In vitro wound healing assays - State of the art

Wound healing is essential for the restoration of the barrier function of the skin. During this process, cells at the wound edges proliferate and migrate, leading to re-epithelialization of the wound surface. Wound healing assays are used to study the molecular mechanisms of wound repair, as well as in the investigation of potential therapeutics and treatments for improved healing. Numerous models of wound healing have been developed in recent years. In this review, we focus on in vitro assays, as they allow a fast, cost-efficient and ethical alternative to animal models. This paper gives a general overview of 2-dimensional (2D) cell monolayer assays by providing a description of injury methods, as well as an evaluation of each assay's strengths and limitations. We include a section reviewing assays performed in 3-dimensional (3D) culture, which employ bioengineered skin models to capture complex wound healing mechanics like cell-matrix interactions and the interplay of different cell types in the healing process. Finally, we discuss in detail available software tools and algorithms for data analysis. © 2016 by De Gruyter

Cell Motility Dynamics: A Novel Segmentation Algorithm to Quantify Multi-Cellular Bright Field Microscopy Images

Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications

Minimizing the storage losses of potatoes under different storage treatments

2021 Fall.Includes bibliographical references.To view the abstract, please see the full text of the document

Outcome measurements following palatal soft tissue graft harvesting : a review

Free gingival graft (FGG) and connective tissue graft (CTG) are two of the most commonly techniques performed in periodontal and peri-implant plastic surgery. Although several outcome measurements have been proposed for evaluation of palatal wound healin

Inflammatory dependent bioresponsive smart transdermal delivery system incorporating susppended nanofibrous mats as a platform for wound healing

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, South Africa Johannesburg 2016The perception of wound healing within the current decade goes beyond the straightforward assertion of the three phases assembling the wound healing cascade. Healing of wounds is a complex process that involves a dynamic series of interactions and reactions and requires a collaboration of the many cell pedigrees, mediators and different tissues. The skin is the largest organ of the body and serves as a protective barrier against foreign objects therefore a loss in its veracity may lead to a decrease quality of life or even death. The primary goal for wound care and treatment is an aesthetically pleasing scar with close to complete functionality at the wound site and rapid wound closure. Attainment of these features requires incorporation of various characteristics such as a moisture retention, absorption and debridgement amongst others. A huge variety of wound dressings are available however not all of these meet the specific requirements of an ideal wound healing device to cover every aspect within the wound healing cascade. Highlighted within this thesis is the design and development of a Bioresponsive transdermal delivery system (BTDS) for wound healing that aims at the incorporation of the significant characteristics for optimal wound management and treatment. Nanobiotechnology is an interdisciplinary field that combines many avenues to revolutionise the development of drug delivery systems specific to wound healing. Delivery systems produced on the nanoscale can encourage the promotion of biologically active new molecular entities that were previously considered underdeveloped by the enhancement of the therapeutic efficacy of wound healing materials. Recent research interest has focused on the development of smart biomaterials. Combining biomaterials that are crucial for wound healing will provide opportunities to synthesize matrices that are inductive to cells and that stimulate and trigger target cell responses crucial to the wound healing process. Stimuli responsive systems provides an attractive, novel and alternate approach to the process of healing by offering an advanced alternative to simple wound dressings as they have the ability to adapt to the surrounding wound environment and regulate the healing process by thermal, chemical, biochemical, electrical and mechanical means on exposure to an external stimulus that triggers the effect. The research focused on the development and characteristic analysis of a complete prototyped device for wound healing incorporating a nanofibrous mat as well as a bioresponsive component to inflammation which could be the first novel prototype developed as an inflammation bioresponsive device for superior wound healing incorporating a nanofibrous mat. The BTDS was synthesized by the attainment of a statistically derived Box- Behnken Design Template, whereby 15 formulations were generated to fabricate a wound healing nanofibrous mats as well as a lyophilized inflammatory dependent matrix. The technique entailed the process of electrospinning for nanofiber formation as well as blending and lyophilization for the inflammatory responsive component. Elucidation of the various polymeric and crosslinker concentrations greatly influenced the properties and characteristics of the system. An endorsement in intensity and conjugation is noted by the FTIR spectra whereby greater shifts in wavelengths from 3260.11cm-1 to 3278.79cm-1 is noted when enhancements in crosslinking bridges is undertaken. Structural morphological analysis revealed the synthesis of smooth, cylindrical, uniformly aligned nanofibres without the presence of nanobeads as well as the formation of a lyophilized matrix having a tough backbone structure at higher concentrations. Upon nanotensile mapping, variation in Young‟s Modulus was observed at 4.25MPa providing flexibility whereas a higher Young‟s Modulus provides rigidity and stiffness to the structure. Determination of the bioresponsive nature was carried out in a stimulated inflammatory environment by utilisation of the Fentons reaction: Fe2+ + H2O2 → Fe3+ + OH∙ + OH- . Results amongst the experimentally derived formulations revealed the reliance of bioactive release on the hyaluronic acid concentration and degradation by hydroxyl radicals present. MDT results obtained depicted a value at 42.39 at a higher hyaluronic concentration and degree of crosslinkage whereas at lower concentrations, MDT values at 33.21 and 35.76 were depicted. In vivo histological examination revealed the healing progression whereby the presence of the nanofibrous mat illucidated a close to complete re-epithelisation and remodelling of the wound site represented by thick, vascular granulation tissue dominated by fibroblasts and extensive collagen deposition. The approach of introducing a topical device for wound management containing both nanotechnology and stimuli responsive techniques provide an innovative and encouraging proposal for wound care to the pharmaceutical industry.MT201

Advanced delivery system for skin and burns therapy : mupirocin as an antibacterial model drug

Trauma to the skin in the form of severe wound, particularly burns, can facilitate colonization of potentially life threatening bacterial infections. To prevent infections of the wounded area, antimicrobial agents are recommended as standard treatment. Topical administration of antimicrobial agents, such as mupirocin, can provide local therapy, while avoiding the risks of systemic administration. Mupirocin-in-liposomes-in hydrogels were proposed as advanced delivery system for this purpose. Up to now, no liposomal mupirocin for topical administration has been reported. Chitosan was selected as hydrogel matrix due to its biodegradability and in-built antimicrobial and wound healing potentials. Phosphatidylcholine liposomes containing mupirocin, namely non-sonicated and sonicated liposomes, were characterized for vesicle size and size distributions. Non-sonicated vesicles entrapped in average 74 and sonicated 49 % of mupirocin calcium, respectively. Sonication reduced the original vesicle size from around 1 micron down to 135 nm. Liposomes (10 %, w/w) were incorporated in chitosan hydrogels and liposomal hydrogels evaluated for their textural properties. Hydrogels were found to exhibit satisfactory adhesiveness and cohesiveness, with corresponding stability profile. Microbiological assessment confirmed antibacterial properties of liposomally entrapped mupirocin incorporated in hydrogels. In vitro and ex vivo (on pig skin) drug release profiles of various formulations containing mupirocin were performed on Franz diffusion cells. Liposomal hydrogels were compared with marketed mupirocin product, Bactroban® cream. The release studies showed that liposomal size affects the release of the incorporated drug. Liposomal hydrogels were shown to provide sustained release of incorporated mupirocin. In conclusion, liposomal hydrogels developed for mupirocin offer the potential to increase retention time and provide sustained release of a drug, which are important parameters for improved treatment of wounds, including burns

Composite alginate and gelatin based bio-polymeric wafers containing silver sulfadiazine for wound healing

Lyophilized wafers comprising sodium alginate (SA) and gelatin (GE) (0/100, 75/25, 50/50, 25/75, 0/100 SA/GE, respectively) with silver sulfadiazine (SSD, 0.1% w/w) have been developed for potential application on infected chronic wounds. Polymer–drug interactions and physical form were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), respectively, while morphological structure was examined using scanning electron microscopy (SEM). Functional characteristics [(mechanical hardness and adhesion using texture analyzer, and swelling capacity)] of blank wafers were determined in order to select the optimal formulations for drug loading. Finally, the in vitro drug dissolution properties of two selected drug loaded wafers were investigated. There was an increase in hardness and a decrease in mucoadhesion with increasing GE content. FTIR showed hydrogen bonding and electrostatic interaction between carboxyl of SA and amide of GE but no interaction between the polymers and drug was observed, with XRD showing that SSD remained crystalline during gel formulation and freeze–drying. The results suggest that 75/25 SA/GE formulations are the ideal formulations due to their uniformity and optimal mucoadhesivity and hydration. The drug loaded wafers showed controlled release of SSD over a 7 h period which is expected to reduce bacterial load within infected wounds

Advanced multi-targeted composite biomaterial dressing for pain and infection control in chronic leg ulcers

This study aimed to develop advanced biomaterial polysaccharide based dressings to manage pain associated with infected chronic leg ulcers in older adults. Composite carrageenan (CARR) and hyaluronic acid (HA) dressings loaded with lidocaine (LID) and AgNPs were formulated as freeze-dried wafers and functionally characterized for porous microstructure (morphology), mechanical strength, moisture handling properties, swelling, adhesion and lidocaine release. Antimicrobial activity of AgNPs was evaluated (turbidity assay) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus whilst cell viability studies (MTT) was performed on normal adult human primary epidermal keratinocyte cells. The wafers were soft, flexible and elegant in appearance. HA affected the wafer structure by increasing the resistance to compression but still possessed a balance between toughness and flexibility to withstand normal stresses and prevent damage to newly formed skin tissue respectively. Water uptake was influenced by HA, whilst equilibrium water content and LID release were similar for all the formulations, showing controlled release up to 6 h. AgNPs loaded CARR/HA wafers were effective in inhibiting the growth of both Gram positive and Gram negative bacteria. MTT assay showed evidence that the AgNPs/ LID loaded wafers did not interfere with cell viability and growth. CARR/HA wafers seem to be a promising system to simultaneously deliver LID and AgNPs, directly to infected chronic leg ulcers