Molecular mechanisms of skin wound healing in non-diabetic and diabetic mice in excision and pressure experimental wounds

Experimental models for chronic skin lesions are excision and pressure ulcer, defined as "open" and "closed" lesions, respectively, only the latter characterized by tissue hypoxia. Moreover, systemic diseases, such as diabetes mellitus, affect wound repair. Thus, models for testing new therapies should be carefully selected according to the expected targets. In this study, we present an extensive and comparative histological, immunohistochemical, and molecular characterization of these two lesions in diabetic (db/db) and non-diabetic (C57BL/6 J) mice. In db/db mice, we found significant reduction in PGP9.5-IR innervation, reduction of capillary network, and reduced expression of NGF receptors. We found an increase in VEGF receptor Kdr expression, and the PI3K-Akt signaling pathway at the core of the altered molecular network. Db/db mice with pressure ulcers showed an impairment in the molecular regulation of hypoxia-related genes (Hif1a, Flt1, and Kdr), while extracellular matrix encoding genes (Itgb3, Timp1, Fn1, Col4a1) were upregulated by hyperglycemia and lesions. Overall, the molecular analysis suggests that db/db mice have a longer inflammatory phase of the wound repair process, delaying the progression toward the proliferation and remodeling phases

Development of a cell-based lab-on-a-chip sensor for detection of oral cancer biomarkers

textOral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. Development of new diagnostic tools incorporating tumor biomarkers could enhance early detection by providing molecular-level insight into the biochemical and cellular changes associated with oral carcinogenesis. The work presented in this doctoral dissertation aims to address this clinical need through the development of new automated cellular analysis methods, incorporating lab-on-a-chip sensor techniques, for examination of molecular and morphological biomarkers associated with oral carcinogenesis. Using the epidermal growth factor receptor (EGFR) as a proof-of-principle biomarker, the sensor system demonstrated capacity to support rapid biomarker analysis in less than one-tenth the time of traditional methods and effectively characterized EGFR biomarker over-expression in oral tumor-derived cell lines. Successful extension from in vitro tumor cell lines to clinically relevant exfoliative brush cytology was demonstrated, providing a non-invasive method for sampling abnormal oral epithelium. Incorporation of exfoliative cytology further helped to define the important assay and imaging parameters necessary for dual molecular and morphological analysis in adherent epithelium. Next, this new sensor assay and method was applied in a small pilot study in order to secure an initial understanding of the diagnostic utility of such biosensor systems in clinical settings. Four cellular features were identified as useful indicators of cancerous or pre-cancerous conditions including, the nuclear area and diameter, nuclear-to-cytoplasm ratio, and EGFR biomarker expression. Further examination using linear regression and ROC curve analysis identified the morphological features as the best predictors of disease while a combination of all features may be ideal for classification of OSCC and pre-malignancy with high sensitivity and specificity. Further testing in a larger sample size is necessary to validate this regression model and the LOC sensor technique, but shows strong promise as a new diagnostic tool for early detection of oral cancer.Chemistry and Biochemistr

Nuclear morphometry, apoptotic and mitotic indices, and tubular differentiation in Libyan breast cancer

Aims of this study were to evaluate the relations of nuclear morphometry, mitotic and apoptotic indices, and tubular differentiation with clinicopathological features and survival rate in Libyan women. The data were compared with corresponding results on Finnish, and Nigerian female breast cancer patients. Histological samples of breast cancer (BC) from 131 patients were retrospectively studied. Mitotic activity indices (MAI and SMI), apoptotic index (AI), and fraction of fields with tubular differentiation (FTD) were estimated. Samples were also studied by computerized nuclear morphometry, such as mean nuclear area (MNA). Demographic and clinicopathological features were analyzed from 234 patients. The Libyan BC was dominantly premenopausal, and aggressive in behavior. There were statistically significant correlations between the mean nuclear area, fraction of fields with tubular differentiation, apoptotic index and proliferative indices, and most clinicopathological features. The highest significances were shown between lymph node status and the proliferative and apoptotic indices (p=0.003 with SMI, and p=0.005 with AI). There were significant associations between clinical stage and SMI and AI (p=0.002 and 0.009, respectively). The most significant associations with grade were observed with MNA and FTD (p<0.0001 and 0.001, respectively). The proliferative differences between Libyan, Nigerian and Finnish populations were prominent. These indices in Libyan were lower than in Nigerian, but higher than in Finnish patients. The Libyan patients’ AI is slightly higher than in Nigeria, but much higher than in Finland. The differences between countries may be associated with the known variation in the distribution of genetic markers in these populations. The results also indicated that morphometric factors can be reliable prognostic indicators in Libyan BC patients.Tämän työn tarkoituksena on arvioida tumamorfometrian, mitoosi- ja apoptoosi-indeksien ja tubulaarisen differentaation suhdetta libyalaisten rintasyöpäpotilaiden kliinispatologisiin piirteisiin ja eloonjäämiseen. Tietoja verrattiin suomalaisiin ja nigerialaisiin rintasyöpää sairastavien naisten tietoihin. Työssä tutkittiin 131 potilaan histologisia rintasyöpänäytteitä retrospektiivisesti. Mitoosiaktiviteetti-indeksit (MAI ja SMI), apop¬toottinen indeksi (AI) ja niiden mikroskooppikenttien osuus, joissa todettiin tubulaarista differentaatiota (FTD) arvioitiin. Myös kasvainsolujen keskimääräinen ala (MAI) arvioitiin tietokoneistettua morfometriaa käyttäen. Libyan rintasyöpäpotilaiden demografisia ja kliinispatologisia piirteitä analysoitiin 234 potilaasta. Libyan rintasyöpä (BC) on etupäässä premenopausaalista ja käyttäytymiseltään agressiivista. MNA, FTD, AI, MAI ja SMI olivat selvästi korrelaatiossa useimpiin kliinispatologisiin tietoihin. Merkittävin suhde todettiin imusolmukestatuksen ja proliferaatioindeksien ja apoptoottisen indeksin välillä (SMI p=0.003, AI p= 0.009). Histologinen gradus korreloi parhaiten MNA:n (p=0.001) ja FTD:n (p=0.001) kanssa. Kasvainten proliferaatioindeksit Libyassa, Nigeriassa ja Suomessa olivat selvästi erilaisia. Libyan indeksit olivat matalampia kuin Nigerian indeksit, mutta korkeampia kuin Suomen indeksit. AI oli hieman matalampi kuin Nigeriassa, mutta selvästi korkeampi kuin Suomessa. Erot maiden välillä voivat liittyä populaatioiden geneettisiin eroihin. Tulokset myös osoittavat, että morfometrisia tekijöitä voidaan käyttää libyalaisten rintasyöpäpotilaiden ennustetekijöinä.Institute of BiomedicineSiirretty Doriast

Computer aided classification of histopathological damage in images of haematoxylin and eosin stained human skin

EngD ThesisExcised human skin can be used as a model to assess the potency, immunogenicity and contact sensitivity of potential therapeutics or cosmetics via the assessment of histological damage. The current method of assessing the damage uses traditional manual histological assessment, which is inherently subjective, time consuming and prone to intra-observer variability. Computer aided analysis has the potential to address issues surrounding traditional histological techniques through the application of quantitative analysis. This thesis describes the development of a computer aided process to assess the immune-mediated structural breakdown of human skin tissue. Research presented includes assessment and optimisation of image acquisition methodologies, development of an image processing and segmentation algorithm, identification and extraction of a novel set of descriptive image features and the evaluation of a selected subset of these features in a classification model. A new segmentation method is presented to identify epidermis tissue from skin with varying degrees of histopathological damage. Combining enhanced colour information with general image intensity information, the fully automated methodology segments the epidermis with a mean specificity of 97.7%, a mean sensitivity of 89.4% and a mean accuracy of 96.5% and segments effectively for different severities of tissue damage. A set of 140 feature measurements containing information about the tissue changes associated with different grades of histopathological skin damage were identified and a wrapper algorithm employed to select a subset of the extracted features, evaluating feature subsets based their prediction error for an independent test set in a Naïve Bayes Classifier. The final classification algorithm classified a 169 image set with an accuracy of 94.1%, of these images 20 were an unseen validation set for which the accuracy was 85.0%. The final classification method has a comparable accuracy to the existing manual method, improved repeatability and reproducibility and does not require an experienced histopathologist

New Frontiers of Skin Tissue Engineering: from the Laboratory to Clinical Practice

This thesis is focused on the key role of extracellular matrix in skin tissue engineering. Firstly, I presented three dimensional structures derived through the physiological secretion of extracellular matrix (ECM), that may be a bioinspired scaffold. I performed a biological characterization of cell-assembled ECMs from three different sub-populations of skin fibroblasts; papillary fibroblasts (Pfi), reticular fibroblasts (Rfi), and dermal papilla fibroblasts (DPfi). Fibroblast sub-populations were cultured with ascorbic acid to promote cell-assembled matrix production for 10 days. Cells were removed and the remaining matrices were characterized. I found that the ECM assembled by Pfi exhibited randomly oriented fibers, associated with highest interfibrillar space, reflecting ECM characteristics which are physiologically present within the papillary dermis. Mass spectrometry followed by immunofluorescence analysis showed that Thrombospondin is preferentially expressed within the DPfi ECM. In another experiment, keratinocytes were seeded on the top of cell depleted ECMs to generate epidermal skin constructs. I found that epidermal constructs grown on DPfi or Pfi matrices exhibited normal basement membrane formation, while Rfi matrices were unable to support membrane formation. Thus, inspiration should be taken from these different ECMs, to design therapeutic biomaterials in skin engineering applications. In the second part, I focused on human decellularized matrix for soft tissue repair and I investigated the biological interaction post-implant of this scaffold. The aims of this second part were to show the clinical results after the application of human decellularized matrix in patients suffering from abdominal hernia and to evaluate the response one year post implant, through morphological analysis of biopsy specimens. Clinical results showed that all the patients revealed a well tolerability of human decellularized matrix. Post-implant morphological results showed cellular repopulation, neo-angiogenesis, minimal inflammatory response and a well-organized collagen matrix in all biopsies. This scaffold can be considered a safe product to treat large abdominal defects

Dynamics, regulation and function of macrophages in skin repair

Tissue repair is a highly dynamic process comprising the sequential phases of inflammation, tissue formation, and maturation. The mechanisms that orchestrate the natural sequence of the wound healing response remain elusive. Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both, tissue injury and repair. In this study the hypothesis was examined whether macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose a mouse model was developed that allows conditional depletion of macrophages during the sequential stages of the repair response by using the inducible diphtheria toxin receptor mouse model in combination with a myeloid cell-specific Cre mouse line. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of a vascularized granulation tissue and showed impaired re-epithelialization. However, recruitment of macrophages during the mid phase of repair, after macrophage depletion was stopped, rescued the impaired healing response and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the mid stage of the repair response (phase of tissue formation) resulted in severe hemorrhages within the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. Taken together, these results demonstrate that macrophages exert distinct functions during the different phases of skin repair, which are crucial to control the natural sequence of repair events. Furthermore, the effect of macrophages on endothelial cell function and wound angiogenesis appeared to be critical. Therefore the impact of macrophage-derived vascular endothelial growth factor-A (VEGF-A) on the outcome of the wound healing response was analyzed, by using conditional gene targeting to specifically deplete VEGF-A expression in myeloid cells. It could be shown that during the early phase of repair, myeloid cell-derived VEGF-A is essential to induce the angiogenic response, in contrast, at later stages of the wound healing response epidermal-derived VEGF-A controls vascular growth. We further showed that myeloid cell-derived VEGF-A is critical for tip cell formation, a process fundamental for vascular sprouting. Collectively, our findings propose novel mechanistic insights on macrophage-mediated repair events after skin injury and potentially might identify new therapeutic targets that can promote wound angiogenesis in impaired wound healing conditions

Added benefits of computer-assisted analysis of Hematoxylin-Eosin stained breast histopathological digital slides

This thesis aims at determining if computer-assisted analysis can be used to better understand pathologists’ perception of mitotic figures on Hematoxylin-Eosin (HE) stained breast histopathological digital slides. It also explores the feasibility of reproducible histologic nuclear atypia scoring by incorporating computer-assisted analysis to cytological scores given by a pathologist. In addition, this thesis investigates the possibility of computer-assisted diagnosis for categorizing HE breast images into different subtypes of cancer or benign masses. In the first study, a data set of 453 mitoses and 265 miscounted non-mitoses within breast cancer digital slides were considered. Different features were extracted from the objects in different channels of eight colour spaces. The findings from the first research study suggested that computer-aided image analysis can provide a better understanding of image-related features related to discrepancies among pathologists in recognition of mitoses. Two tasks done routinely by the pathologists are making diagnosis and grading the breast cancer. In the second study, a new tool for reproducible nuclear atypia scoring in breast cancer histological images was proposed. The third study proposed and tested MuDeRN (MUlti-category classification of breast histopathological image using DEep Residual Networks), which is a framework for classifying hematoxylin-eosin stained breast digital slides either as benign or cancer, and then categorizing cancer and benign cases into four different subtypes each. The studies indicated that computer-assisted analysis can aid in both nuclear grading (COMPASS) and breast cancer diagnosis (MuDeRN). The results could be used to improve current status of breast cancer prognosis estimation through reducing the inter-pathologist disagreement in counting mitotic figures and reproducible nuclear grading. It can also improve providing a second opinion to the pathologist for making a diagnosis

Nanotechnology applied to translational oncology: Developing tools for liquid biopsy

Liquid biopsy represents a powerful tool to support precision medicine, allowing the study of the subset of circulating components that derived from cancer tissue. Among all these circulating materials, the Circulating tumour cells (CTCs) represent one of the most promising biomarkers. However, the evaluation of CTCs has not been incorporated yet into current clinical guidelines for treatment decision. This might be due to CTCs are infrequent, appearing at an estimated level of one against the background of millions of surrounding normal peripheral mononuclear blood cells (PBMCs). The objective of this thesis project is to develop innovative nanoparticles that can address two of the critical points that make challenging the use of CTCs in translational studies of breast cancer: ex vivo culture and isolation. Nanoemulsions composed by a combination of lipids with potential to improve cell viability were formulated. The use of proliferative nanoemulsions (NEs) was successfully translated to ex vivo CTC cultures from metastatic breast cancer patients to expand these cells for their characterization. The analysis of these cells in culture not only showed that the precursor cells had mesenchymal and stem features but also it was determined that the capability of CTCs to grow ex vivo using the established protocol is a predictive factor in metastatic breast cancer. Finally, the NEs were functionalized with peptides (Pept-NEs) to endow them with specific recognition capabilities and it was confirmed that Pept-NEs can be immobilized on surfaces for their use as a potential isolation system