PLASTIC Reconstructive Surgery focuses on the reconstruction of tissue defects caused by trauma, burns, congenital deformity, vascular failure or as the result of tumor excision. Based on specific features of the defect, the most appropriate strategy is selected in order to reconstruct tissues as authentic as possible. State-of-the art reconstructive strategies should optimally restore function and aesthetics (e.g., for deep burns in the face) and simultaneously aim for minimal donor site morbidity.

In this context cell-and tissue engineering and regenerative medicine were heralded as promising strategies for tissue repair two decades ago but so far only few clinical therapeutic options evolved and no autogenic engineered tissues could be developed. Reason is the complex molecular biology of cells and genes that should be matched with the macroscopic clinical requirements for tissue repair such as neovascularization and tissue rejection of non-autogenic tissues. We need to understand key features of the regular wound repair processes and take that knowledge to threedimensional tissue repair.

The response of tissues to injury forms the foundation of all reconstructive procedures. The intricate wound healing and tissue repair process involves the complex interplay of numeral cells, proteins and humeral factors. Growth factors are secreted by all cells orchestrating the several phases of wound repair. These proteins seem to play the role of directors of healing, messengers to signal other cells and inducers of cellular migration and proliferation. Topical administration of recombinant growth factors as proteins have major shortcomings such as short shelf life, low bioavailibility, enzymatic inactivaton by proteinases in the wound and inefficient delivery to target cells.Gene therapy differs from recombinant therapy in its delivery of the template for the protein rather than the protein itself. These genes are integrated into cells, turning the cells into ‘mini-factories’ of the growth factor. The major challenges facing wound repair then consist in identifying an appropriate gene that is effective in wound healing and then make that gene expressed by cells in the wound at clinical beneficial levels.

Wound repair and angiogenesis

Guided by the lack of appropriate autologous strategies in many clinical tissue reconstructions, our aim is to generate custom-made vascularised three-dimensional tissue constructs built up by autologous donor cells. We focus on ex vivo gene transfer using primarily autologous cultured cells. The advantage of ex-vivo strategies is the synergistic impact of the cultivated cell substrate and the expressed growth factors on the wound microenvironment. Ex vivo gene transfer techniques may form the backbone in the development of ‘smart’ biomaterials by supplying the bioinformation content that may induce cell migration and proliferation, angiogenesis and tissue integration.

With the cooperation of Dr Eriksson’s laboratory in Boston, we integrated the standardized wet wound healing model and the novel flexible transparent wound chambers that function as mini-incubators. We established protocols to cultivate cell suspension cultures of the progenitor fraction of p63+ basal cell keratinocytes, fibroblasts and endothelial progenitor cells (identified by a set of primers). All these cell cultures are grown in serum-limited conditions.

Proteinomic studies using protein chips, allowed us to analyze growth factor profiles from clinical and research wound fluids. In all our actual research protocols, we quantify concentrations of VEGF, EGF, bFGF, TGF-ß1, IGF-1,HGF, MMP1, MMP9, TIMP1,TIMP2 and MT1- MMP. All these proteins play an essential role in our aim to generate a three-dimensional vascularized tissue construct.

3-D vascularized tissue engineering

Angiogenesis is induced by VEGF 165-expressing cell cultures and endothelial progenitor cells (EPCs).After having identified human and porcine EPCs by PCR with multiple primers, immunohistochemistry, and LDL uptake assays, we actually perform in vitro and in vivo angiogenesis models to analyze the development of endothelial networks in various 3-D substrates. We introduced these well defined EPCs into our standardized full thickness porcine wound model.

We cultivated an autogenic matrix laminated with fibroblast sheets and covered with VEGF-expressing basal cell keratinocytes. We now analyze cell behavior and lamination properties. In our ‘Millefeuille approach’ (TERMIS 2007), we use these confluent fibroblast sheets as template, covered with keratinocyte layers and filled with EPCs to trigger angiogenesis: 3-D vasculaires tissue layers as a scaffold for tissue engineering.

Lipoaspirate-derived mesenchymal stem cell fractions are produced (fatty tissues are harvested easily by a plastic surgeon) to cultivate chondrocytes and adipocytes using appropriate culture conditions. The adipocytes are cultured to obtain adipose tissue layers which are elementary to define the subcutis in full thickness skin; for a reconstructive surgeon these adipose layers could also serve as substrate for breast reconstruction or scar treatment. The chondrocytes serve in tissue engineering protocols to generate a cartilage framework that may be used for nose and ear reconstructions.

For such 3-dimensional constructs we need a 3-dimensional shaped biomatrix as a template. We actually work with rapid prototyping technologies that translate our clinical CT data into three-dimensional bioplotted templates. These custom-made porous templates are further treated with ex vivo gene transfer protocols, using autogenic cells that gradually invade the template while secreting pro-angiogenic growth factors which induce and coordinate matrix deposition and angiogenesis.

Since the appropriate cocktail of cells and growth factors may strongly induce angiogenesis and tissue growth, we must be able to regulate timing and gene overexpression levels to avoid progression of healing towards hypertrophy, sclerosis or even cancerogenesis. We integrated a tetracycline- inducible gene switch into our VEGF expressing plasmids.

When tetracycline was added into the FTW through the wound chamber, expression of VEGF started. Cell suspension cultures of VEGF-overexpressing BCKs under regulation of a TC-inducible gene switch enhanced fibronectin deposition, endothelial cell tubuli formation and accelerated reepithelialization of FTW. The effects may be explained by the complex crosstalk between KCs, FBs, the ECM, VEGF and ECs.

The actual multidisciplinary approach brings together the molecular biology of cells and genes, the material sciences of resorbable matrices and scaffolds and the clinical expertise of tissues and wound repair and might well lead to the cultivation and production of a veritable authentic and autologous full thickness tissue equivalents, improving functional and esthetic outcome, social reintegration, pain relief and quality of life.

Jan Jeroen Vranckx MD, PhD, FCCP

Dept Plastic, Reconstructive Surgery, Burn Centre
PI. Laboratory of Plastic Surgery & Tissue Engineering Research,
KUL Leuven University Hospitals, Herestraat 49
Leuven, Belgium

jan.vranckx@uz.kuleuven.ac.be
32 16 348722