Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, June 1999. / Includes bibliographical references. Also available on the Internet.

No description available.

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.

No description available.

A dissertation submitted to the Faculty of Science University of the Witwatersrand, Johannesburg for the Degree of Master of Science. Johannesburg 1992 / 1. Beder, J.B. Thornley, A.L. and Veale.R, Human keratinocyte culture techniques using foetal, neonatal and adult keratinocytes with particular reference to extensive bum wounds. This paper was presented at the South African Burn Society Biannual Congress held at Berg en Dal, Kruger National Park, from 31 August to 1 September 1989. 2. Beder, J.B., Thornley, A.L. and Veale, R. Human keratinocyte culture techniques in extensive burn wounds. The Journal of Trauma and Emergency Medicine. 1990 Jan/Feb; 7 (1); 29-32. 3. Beder J.B. Cultured allografts. A letter appearing in the Continuing Medical Education Journal (CEM). 1990 Jan; 8(1): 48. 4. Beder, J.B. Problems and attempted solutions associated with the application of cultured epidermal grafts to full thickness burns in extensively burnt patients. This paper was presented in the form of a poster at the Association of Surgeons of South Africa Biannual Congress held in Bloemfontein from May 1-4 1990. 5. Beder, J.B., Thornley, A.L., Veale R., Kadwa, A.M., Scott, E. Healing chronic leg ulcers cultured keratinocyte allografts. This paper was presented at the annual Congress of The Surgical Research Society of Southern Africa held in Bloemfontein from 29 April to 1 May 1990. This paper was the runner-up for the best paper presented at the above congress. 6. The abstract of the paper mentioned in 5. above was published in the South African Journal of Surgery 1990 Sep; 28 (3): 123. 7. Beder, J.B., Healing full thickness burns with various types of cultured epidermal grafts - a comparative study. This paper was presented at the Z4th annual meeting of the British Burn Association held at the post-graduate centre, Stock Mandeville, Aylesbury, Bucks. England, United Kingdom, on the 10th May 1991. 8. Beder, J.B, J.B. A comparative study of various keratinocyte cell lines used in the synthesis of confluent stratified keratinocyte sheets. This paper was presented at the skin culture meeting, a satellite meeting of the 24th annual meeting of the British Burn Association, held at the post-graduate centre, Stoke-Mandeville, Aylesbury, Bucks, England, United Kingdom, on the 8th May 1991. 9. Beder, J.B. Healing chronic leg ulcers using cultured keratinocyte allografts. This paper was presented at the skin culture meeting of the British Burn Association held at the post-graduate centre, Stoke- Mandeville, Aylesbury, Bucks, England, United Kingdom on the 8th May 1991. 10. Beder, J.B., Thornley, A.L. and Veale, R. Healing chronic ulcers and other skin defects, using cultured keratinocyte allografts - including an assessment of their survival. Submitted for publication to the British Medical Journal. 11. Beder, J.B. The preparation and grafting of cultured epidermal sheets. Submitted for publication to the South African Journal of Surgery. 13. Beder, J.B., Thoenley, A.L. and Veale. R. Healing full thickness burns with cultured epidermal grafts - a comparative study. Submitted for publication to Transplantation.

Bioengineered skin substitutes have been developed to treat burn and non-healing wounds; however limitations still hinder their clinical success rates. Optimizing these current design strategies requires an understanding of how biochemical and topographical features of the native tissue modulate keratinocyte processes involved in tissue functionality. In this thesis, a novel bioengineered skin substitute was developed that contains a microfabricated basal lamina analog that recapitulates the native microenvironment found at the dermal-epidermal junction (DEJ). In native skin, this microenvironment consists of both biochemical and topographical cues which play critical roles in maintaining tissue architecture and overall homeostasis with the external environment. Therefore, we hypothesize that microfabricated basal lamina analogs with extracellular matrix cues and three-dimensional features that mimics the cellular microenvironment of the DEJ will promote enhanced epithelialization and increase epidermal stem cell clustering on the surface of bioengineered skin substitutes. We determined that the extracellular matrix protein fibronectin (FN) found in the cellular microenvironment of the DEJ enhanced keratinocyte attachment, proliferation, and epithelialization of a collagen based basal lamina analog. It was also found that the collagen material used to create the basal lamina analog as well as the FN conjugation strategy to this material significantly influenced the bioactivity of FN and its ability to modulate keratinocyte functions through integrin based mechanism. To investigate spatial tissue organization and the role it plays in the cellular microenvironment of the DEJ on epithelialization and epidermal stem cell localization, we used photolithography coupled with materials processing techniques to create microfabricated basal lamina analogs. It was determined that epidermal thicknesses found in narrow channels of microfabricated basal lamina analogs (50 µm and 100 µm widths with 200 µm depths) were similar to cultures on de-epithelialized acellular dermis and native foreskin tissues after 7 days of in vitro culture. We also determined that the microfabricated basal lamina analogs created an epidermal stem cell niche that promoted epidermal stem cell clustering in the channels which is critical for longevity of the tissue. Overall, we developed a platform technology that was specifically used to produce a highly functional bioengineered skin substitute with regenerative capacity that mimics native skin. We anticipate through the use of this technology, we can further improve bioengineered skin substitutes by incorporating epidermal structures of native skin including hair follicles and sweat glands as well as improve overall cosmetic appearance. Additionally, this novel bioengineered skin substitute can serve as a model system to further our understanding of pathological conditions and diseases of the skin as well as facilitate robust preclinical screenings of epidermal responses to new therapeutic agents as well as to cosmetic and chemical products.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to an embryonic state. Therefore iPSCs represent an extremely valuable tool for modelling disease and organ toxicity, with enormous potential in veterinary medicine. Several equine diseases are currently untreatable and can result in euthanasia on medical grounds. In contrast to humans, in vitro models for cellular research in equine do not exist. Therefore it has been necessary to explore the use of stem cells in constructing cell based equine models. Pluripotent stem cell populations are of great interest in this field given their ability to form the three germ layers found in the developing embryo. While a promising notion, the isolation of equine embryonic stem cells has thus far proved elusive and therefore it has been necessary to explore other pluripotent stem cell populations. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but studies in humans showed that other cell types such as keratinocytes were more amenable to reprogramming and generated iPSCs with much higher efficiency; whether this may be also the case in other species has not been investigated. Moreover, iPSC lines reported so far from domestic species, including the horse, depended on complex culture conditions for growth, including feeder layers and media supplementation with several growth factors. Although a promising alternative to fibroblast for generation of induced pluripotent stem cells there is dearth in literature on equine keratinocyte culture techniques. In this work I am reporting a novel approach to generate equine iPSCs lines from keratinocytes. Skin biopsies were used to derive keratinocyte cultures. The three dimensional culture systems were developed for robust culture of equine keratinocytes. These cells were then transduced with retroviral constructs coding for murine Oct-4, Sox-2, c-Myc and Klf-4 sequences, following the original Yamanaka protocol. Following transduction, tight cell colonies with sharp boundaries staining positive for alkaline phosphatase resembling previously reported human iPSCs were generated. The reprogrammed cells were successfully maintained in feeder free and serum free conditions with LIF supplementation. Immunochemistry and qPCR analyses revealed the equine iPSCs lines expressed pluripotency markers expressed in equine embryonic stages including, OCT4, SOX2, SSEA1, LIN 28, NANOG, REX1 and DNMT3B. Equine iPSCs were able to form embryoid bodies and differentiate into derivatives of the three germ layers in vitro. Equine iPSCs were pluripotent in vivo as demonstrated by the formation of teratoma consisting of tissue derivatives of all three lineages such as bone, cartilage, pulmonary epithelium and mature neurons in SCID mice. Importantly, equine iPSCs should not only have the ability to differentiate in a non-directed manner. Therefore, the ability for efficient and directed cellular differentiation was analysed. Equine iPSCs were successfully induced to differentiate into neurospheres forming extensive neuronal projections and synapses. Equine iPSCs were differentiated to neurons using a novel and robust approach. The neurons expressed FOXG1, TUBB3 at induction before ISL1 up regulation, a potent and specific inducer of motor neurons, during terminal differentiation. The neurons tested could fire multiple action potentials and also induce TTX –sensitive action potentials. The iPSC line that showed in vivo differentiation in bone and cartilage was tested for directed differentiation into bone and results were compared to equine mesenchymal stem cells. This study provides the first demonstration of the potential of iPSCs in equine biomedicine. The ability to derive iPSC cells capable of direct differentiation in vitro opens the way for new and exciting applications in equine regenerative medicine.

<p>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.<strong> </strong>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.</p><p>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.</p><p>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.</p><p>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.</p>

abstract / Dentistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Eugene G. Mueller, Dept. of Chemistry & Biochemistry. Includes bibliographical references.