Francine Goulet

Skin equivalent produced with human collagen

SummarySeveral studies have recently been conducted on cultured skin equivalent (SE), prepared using human keratinocytes seeded on various types of dermal equivalents (DE). We previously showed the advantages of our anchorage method in preventing the severe surface reduction of DE due to fibroblast contractile properties in vitro. A new anchored human SE was established in our laboratory in order to obtain a bioengineered tissue that would possess the appropriate histological and biological properties. In order to compare the effects of different collagen origins on the evolution of SE in vitro, human keratinocytes were seeded on three types of anchored DE. A comparative study was carried out between bovine SE (bSE), human SE (hSE), and human skin equivalent containing additional dermal matrix components (hSE +). Immunohistological analysis showed that hSE and hSE+ presented good structural organization, including the deposition of several basement membrane constituents. Higher amounts of transglutaminase, ceramides, and keratin 1 were detected in the epidermal layers of all SE when cultured at the air-liquid interface. However, a 92 kDa gelatinase activity was higher in bovine skin equivalent (bSE) compared to hSE cultures. The use of human collagens comparatively to bovine collagen as SE matricial component delayed the degradation of the dermal layer in culture.

Tissue-engineered human skin substitutes developed from collagen- populated hydrated gels: clinical and fundamental applications

The field of tissue engineering has opened several avenues in biomedical sciences, through ongoing progress. Skin substitutes are currently optimised for clinical as well as fundamental applications. The paper reviews the development of collagen-populated hydrated gels for their eventual use as a therapeutic option for the treatment of burn patients or chronic wounds: tools for pharmacological and toxicological studies, and cutaneous models for in vitro studies. These skin substitutes are produced by culturing keratinocytes on a matured dermal equivalent composed of fibroblasts included in a collagen gel. New biotechnological approaches have been developed to prevent contraction (anchoring devices) and promote epithelial cell differentiation. The impact of dermo-epidermal interactions on the differentiation and organisation of bio-engineered skin tissues has been demonstrated with human skin cells. Human skin substitutes have been adapted for percutaneous absorption studies and toxicity assessment. The evolution of these human skin substitutes has been monitored in vivo in preclinical studies showing promising results. These substitutes could also serve as in vitro models for better understanding of the immunological response and healing mechanism in human skin. Thus, such human skin substitutes present various advantages and are leading to the development of other bio-engineered tissues, such as blood vessels, ligaments and bronchi.

Stimulation of human keratinocyte proliferation through growth factor exchanges with dermal fibroblasts in vitro

Progress in biotechnology has led to new therapeutic approaches in various fields of human health care, such as the autologous grafting of cultured epidermal cell sheets on burned patients. These cultures depend on various parameters but growth factors are of paramount importance. Cutaneous cells are known to secrete various growth factors in vivo, although only a few have been identified. The aim of this study was to determine if such factors are secreted from human cutaneous cells in culture, to evaluate their effects on epidermal cell proliferation in vitro and to analyse them on SDS-PAGE. Human skin fibroblasts and keratinocytes were co-cultured for 8-10 days using a Costar trans-filter system. Dermo-epidermal cooperation was observed in such a co-culture system through the exchange of secretion products in the culture medium. Epidermal cell growth and metabolic activities were highly stimulated in co-culture (2-fold and 1.5-fold, respectively, P < 0.02) compared to the control. The de novo synthesis of secretion products, notably of a protein of about 40 kDa, was specifically induced in co-culture. The identification of new keratinocyte growth factors could accelerate graftable epidermal sheet production in vitro for human wound coverage and possibly enhance wound healing in vivo.

Establishment and Characterization of a New Cell Line Derived from a Human Primary Breast Carcinoma

A new cell line, designated HDQ-P1, was successfully established from a primary ductal infiltrating mammary carcinoma by using a 3T3 feeder layer lethally irradiated to 60 Gy. The HDQ-P1 cells have been grown in culture for over 115 passages and have a doubling time of 60 hours. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, telomerase expression, tumor antigen expression, xenograft implantation into nude mice, colony formation in soft agar, TP53 sequencing, and gene copy number of C-MYC, C-ERBB-2, and C-H-RAS oncogenes. The epithelial nature of this cell line was confirmed by ultrastructural analysis, expression of cytokeratins, and epithelial membrane antigen. The HDQ-P1 cells possess an extensively rearranged and polyploid karyotype, with an average of 20 recurrent marker chromosomes. Scatchard analysis demonstrated that both primary tumor and HDQ-P1 cells were estrogen- and progesterone-receptor negative. The HDQ-P1 cells had the same expression of human telomerase reverse transcriptase as other established breast cancer cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Direct DNA sequencing showed a point mutation which yielded to a stop codon at the amino acid 213 in exon 6 of the TP53 gene. A five-fold amplification of C-MYC was observed in HDQ-P1 cells. No amplification of C-ERBB-2 and C-H-RAS genes were observed. This cell line presents unique characteristics and may prove to be a good experimental model for investigating breast cancer biology.

PRODUCTION OF TISSUE-ENGINEERED THREE-DIMENSIONAL HUMAN BRONCHIAL MODELS

SummaryWe have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serium-free medium supplemented with retinoic acid (5×10−8M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models with be useful for studying human bronchial properites in vitro.

Grafting on nude mice of living skin equivalents produced using human collagens

Autologous epidermal transplantation for human burn management is an example of a significant breakthrough in tissue engineering. However, the main drawback with this treatment remains the fragility of these grafts during and after surgery. A new human bilayered skin equivalent (hSE) was produced in our laboratory to overcome this problem. The aim of the present work was to study skin regeneration after hSE grafting onto nude mice. A comparative study was carried out over a period of 90 days, between anchored bovine skin equivalent, hSE and hSE+, the latter containing additional matrix components included at concentrations similar to those in human skin in vivo. The addition of a dermal layer to the epidermal sheet led to successful graft take, enhanced healing, and provided mechanical resistance to the grafts after transplantation. In situ analysis of the grafts showed good ultrastructural organization, including the deposition of a continuous basement membrane 1 week after surgery.

MULTISTEP PRODUCTION OF BIOENGINEERED SKIN SUBSTITUTES: SEQUENTIAL MODULATION OF CULTURE CONDITIONS

SummaryMany studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboïdal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer

The main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.

Engineering Human Tissues for in Vivo Applications

Tissue engineering is a rapidly developing field. This technology could offer a new alternative for wound repair and organ replacement. It is based on the ability of living cells, with or without biomaterials, to be assembled as three-dimensional tissues. The in vivo applications extend from specialized dressings that improve host tissue repair (e.g., ulcer) to permanent grafts that restore the function of the tissue (e.g., skin grafting for burn patients). The presence of living cells in grafts has the advantages of potential self-renewal and regeneration after wounding by migration of the living cells. Depending on the application foreseen, either allogeneic or autologous grafts will be indicated. The challenges in the production and distribution of these two types of tissue-engineered products differ. In the case of allogeneic tissues, cell banks can be generated. Particular attention should be devoted to the absence of infectious agents in the cell banks in order to avoid one of the main drawbacks in organ transplantation—the transmission of diseases (e.g., AIDS, hepatitis C). Although some allogeneic tissues from mesenchymal cell types may be transplanted without rejection, the tissues containing epithelial and/or endothelial cells must be autologous. For autologous tissue grafts, special care has to be taken to avoid the introduction of infectious agents during the culture period, but there is no need for extensive viral testing of the cells and biopsy (e.g., for AIDS). During cell expansion in culture, the cellular morphology assessment by phase contrast microscopy, cell viability and number must be evaluated. The characterization of cell types present in the cultures (using known differentiation markers) will be particularly important when producing cell banks that could be stored frozen in liquid nitrogen. Upon thawing, a high cell viability must be obtained to insure the high quality of the starting material in the production of engineered tissues. If pluripotent stem cells are chosen as the cell source, the extent of differentiation into the adequate cell type must be carefully monitored with several differentiation markers.

Tissue Engineering of ACL Replacements

ACL injuries affect a large number of people, and such injuries lead to joint instability and increased risk for degenerative joint disease later in life. Reconstruction of the damaged joint using an in vitro engineered ACL replacement construct is becoming more of a reality and avoids the risk of allografts or the donor site morbidity associated with autografts. Whereas progress has been made in generating engineered tissues for ACL replacement in model systems, several challenges still remain. These include the choice of a suitable scaffold (components of normal tissue or biodegradable materials), the density and differentiation state of appropriate cells, the biomechanical environment required for optimal in vitro generation, parameters required for successful engraftment, and the biologic diversity of the host. Whereas the solutions to these challenges are being addressed by various groups and progress is being made, there are still gaps in our understanding of how to generate, implant, and ensure the success of these engineered tissues. This review discusses the current status of engineering ACL replacements and the challenges remaining.