescence of NADH and FAD. The area of the wounds can also be monitored simultaneously. The spatiotemporal mitochondrial redox ratio changes can give information on the status of wound healing online. This device was utilized to study diabetic wounds and the effect of photo-biomodulation on the wound healing progress. I have also utilized the optical cryo-imaging system to study the three-dimensional (3D) mitochondrial redox state of kidneys, hearts, livers, and wound biopsies of the small animal models of various injuries. For example, cryo-imaging was conducted on irradiated rat hearts during ischemia-reperfusion (IR) to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. Also, I developed a 3D image processing tool that can segment and quantify the medullary versus the cortical redox state in the kidneys of animal injury models. 2) 3D Vascular-Metabolic Imaging (VMI). I have designed VMI, an image processing algorithm that segments vascular networks from intrinsic fluorescence. VMI allows the simultaneous acquisition of vasculature and metabolism in multiple organs. I demonstrate that this technique provides the vascular network of the whole organ without the need for a contrast agent. A proof validation has performed using TdTomato fluorescence expressing endothelium. The VMI also showed convincing evidence for the “minimum work” hypothesis in the vascular network by following Murray’s law. For a proof-of-concept, I have also utilized a partial body irradiation model that VMI can provide information on radiation-induced vascular regression. 3) Time-lapse fluorescence microscopy. I have utilized fluorescence microscopy to quantify the dynamics of cellular reactive oxygen species (ROS) concentration. ROS is imaged and quantified under oxygen or metabolic stress conditions in cells in vitro. This approach enabled me to study the sensitivity of retinal endothelial cells and pericytes to stress under high glucose conditions. In short, I developed and utilized optical bio-instrumentation and image processing tools to be able to detect metabolic and vascular information about different diseases

Mobile Wound Assessment and 3D Modeling from a Single Image

The prevalence of camera-enabled mobile phones have made mobile wound assessment a viable treatment option for millions of previously difficult to reach patients. We have designed a complete mobile wound assessment platform to ameliorate the many challenges related to chronic wound care. Chronic wounds and infections are the most severe, costly and fatal types of wounds, placing them at the center of mobile wound assessment. Wound physicians assess thousands of single-view wound images from all over the world, and it may be difficult to determine the location of the wound on the body, for example, if the wound is taken at close range. In our solution, end-users capture an image of the wound by taking a picture with their mobile camera. The wound image is segmented and classified using modern convolution neural networks, and is stored securely in the cloud for remote tracking. We use an interactive semi-automated approach to allow users to specify the location of the wound on the body. To accomplish this we have created, to the best our knowledge, the first 3D human surface anatomy labeling system, based off the current NYU and Anatomy Mapper labeling systems. To interactively view wounds in 3D, we have presented an efficient projective texture mapping algorithm for texturing wounds onto a 3D human anatomy model. In so doing, we have demonstrated an approach to 3D wound reconstruction that works even for a single wound image

Dynamic hypoxic pre-conditioning of cells seeded in tissue-engineered scaffold to improve neovascularisation

Introduction: Tissue engineering (TE) is the potential solution to the global shortage of tissue and organs. However, the lack of adequate angiogenesis to TE scaffolds during the initial stages of implantation has hindered its success in vivo. Mesenchymal stem cells (MSC) have the most established track record for translational regenerative therapy and have been widely used in combination with TE scaffolds. Hypoxia is one of the main potentiators for upregulating angiogenic factors in MSC. However, fine-tuning their cellular function and behaviour is still not fully understood. This study aims to help increase the understanding of this process by determining the effects of in vitro hypoxic conditioning on enhancement of angiogenesis of MSC for the purpose of pre-clinical translational for TE application. Methods: The angiogenic potential of 3 different tissue sources (bone marrow, umbilical cord and adipose) MSC were initially determined for downstream pre-clinical application. We established the appropriate regime for in vitro dynamic hypoxia conditions in 2D and 3D hydrogel to enhance MSC angiogenic pathway using real-time continuous oxygen sensors and angiogenic cytokine profiling. Cell metabolism and proliferation effects were also evaluated using intravital Realtime-glo, D-luciferin (on transduced MSC) and microscopic Live-Dead stain techniques. We optimised seeding of cells on the tissue engineered dermal (INTEGRA®) for in vivo translational purpose and used targeted in vitro and ex vivo angiogenesis assays, which helped to determine aspects of the MSC conditioned media on endothelial migration, proliferation, morphogenesis and matrix degradation. Finally, the functional reproducibility of the in vitro angiogenic response was assessed using in vivo angiogenesis CAM assay and murine diabetic wound healing models. Results: Adipose derived MSC (adMSC) were found to have the most angiogenic potential in response to hypoxic conditioning. Dynamic hypoxia (DH) regime of changing oxygen levels from 21% to 1% when transitioning from T-flask subculture to multiwell plate seeding was most effective at eliciting pro-angiogenic response from adMSC for both in vitro 2D and 3D models compared to controls using static normoxia (21% oxygen) and static hypoxia (1%). Low seeding density of adMSC was found to be the most appropriate to ensure optimised cell adherence and survival post-seeding on TE dermal scaffold (INTEGRA®). It also minimised on localised hypoxic gradient induced oxidative stress by the seeded cells when compared to high seeding density techniques found on non-invasive oxygen monitoring. Conditioned media from DH seeded adMSC was shown to have enhanced angiogenic proteomic profile compared to the controls. In vitro angiogenesis assays showed better human endothelial cell migration and morphogenesis in scratch assay and tubular formation assay compared to controls. Preliminary ex vivo organ assay results using novel human umbilical arterial rings showed better endothelial out-sprouting and migration through embedded matrix compared to controls. Results from in vivo transplantation of adMSC seeded INTEGRA® scaffold showed a mixed response in the CAM assay, highlighting an unaccounted scaffold effect from INTEGRA® from the host. Histological sections showed increased vascular and host tissue infiltration into the scaffold. When evaluating the functional angiogenesis in murine wound healing models, although DH adMSC seeded scaffolds showed non-statistically significant increased rate of wound closure, there was significantly greater vessel density within the scaffold on histological evaluation in this group compared to controls. Conclusion: The results provide a better comprehension of how cells behave in 2D and 3D environments when cultured in dynamically changing oxygen environments. The study addresses important issues, such as the effects of chronic hypoxia on MSC, and how dynamic hypoxia can enhance angiogenic signalling. It also offers a crucial understanding of the in vitro oxygen culture environments for future research applications. Further insight into cell-scaffold interaction during in vivo transplantation was also established. The importance of having an appropriate in vivo model to determine if such in vitro angiogenic enhancement would translate to functionally improving neoangiogenesis and subsequent tissue regeneration in vivo was also highlighted in this study. Improving and advancing research into optimising and evaluating the in vitro environment for clinical application will undoubtedly have a huge impact on the future of cell therapy for regenerative medicine purposes

Viewing life without labels under optical microscopes

Optical microscopes today have pushed the limits of speed, quality, and observable space in biological specimens revolutionizing how we view life today. Further, specific labeling of samples for imaging has provided insight into how life functions. This enabled label-based microscopy to percolate and integrate into mainstream life science research. However, the use of labelfree microscopy has been mostly limited, resulting in testing for bio-application but not bio-integration. To enable bio-integration, such microscopes need to be evaluated for their timeliness to answer biological questions uniquely and establish a long-term growth prospect. The article presents key label-free optical microscopes and discusses their integrative potential in life science research for the unperturbed analysis of biological samples