Current research projects

The main goal of our research is the in vivo construction of an autologous skin substitute that ideally approximates the qualities of human skin and that can be used in procedures of skin replacement in reconstructive and plastic surgery.

• Project 1: Engineering autologous dermo-epidermal skin grafts
  
• Project 2: Maintaining the keratinocyte stem cell compartment in cell culture
• Project 3: Identifying novel sources of human keratinocytes stem cells
• Project 4: Optimizing the biomatrix

• Project 5: Engineering pre-vascularized skin grafts
• Project 6: Colour adjusted engineered autologous skin composites
• Project 7: Clinical Phase I and Phase II trials
• Project 8: Use of amniocytes for the production of cartilage and skin substitutes
• Project 9: Raman profiles of amniotic, fetal and adult cells
• Project 10: Introduction of rete ridges in fetal dermo-epidermal skin substitutes

Our goal is to develop autologous dermo-epidermal skin composites (full thickness skin analogues) that can be used clinically to cover skin defects of any origin in one single surgical intervention. To achieve that we are testing different biodegradable matrices which serve as scaffolds with regard to their population by keratinocytes, melanocytes, endothelial cells and fibroblasts.

Epidermal regeneration in native and in vitro engineered skin, indispensably requires the presence of self renewing keratinocyte stem cells. To identify markers specific for keratinocyte stem cells, we screened a series of antibodies for their exclusive binding to the human hair follicle bulge (where those stem cells are known to have their niche). In a second step these antibodies were used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. (Pontiggia 2009).

To determine whether human sweat glands harbour keratinocyte stem cells (in analogy to the hair follicle bulge region), we developed engineered skin grafts containing human sweat gland cells. We demonstrated, the capability of human eccrine epithelial sweat gland cells to form a stratified interfollicular epidermis substitute on collagen hydrogels both, in vitro and in vivo. From these data we conclude that in analogy to the human hair follicle bulge region, also human eccrine sweat glands harbour potential keratinocyte stem cells (Biedermann 2010).

The bio-engineering of skin for the generation of large transplantable dermo-epidermal skin replacements is dependent on a three-dimensional matrix that supports the biological function of skin cells and provides mechanical properties to allow for surgery. Until now, collagen type I hydrogels promise the best biological functionality but their mechanical weakness and tendency to contract and degrade do not allow for the generation of large transplants. We have proven that by plastic compression, collagen hydrogels can acquire mechanical and biological stability, while maintaining excellent biological functions. Cultured dermo-epidermal skin grafts based on compressed collagen hydrogels can be handled easily in clinically relevant sizes, do degrade at an appropriate rate, and give rise to near normal homeostatic skin (Braziulis 2012).

Initial take, development, and function of transplanted engineered tissue substitutes are crucially dependent on rapid and adequate blood perfusion. Therefore, the development of rapidly and efficiently vascularized tissue grafts is vital for tissue engineering and regenerative medicine. Our goal is to develop pre-vascularized human skin grafts and to evaluate the effects of pre-vascularization on skin regeneration in vivo. We were able to generate a network of highly organotypic, branching, lumen-forming capillaries in engineered skin substitutes. After transplantation onto immuno-incompetent rats, histological analyses of the pre-vascularized skin substitutes showed that the engineered human vessels quickly connected to the vasculature of the recipient animal. This became obvious because the bio-engineered capillaries of human origin contained red blood cells of rat origin, and rapidly supplied the graft with oxygen and nutrients. (Montaño 2010 and Luginbühl 2012, manuscript in preparation).

Although current tissue engineered autologous dermo-epidermal substitutes show a multilayered epidermal structure with an excellent mechanical protective function, no structured approach to melanocytic integration has been aimed for to date. This leads not only to insufficient biological protective function against radiation but also to randomly hypo- or hyperpigmented transplanted areas on the patient. Our aim is to systematically add melanocytes to autologous substitutes to achieve an additional protective function as well as an aesthetic improvement of autologous dermo-epideramal skin substitutes.

After more than 11 years of research the Tissue Biology Research Unit (TBRU) of the Department of Surgery of the Children's Hospital in Zurich has come up with new skin grafts for Regenerative and Transplantation Medicine. The complex, bioengineered skin grafts resemble the properties of normal human skin as closely as possible. The close collaboration between the TBRU and the Pediatric Burn Center of the Department of Surgery of the Children's Hospital in Zurich made it possible that the above-mentioned skin grafts can be transplanted in just one surgical intervention. The new skin grafts are intended to be applied in clinical disciplines, such as Burn and Plastic Surgery and Dermatology, in Zurich and in Europe. The development of the grafts was, and is, based on findings gained by the knowledge and methods of basic research in cell and molecular biology. The Clinical Trials Phase I started in 2014 by the EuroSkinGraft, EU FP7 Consortium (coordinated by the TBRU) and the Clinical Research Priority Program (CRPP) of the University of Zurich and in close collaboration with the Pediatric Burn Center, the Clinical Trial Center (CTC) in Zurich, and the Swiss Center for Regenerative Medicine (SCRM) in Zurich.

Spina bifida is a severe neurological malformation. If untreated, it leads to considerable physical and mental disabilities. Today, fetal surgery is the state-of-the-art treatment option to yield the best possible outcomes minimizing mechanical and/or neurotoxic and degenerative damage throughout gestation. In case of large defects, when the primary skin closure is not feasible, the availability of autologous skin and cartilage substitutes would be a great help. One limitation regarding the clinical application of fetal skin grafts in clinics for prenatal closure of spina bifida lesions is the necessity to conduct two separate fetal operations. First to obtain the fetal skin sample from the fetus, and second to close the spina bifida defect using laboratory-grown skin. Therefore, our principal goal is to develop fetal dermo-epidermal skin substitutes using pluripotent stem cells derived from amniotic fluid samples. Amniotic fluid can be easily obtained in relatively large quantities during frequently performed diagnostic amniocenteses. We want to isolate, identify and sort amniotic fluid stem cells by FACS. We want to culture and differentiate them into chondrocytes, fibroblasts and keratinocytes. We may become able to produce cartilage and dermo-epidermal skin substitutes with the aim of transplantation in an in vivo model and in the future to apply combined cartilage/skin in fetal surgery.

Raman spectroscopy and optical trapping are emerging tools in regenerative medicine which allow to discriminate, classify and eventually sort the different amniotic cell types in a label-free and non-invasive manner. Therefore, we want to establish a data base of the Raman molecular signatures of fetal and adult fibroblasts, keratinocytes, chondrocytes and endothelial cells as a sort of reference cell populations. This will allow to study the bio-chemical changes of skin and cartilage during development. Subsequently, we want to categorize the heterogeneous composition of the amniotic fluid cell pool into clusters and compare them with the data base. This will allow to discriminate and classify and eventually sort the different amniotic cell types in a label-free and non-invasive manner as alternative to FACS and MACS.

In this project we address another difficulty in the clinical application of fetal skin grafts for prenatal closure of spina bifida lesions: relatively fragile dermo-epidermal skin substitutes have to sustain a particular mechanical stress especially in case of fetoscopic surgery where the substitute has to be furled and pushed trough the fetoscope. One of the reason of fragility of skin substitutes consists in the absence of rete ridges which forms the typical wave-shaped structure of the natural dermo-epidermal junction. Rete ridges increase the surface area of the epidermal-dermal interface, thus providing structural integrity and mechanical strength. They also increase the capillary-epidermal surface area to improve the nutrient supply to the avascular epidermis. Using very small fetal tissue left-over biopsies we want to obtain fetal fibroblasts, endothelial cells and keratinocytes for the assembly of dermo-epidermal fetal skin substitutes. We want to recreate rete ridges by means of pistons which impress the wave-shaped structure on the collagen matrix during plastic compression of the dermal component. The bioengineered skin substitutes will be raised in vitro or in vivo on nude rats. They will be analysed by histological, whole-mount stainings and confocal microscopy. The vascularization pattern will be studied and the shear-stress resistance will be measured.