Sophie Böttcher-Haberzeth

Tissue engineering of skin

The engineering of skin substitutes and their application on human patients has become a reality. However, cell biologists, biochemists, technical engineers, and surgeons are still struggling with the generation of complex skin substitutes that can readily be transplanted in large quantities, possibly in only one surgical intervention and without significant scarring. Constructing a dermo-epidermal substitute that rapidly vascularizes, optimally supports a stratifying epidermal graft on a biodegradable matrix, and that can be conveniently handled by the surgeon, is now the ambitious goal. After all, this goal has to be reached coping with strict safety requirements and the harsh rules of the economic market.

Markers to Evaluate the Quality and Self-Renewing Potential of Engineered Human Skin Substitutes In Vitro and after Transplantation

We screened a series of antibodies for their exclusive binding to the human hair follicle bulge. In a second step these antibodies were to be used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. Of all the antibodies screened, we identified only one, designated C8/144B, that exclusively recognized the hair follicle bulge. However, C8/144B-binding cells were never detected in the human epidermal stratum basale. In the bulge C8/144B-binding cells gave rise to cytokeratin 19-positive cells, which were also tracked in the outer root sheath between bulge and the hair follicle matrix. Remarkably, cytokeratin 19-expressing cells were never detected in the hair follicle infundibulum. Yet, cytokeratin 19-expressing keratinocytes were found in the epidermal stratum basale of normal skin as a subpopulation of cytokeratin 15-positive (not C8/144B-positive) basal keratinocytes. Cytokeratin 19/cytokeratin 15-positive keratinocytes decreased significantly with age. We suggest that cytokeratin 19-expressing cells represent a subpopulation of basal keratinocytes in neonates and young children (up to 1.5 years) that is particularly adapted to the lateral expansion of growing skin. Our data show that cytokeratin 19 in combination with cytokeratin 15 is an important marker to routinely monitor epidermal homeostasis and (at least indirectly) the self-renewing potential of engineered skin.

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells.

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipients vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.

Human Eccrine Sweat Gland Cells Turn into Melanin-Uptaking Keratinocytes in Dermo-Epidermal Skin Substitutes

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention

Extensive full‐thickness skin loss, associated with deep burns or other traumata, represents a major clinical problem that is far from being solved. A promising approach to treat large skin defects is the use of tissue‐engineered full‐thickness skin analogues with nearly normal anatomy and function. In addition to excellent biological properties, such skin substitutes should exhibit optimal structural and mechanical features. This study aimed to test novel dermo‐epidermal skin substitutes based on collagen type I hydrogels, physically strengthened by two types of polymeric net‐like meshes. One mesh has already been used in clinical trials for treating inguinal hernia; the second one is new but consists of a FDA‐approved polymer. Both meshes were integrated into collagen type I hydrogels and dermo‐epidermal skin substitutes were generated. Skin substitutes were transplanted onto immuno‐incompetent rats and analyzed after distinct time periods. The skin substitutes homogeneously developed into a well‐stratified epidermis over the entire surface of the grafts. The epidermis deposited a continuous basement membrane and dermo‐epidermal junction, displayed a well‐defined basal cell layer, about 10 suprabasal strata and a stratum corneum. Additionally, the dermal component of the grafts was well vascularized. Copyright

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

Characterization of pigmented dermo-epidermal skin substitutes in a long-term in vivo assay

In our laboratory, we have been using human pigmented dermo‐epidermal skin substitutes for short‐term experiments since several years. Little is known, however, about the long‐term biology of such constructs after transplantation. We constructed human, melanocyte‐containing dermo‐epidermal skin substitutes of different (light and dark) pigmentation types and studied them in a long‐term animal experiment. Developmental and maturational stages of the epidermal and dermal compartment as well as signs of homoeostasis were analysed 15 weeks after transplantation. Keratinocytes, melanocytes and fibroblasts from human skin biopsies were isolated and assembled into dermo‐epidermal skin substitutes. These were transplanted onto immuno‐incompetent rats and investigated 15 weeks after transplantation. Chromameter evaluation showed a consistent skin colour between 3 and 4 months after transplantation. Melanocytes resided in the epidermal basal layer in physiological numbers and melanin accumulated in keratinocytes in a supranuclear position. Skin substitutes showed a mature epidermis in a homoeostatic state and the presence of dermal components such as Fibrillin and Tropoelastin suggested advanced maturation. Overall, pigmented dermo‐epidermal skin substitutes show a promising development towards achieving near‐normal skin characteristics and epidermal and dermal tissue homoeostasis. In particular, melanocytes function correctly over several months whilst remaining in a physiological, epidermal position and yield a pigmentation resembling original donor skin colour.

Novel Treatment for Massive Lower Extremity Avulsion Injuries in Children: Slow, but Effective with Good Cosmesis

BACKGROUND Extended avulsion injuries are associated with significant loss of skin and subcutaneous fat, leaving the reconstructive surgeon with the challenge of substituting all tissues lost in the best possible way. We wanted to test whether the combined use of a Vacuum Assisted Closure system (VAC) and Integra Dermal Regeneration Template (IDRT) matched the required treatment profile encompassing initial control of infection, remodeling of body contours, and reconstruction of near normal skin. MATERIALS AND METHODS 4 children with massive lower extremity avulsion injuries were treated with early necrosectomy, VAC application for 3-5 weeks for wound cleansing and wound bed conditioning, subsequent implantation of IDRT, and finally autologous split thickness skin grafting (STSG) for definitive wound closure. Thereafter, a standard rehabilitation program was used. The key parameters of VAC and IDRT application, take rates of IDRT and STSG, complications, length of stay, and final outcome were recorded. RESULTS In all patients, early removal of necrosis and infection control was successfully achieved. Continuous VAC application fostered the formation of a several millimeters thick new tissue layer partly compensating for the lost hypodermis. IDRT implantation and subsequent STSG yielded take rates of nearly 100% and both functionally and cosmetically excellent long-term results. There were no major complications. CONCLUSION The combination of VAC and IDRT in children with massive leg avulsion injuries is feasible, safe, and delivers high-quality long-term outcomes that appear to justify the multiple operative procedures, the long hospitalization times, and the comparatively high costs entailed.

Osmotic expanders in children: No filling – no control – no problem?

BACKGROUND Self-filling, hydrogel-based osmotic tissue expanders have been successfully used for several years, mainly in adult patients. We wanted to test this novel device in pediatric plastic and reconstructive surgery. MATERIAL AND METHODS Between November 2004 and September 2009, we implanted 53 osmotic tissue expanders following standard surgical principles in a total of 30 children and adolescents with burn scars, congenital nevi, alopecia, or foot deformities. RESULTS All expanders reached their predicted volume within 6 weeks and 51 (96.2%) produced a sufficient amount of additional skin for the intended coverage of the defect. A serious infection precluding the planned reconstructive procedure occurred with 2 expanders (3.8%). Minor complications occurred at 6 implantation sites (11.4%), and consisted of small necrotic areas and perforations (n = 3) and minor infections (n = 3). These problems could be controlled and did not interfere with the subsequent plasty. The final results recorded at the last follow-up (mean: 21 months, range: 9-48 months) were rated as excellent in 25, good in 19, moderate in 6, and poor in 1 patient. CONCLUSION This is apparently the largest pediatric series in which self-filling expanders have been used. The data obtained indicates that self-filling expanders can be safely and effectively used for various plastic, reconstructive and orthopedic procedures in children and adolescents. The fact that numerous painful and distressing filling sessions are obviated with these expanders is particularly beneficial for those children too young to understand and cooperate. Moreover, this approach minimizes the risk of infection and lowers costs.

The effect of wound dressings on a bio-engineered human dermo-epidermal skin substitute in a rat model

Autologous bio-engineered dermo-epidermal skin substitutes are a promising treatment for large skin defects such as burns. For their successful clinical application, the graft dressing must protect and support the keratinocyte layer and, in many cases, possess antimicrobial properties. However, silver in many antimicrobial dressings may inhibit keratinocyte growth and differentiation. The purpose of our study was to evaluate the effect of various wound dressings on the healing of a human hydrogel-based dermo-epidermal skin substitute in preparation for the first-in-human clinical trials. Human dermo-epidermal skin substitutes approved for clinical trials were produced under good manufacturing practice conditions, transplanted onto immuno-incompetent rats, and dressed with either Vaseline Gauze™ (Kendall Medtronic, Minneapolis, USA), Suprathel® (PolyMedics Innovations GmbH, Denkendorf, Germany), Urgotul® SSD (Urgo Medical, Shepshed, United Kingdom), Mepilex® AG (Mölnlycke Health Care, Gothenburg, Sweden), or Acticoat™ (Smith&Nephew, Baar, Switzerland). Grafts were assessed clinically for take, epithelialization, and infection at 10 and 21 days post-transplantation, and histologically at 21 days. There were three subjects each in the Vaseline Gauze™ and Suprathel® groups, and four subjects each in the Urgotul® SSD, Mepilex® AG, and Acticoat™ groups. For all samples, the take rate was 100% and the expected keratinocyte number, epithelialization and epidermal stratification were observed. All of the dressings in the current study were well tolerated by our human dermo-epidermal skin substitute. The tolerance of the silver-based dressings is particularly relevant given the high risk of bacterial contamination with large skin defects, and provides pivotal information as we embark on clinical trials for this novel skin substitute.