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Applications of human skin in vitro
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chronic wounds are a substantial problem in today’s health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal  keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic wound that fails to re-epithelialise. The wound healing process constitutes an example of dynamic reciprocity in tissue where cellular changes take place on cues from the extracellular matrix and vice versa when tissue homeostasis is disturbed. The extracellular matrix provides a structural context for the resident cells and the epidermal keratinocytes, and a functioning interplay between the two tissue compartments is crucial for successful wound healing to take place. Work included in this thesis has applied viable human full thickness skin in vitro to investigate the re-epithelialisation process and barrier function of intact skin.The use of full thickness skin in vitro can take into account the contextual aspect of the process where the epidermal keratinocytes are activated and obtain a migratory phenotype, and are continuously dependent on the cues from the extracellular matrix and support of the dermis. When utilising skin for studies on re-epithelialisation, circular standardised full thickness wounds were created and cultured  for up to four weeks in tissue culture. In paper I, the organisation of a thick neoepidermis was investigated in the in vitro wound healing model when resident cells were provided with a porous suspended three dimensional gelatin scaffold. In paper II we investigated the use of a fluorescent staining conventionally used for proliferation studies to facilitate the tracing of transplanted epidermal cells in in vitro  wounds, in order to improve and expand the use of the model. In paper III the model was utilised to investigate the treatment approach of acidification of wounds to evaluate the suitability of such intervention in regards to keratinocyte function and re-epithelialisation. Studies on re-epithelialisation with the aid of the in vitro wound healing model provided insight in neoepidermal structure with porous gelatin scaffolding in the wound, a novel methodological approach to tracing cells and response to constrained wound healing environment. In paper IV, intact human skin was evaluated for modelling the cytotoxic response after exposure to a known irritant compound. To study barrier function, intact skin was exposed to irritants by restricting exposure topically, and full thickness skin in vitro was found suitable for modelling cytotoxicity responses. Employing human full thickness skin in vitro makes use of the actual target tissue of interest with epidermal and dermal cells, and full barrier function.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 99 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1493
National Category
Clinical Medicine Clinical Science
Identifiers
URN: urn:nbn:se:liu:diva-123313DOI: 10.3384/diss.diva-123313ISBN: 978-91-7685-895-0 (print)OAI: oai:DiVA.org:liu-123313DiVA: diva2:881379
Public defence
2016-01-29, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2015-12-10 Created: 2015-12-10 Last updated: 2015-12-18Bibliographically approved
List of papers
1. Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin
Open this publication in new window or tab >>Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin
2015 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 10, no 6, e0128093- p.Article in journal (Refereed) Published
Abstract [en]

The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.

Place, publisher, year, edition, pages
Public Library of Science, 2015
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-120232 (URN)10.1371/journal.pone.0128093 (DOI)000355979500074 ()26061630 (PubMedID)
Available from: 2015-07-21 Created: 2015-07-20 Last updated: 2016-03-09
2. Tracing human keratinocytes and melanocytes with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining
Open this publication in new window or tab >>Tracing human keratinocytes and melanocytes with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining
2015 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Burn treatment and conditions of hypopigmentation may require autologous transplantation of keratinocytes and melanocytes. The tracing of transplanted cells presents a challenge. We report a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localising cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. CFSE-stained keratinocytes and CFSE-stained melanocytes were transplanted to human full thickness in vitro wounds either as cell suspension for keratinocytes, or with the aid of  macroporous gelatin microcarriers for both cells types in single and co-culture. Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated, and proliferation of the cells cultured on microcarriers was measured with flow cytometry. Wounds with transplanted cells were harvested after seven, 14 and 21 days in culture, cryosectioned and investigated using fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish double stained keratinocytes. The CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were traced in tissue sections after 21 days and wound re-epithelialisation was not affected. We propose a novel application of CFSE-staining in transplantation studies here presented with primary human keratinocytes and melanocytes.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-123310 (URN)
Available from: 2015-12-10 Created: 2015-12-10 Last updated: 2015-12-10Bibliographically approved
3. Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds
Open this publication in new window or tab >>Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds
2015 (English)In: Journal of Plastic Surgery and Hand Surgery, ISSN 2000-656X, E-ISSN 2000-6764, Vol. 49, no 6, 346-352 p.Article in journal (Refereed) Published
Abstract [en]

Background: Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. Aim: The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. Results:In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. Conclusion: The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing.

Place, publisher, year, edition, pages
Taylor & Francis, 2015
Keyword
Keratinocyte; pH; re-epithelialisation; wound model
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-123140 (URN)10.3109/2000656X.2015.1053397 (DOI)000364409400006 ()26051107 (PubMedID)
Available from: 2015-12-07 Created: 2015-12-04 Last updated: 2015-12-10Bibliographically approved
4. Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds
Open this publication in new window or tab >>Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds
2016 (English)In: Toxicology Mechanisms and Methods, ISSN 1537-6516, E-ISSN 1537-6524, Vol. 26, no 2, 82-87 p.Article in journal (Refereed) Published
Abstract [en]

Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R2 = 0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement.

Place, publisher, year, edition, pages
Taylor & Francis, 2016
Keyword
Human full thickness skin, in vitro model, SDS
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-123312 (URN)10.3109/15376516.2015.1091537 (DOI)000373528000002 ()26446981 (PubMedID)
Available from: 2015-12-10 Created: 2015-12-10 Last updated: 2016-05-03Bibliographically approved

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