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Keratinocytes in tissue engineering of human skin: invitro and in vivo studies
Linköping University, Department of Biomedicine and Surgery, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences.
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Full thickness wounds, such as deep burns, need restoration of both the dermal and epidermal layers of the skin. In normal wound healing, re-epithelialization occurs by migration and proliferation of keratinocytes from the wound edges and by differentiation of stem cells from remaining hair follicles. Restoration of dermis occurs by influx of growth factors secreted by macrophages, platelets, and fibroblasts; by fibroblast proliferation and subsequent synthesis and remodeling of collagenous dermal matrix. In the case of full-thickness acute burn injuries and chronic wounds (e.g. pressure ulcers, venous ulcers and diabetic foot ulcers), these processes are defective. With the principles of tissue engineering in mind (to correct, improve and maintain tissues and their functions), researchers have developed promising materials and methods to make it possible to restore either the dermal (Integra® DRT, Alloderm®) or the epidermal layer (split thickness skin grafts (STSG), cultured epithelial autografts (CEA), autologous keratinocytes in single cell suspension). It is now well established that superior results are obtained if both dermal and epidermal components are combined, for example in a bilayered skin equivalent. Apligraf® is recommended for use on venous ulcers and is the only bilayered living skin equivalent currently approved by the FDA. Studies on different factors affecting the wound healing capacity as well as techniques in use provide valuable information for further development.

In this licentiate thesis, we evaluated different transplantation techniques for delivering cultured human keratinocytes in single cell suspension, a measure becoming more frequently used in addition to STSG and CEA for restoring the epidermal layer of the skin. We found that the pressure device, commonly used to spray cell suspension onto the wound with pressures as high as 200 kPa, killed around 0% of the cells. In comparison, an ordinary syringe with the attachment of a spray nozzle showed almost 90% viable cells post transplantation and provided an equally good distribution of the cell suspension.

We also studied different silver containing dressings regarding silver accumulation in human skin. In addition, we graded the re-epithelialization to evaluate whether the dressings caused any delay in the wound healing process. We found that the silver dressings tested, with few exceptions, caused dermal accumulation of silver, primarily aggregated around blood vessels. We could also show that most of the dressings had negative effect on the re-epithelialization.

For the restoration of the dermal layer of the skin, Integra® DRT functions as a scaffold for guided tissue regeneration of the dermis. We had the possibility to study a case of necrotizing fasciitis were the treatment consisted of the use of Integra® DTR together with sub-atmospheric pressure (after initial surgical debridement) and later transplantation of split thickness skin grafts. This measure proved to be safe as well as giving satisfactory pliable and aesthetically acceptable result.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2008. , 49 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 87
Keyword [en]
Keratinocytes tissue engineering
National Category
Surgery
Identifiers
URN: urn:nbn:se:liu:diva-21283ISBN: 978-91-7393-779-5 (print)OAI: oai:DiVA.org:liu-21283DiVA: diva2:240943
Presentation
2008-11-21, Hagdahlssalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2009-10-06 Created: 2009-09-30 Last updated: 2009-10-06Bibliographically approved
List of papers
1. Transplantation of cultured human keratinocytes in single cell suspension: a comparative in vitro study of different application techniques
Open this publication in new window or tab >>Transplantation of cultured human keratinocytes in single cell suspension: a comparative in vitro study of different application techniques
2008 (English)In: Burns, ISSN 0305-4179, E-ISSN 1879-1409, Vol. 34, no 2, 212-219 p.Article in journal (Refereed) Published
Abstract [en]

Transplantation of autologous cultured keratinocytes in single cell suspension is useful in the treatment of burns. The reduced time needed for culture, and the fact that keratinocytes in suspension can be transported from the laboratory to the patient in small vials, thus reducing the costs involved and be stored (frozen) in the clinic for transplantation when the wound surfaces are ready, makes it appealing. We found few published data in the literature about actual cell survival after transplantation of keratinocytes in single cell suspension and so did a comparative in vitro study, considering commonly used application techniques. Human primary keratinocytes were transplanted in vitro in a standard manner using different techniques. Keratinocytes were counted before and after transplantation, were subsequently allowed to proliferate, and counted again on days 4, 8, and 14 by vital staining. Cell survival varied, ranging from 47% to >90%, depending on the technique. However, the proliferation assays showed that the differences in numbers diminished after 8 days of culture. Our findings indicate that a great number of cells die during transplantation but that this effect is diminished if cells are allowed to proliferate in an optimal milieu. A burned patient’s wounds cannot be regarded as the optimal milieu, and using less harsh methods of transplantation may increase the take rate and wound closing properties of autologous keratinocytes transplanted in a single cell suspension.

Place, publisher, year, edition, pages
Institutionen för klinisk och experimentell medicin, 2008
Keyword
Burns, Cell culture, In vitro, Keratinocytes, Transplantation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-11214 (URN)10.1016/j.burns.2007.03.008 (DOI)
Note
Original publication: Camilla Fredriksson, Gunnar Kratz and Fredrik Huss, Transplantation of cultured human keratinocytes in single cell suspension: a comparative in vitro study of different application techniques, 2008, Burns, (34), 2, 212-219. http://dx.doi.org/10.1016/j.burns.2007.03.008. Copyright: Elsevier B.V., http://www.elsevier.com/Available from: 2008-03-07 Created: 2008-03-07 Last updated: 2017-12-13Bibliographically approved
2. Accumulation of Silver and Delayed Re-epithelialization in Normal Human Skin: An ex-vivo Study of Different Silver Dressings
Open this publication in new window or tab >>Accumulation of Silver and Delayed Re-epithelialization in Normal Human Skin: An ex-vivo Study of Different Silver Dressings
2009 (English)In: WOUNDS-A COMPENDIUM OF CLINICAL RESEARCH AND PRACTICE, ISSN 1044-7946, Vol. 21, no 5, 116-123 p.Article in journal (Refereed) Published
Abstract [en]

Silver is commonly used in wound dressings and topical formulations to assist in the management of wounds that are infected or at risk of becoming infected. They provide potent broad-spectrum antimicrobial activity, but should not cause sustained staining of the skin, dermal or systemic accumulation of silver, or discomfort to the patient. However, clinicians and healthcare personnel have been concerned about topical staining of the skin and complaints of additional pain from patients treated with certain silver dressings. Some delay in re-epithelialization has also been noticed and reported. The reasons for this are not clear, and the authors believed further study regarding the possible effects of silver accumulation and silver dressings effect on re-epithelialization was required. The authors studied possible silver accumulation and re-epithelialization in normal human dermal skin. The results showed that most of the dressings or treatments discolored the wound surface and that there was a dermal accumulation of what were assumed to be silver particles. Varying grades of accumulation were found in deep dermal tissue, particularly around blood vessels, depending on the dressing used. The results also indicated that all of the tested products delayed re-epithelialization in this model.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-19124 (URN)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2017-01-13Bibliographically approved
3. A Novel Concept for Treating Large Necrotizing Fasciitis Wounds With Bilayer Dermal Matrix, Split-thickness Skin Grafts, and Negative Pressure Wound Therapy
Open this publication in new window or tab >>A Novel Concept for Treating Large Necrotizing Fasciitis Wounds With Bilayer Dermal Matrix, Split-thickness Skin Grafts, and Negative Pressure Wound Therapy
2009 (English)In: Wounds (King of Prussia, Pa.), ISSN 1044-7946, E-ISSN 1943-2704, Vol. 21, no 8, 215-220 p.Article in journal (Refereed) Published
Abstract [en]

Treatment of necrotizing fasciitis (NF) includes radical surgical debridement often resulting in large wounds that need to be closed with methods including split-thickness skin grafts (STSG), local flaps, or guided tissue regeneration procedures. In this case report, a 45 year-old Caucasian male was surgically treated for a benign left groin hernia, developed NF, and was transferred to the authors burn unit. The wound was treated initially with wide debridement and with a brief delay before finally closing the wound. A collagen matrix such as Integra (R) Dermal Regeneration Template (Integra LifeSciences, Plainsboro, NJ) in combination with STSG and negative pressure wound treatment, can provide fast recovery resulting in pliable, functional skin.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20581 (URN)000269472900006 ()
Note

Funding text: "We express our sincere gratitude to Mrs. Kristina Briheim and Mrs. Anita Lonn, Senior Laboratory Technicians at the Laboratory for Experimental Plastic Surgery, Institute of Biomedicine and Surgery, Faculty Of Health Sciences, Linkoping Universitet, Linkoping, Sweden."

Available from: 2009-09-15 Created: 2009-09-15 Last updated: 2017-12-13Bibliographically approved

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