M Rouabhia

Publications (42)132.1 Total impact

• Article: Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes.

  A Semlali, J Chakir, J-P Goulet, W Chmielewski, M Rouabhia
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  ABSTRACT: Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl-xL and caspase-3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl-xL proteins and caspase-3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.
  Journal of Periodontal Research 04/2011; 46(5):533-41. · 1.69 Impact Factor
• Article: Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa.

  A Semlali, K P Leung, S Curt, M Rouabhia
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  ABSTRACT: We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.
  Peptides 02/2011; 32(5):859-67. · 2.43 Impact Factor
• Article: Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects.

  A Semlali, E Jacques, M Rouabhia, J Milot, M Laviolette, J Chakir
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  ABSTRACT: Bronchial epithelium is considered a key player in coordinating airway wall remodelling. The function of epithelial cells can be modulated by the underlying fibroblasts through autocrine and paracrine mechanisms. To investigate the effect of phenotypic changes in bronchial fibroblasts from asthmatic subjects on epithelial cell proliferation. Epithelial cells and fibroblasts derived from bronchial biopsies of asthmatic and healthy controls were cultured in an engineered model. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT). Epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitors p21 and p27 were measured by western blots. Total and active forms of transforming growth factor (TGF)-β₁ were measured using ELISA and bioassay. TGF-β was inhibited using a recombinant TGF-β soluble receptor II protein. Proliferation of epithelial cells from asthmatics (AE) is increased when cells were cultured with fibroblasts from normal controls (NF). Fibroblasts from asthmatics (AF) significantly decreased the proliferation of epithelial cells from healthy subjects (NE). Activation of p21, p27, EGFR and TGF-β₁ reflects the proliferation data by decreasing in AE cultured with NF and increasing in NE cultured with AF. Neutralization of TGF-β increased proliferation of epithelial cells cultured in the asthmatic model. Fibroblasts from asthmatic subjects regulate epithelial cell prolifearation, and TGF-β signalling may represent one of the pathway involved in these interactions.
  Allergy 04/2010; 65(11):1438-45. · 6.27 Impact Factor
• Article: Development of an engineering autologous palatal mucosa-like tissue for potential clinical applications.

  C Luitaud, C Laflamme, A Semlali, S Saidi, G Grenier, A Zakrzewski, M Rouabhia
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  ABSTRACT: The goal of this study was to optimize key processes in recreating functional and viable palatal mucosa-like tissue that would be easy to handle and would promote wound healing. Normal human gingival fibroblasts and epithelial cells and a clinically useful biomaterial, CollaTape, were used. Structural and ultrastructural analyses showed that the gingival fibroblasts and epithelial cells adhered to the biomaterial and proliferated. Following a 6-day culture, using 10(5) fibroblasts and 10(6) epithelial cells, a well-organized palatal mucosa-like tissue was engineered. The engineered epithelium displayed various layers, including a stratum corneum, and contained cytokeratin 16-positive cells located in the supra-basal layer. This palatal mucosa-like engineered tissue was designed to meet a variety of surgical needs. The biodegradable collagen membrane (CollaTape) contributed to the flexibility of the engineered tissue. This engineered innovative tissue may contribute to the reconstruction of oral soft-tissue defects secondary to trauma, congenital defects, and acquired diseases.
  Journal of Biomedical Materials Research Part B Applied Biomaterials 12/2007; 83(2):554-61. · 2.15 Impact Factor
• Article: Porphyromonas gingivalis-epithelial cell interactions in periodontitis.

  E Andrian, D Grenier, M Rouabhia
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  ABSTRACT: Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.
  Journal of Dental Research 06/2006; 85(5):392-403. · 3.49 Impact Factor
• Article: Porphyromonas gingivalis gingipains mediate the shedding of syndecan-1 from the surface of gingival epithelial cells.

  E Andrian, D Grenier, M Rouabhia
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  ABSTRACT: Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan-1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan-1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan-1 detected in the culture medium increased significantly in a time-dependent manner. However, neither a heat-inactivated supernatant nor a supernatant from a gingipain-deficient mutant had a significant effect on syndecan-1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.
  Oral Microbiology and Immunology 05/2006; 21(2):123-8. · 2.81 Impact Factor
• Article: Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

  N Chouinard, J P Therrien, D L Mitchell, M Robert, R Drouin, M Rouabhia
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  ABSTRACT: Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.
  Biochemistry and Cell Biology 02/2001; 79(4):507-15. · 2.67 Impact Factor
• Article: Decreased capacity of asthmatic bronchial fibroblasts to degrade collagen.

  R Laliberté, M Rouabhia, M Bossé, J Chakir
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  ABSTRACT: The mechanisms of fibrillar collagen accumulation in asthmatic bronchi remain unclear, an imbalance between synthesis and degradation of collagen may be implicated in this process. The aim of this study was to compare the capacities of normal (BNF) and asthmatic (BAF) bronchial fibroblasts to degrade collagen. Metalloproteinases and their inhibitors were measured by ELISA, types I and III procollagen synthesis was determined by liquid RIA and, finally, zymography was used to assess the presence of active and latent forms of MMPs. The capacity of fibroblasts to degrade collagen coated onto latex beads was evaluated by flow cytometry. Our results showed that MMP-2 secretion was significantly higher in BNF when compared to BAF and this was confirmed by gelatin zymography. In BNF culture, TIMP-1 and MMP-1 secretions positively correlated with types I and III procollagen synthesis. However, in BAF, this correlation was negative. This suggests that a balance exists between collagen synthesis and degradation in BNF and that this balance is compromised in BAF. On the other hand, BAF did show significantly reduced capacity to degrade collagen when compared to that of BNF. This reduced phagocytic activity was not associated with a decrease in collagen receptor expression. This study establishes for the first time that a relationship exists between metalloproteinases enzyme dysregulation and the reduced capacity of asthmatic bronchial fibroblast to degrade collagen. These events may shed light on why accumulation of collagen can be observed in asthmatic airways.
  Matrix Biology 02/2001; 19(8):743-53. · 3.30 Impact Factor
• Article: Bronchial mucosa produced by tissue engineering: a new tool to study cellular interactions in asthma.

  J Chakir, N Pagé, Q Hamid, M Laviolette, L P Boulet, M Rouabhia
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  ABSTRACT: The use of fiberoptic bronchial biopsies has improved our understanding of the immunopathology of asthma. However, this approach offers a limited ability to perform mechanistic studies observing cell-cell and cell-matrix interactions, which are a key issue in the study of airway remodeling. Tissue engineering is a technique that combines the use of biology and engineering expertise to generate a limitless amount of tissue from small samples. This technology allows for the study of cell interactions under conditions as close as possible to the natural environment. The aim of this study was to evaluate the feasibility of an engineered human bronchial mucosa as a model to study cellular interactions in asthma. Human bronchial fibroblasts from normal and asthmatic donors were incorporated into collagen gel. Bronchial epithelial cells were seeded over this gel and then cultured in an air-liquid interface in the presence or the absence of T lymphocytes. Biopsy specimens from these engineered mucosa were taken for structural and ultrastructural analysis, and T lymphocytes were harvested and used to localize IL-5. Histologic analysis showed that engineered mucosa with normal bronchial cells presented a pseudostratified ciliated epithelium with the presence of mucus secretory cells. The electron microscopy analysis confirmed these histologic results. These features were comparable with those observed in normal bronchial tissues. However, in engineered mucosa from asthmatic subjects, the tissue structure was disorganized, particularly the epithelial cell arrangement. The percentage of IL-5(+) lymphocytes was significantly (P =.03) higher in engineered bronchial mucosa from asthmatic subjects (87% +/- 2%) compared with mucosa from normal volunteers (2% +/- 0.3%). Using tissue engineering, we produced an in vitro model of bronchial mucosa from normal and asthmatic subjects. These models could be a valuable tool to better understand key mechanisms involved in inflammation and airway repair.
  Journal of Allergy and Clinical Immunology 02/2001; 107(1):36-40. · 11.00 Impact Factor
• Article: The efficacy of a broad-spectrum sunscreen to protect engineered human skin from tissue and DNA damage induced by solar ultraviolet exposure.

  V Bissonauth, R Drouin, D L Mitchell, M Rhainds, J Claveau, M Rouabhia
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  ABSTRACT: Sunscreens are known to protect against sunlight-induced erythema and sunburn, but their efficiency at protecting against skin cancer is still a matter of debate. Specifically, the capacity of sunscreens to prevent or reduce tissue and DNA damage has not been thoroughly investigated. The present study was undertaken to assess the ability of a chemical broad-spectrum sunscreen to protect human skin against tissue and DNA damage after solar UV radiation. Engineered human skin was generated and either treated or not with a broad-spectrum SPF 30 sunscreen and exposed to increasing doses of simulated sunlight (SSL). Immediately after irradiation, histological, immunohistochemical, and molecular quantitative analyses were performed. The unprotected irradiated engineered human skin showed significant epidermal disorganization accompanied by a complete absence of laminin deposition. The sunscreen prevented SSL-induced epidermal damage at low doses and allowed laminin deposition at almost all SSL doses tested. The frequencies of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and photooxidative lesions measured by alkaline gel electrophoresis and radioimmunoassay were significantly reduced by the sunscreen. Thus, tissue and DNA damage may provide excellent quantitative end points for assessing the photoprotective efficacy of sunscreens.
  Clinical Cancer Research 11/2000; 6(10):4128-35. · 7.74 Impact Factor
• Article: Effects of all-trans retinoic acid on UVB-irradiated human skin substitute.

  N Chouinard, M Rouabhia
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  ABSTRACT: Retinoids are frequently used for treatment of photodamaged skin. We wished to find out whether photodamage could be attenuated by applying all-trans retinoic acid (RA) during repetitive irradiation. For this purpose, we used human cutaneous cells and tissue: pure monolayer cultures containing either keratinocytes or fibroblasts, and human skin substitute (SS) containing both cell types. All cultures were exposed to 8 mJ/cm(2) of UVB and were immediately treated with RA (0, 1.5, or 3 microM). The irradiation and RA treatment protocol was repeated until the cells of the nonirradiated culture had reached confluence. In the irradiated SS, RA preserved the structure (epidermal stratification and differentiation) and ultrastructure (well-organized intermediate filaments and desmosomes) in a state comparable to that observed in nonirradiated SS. As well RA maintained secretion of basement membrane components (laminin and type-IV collagen). Following irradiation, cutaneous cells also displayed more proliferative capacity when SS was treated. In the irradiated monolayer cultures, RA maintained the proliferative capacity of fibroblasts and decreased their differentiation whereas the opposite effect was seen on keratinocytes. In conclusion, RA clearly helps protect human skin against photodamage induced by repeated exposure to UVB.
  Journal of Cellular Physiology 11/1999; 181(1):14-23. · 3.87 Impact Factor
• Article: Harmful effects of UVA on the structure and barrier function of engineered human cutaneous tissues.

  M Robert, V Bissonauth, G Ross, M Rouabhia
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  ABSTRACT: To investigate the effects of a single UVA exposure on engineered human cutaneous tissues. Skin equivalents (SE) were obtained by culturing keratinocytes on fibroblast-populated collagen gels; epidermal equivalents (EE) were obtained by seeding keratinocytes on non-populated collagen gels. After maturation and differentiation of the epidermis, SE and EE were exposed to 50 or 100 J/cm2 UVA. Structural damage and total epidermal lipids were analysed and diffusion of radioactive oestradiol was monitored 24 and 72h post-irradiation. Twenty-four hours after UVA irradiation, a disorganization of the living epidermis is observed. UVA also significantly reduced the skin barrier function and led to an increase in phospholipid and in a decrease of ceramide levels. However, both the structure and the barrier function of SE were recovered 72 h post-irradiation, thereby suggesting that an intrinsic repair process might exist within the irradiated SE. This study provides a strong evidence that UVA radiation alters both the epidermal and dermal structures, the synthesis of epidermal lipids, and the permeability of the human skin.
  International Journal of Radiation Biology 04/1999; 75(3):317-26. · 2.28 Impact Factor
• Article: Serum matrix metalloproteinase-9:Tissue inhibitor of metalloproteinase-1 ratio correlates with steroid responsiveness in moderate to severe asthma.

  M Bossé, J Chakir, M Rouabhia, L P Boulet, M Audette, M Laviolette
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  ABSTRACT: Asthma presents a variable clinical response to corticosteroids (CS). Because CS more likely act on inflammation than on tissue remodeling, the presence of bronchial structural changes in certain asthmatics may explain their limited clinical response to CS. Matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), are, respectively, involved in tissue inflammatory processes and fibrogenic processes. Previous reports have suggested that MMP-9:TIMP-1 ratio may reflect the balance between these two processes in various diseases. This study evaluated the relation of this ratio and the response to CS in severe asthma. Twenty asthmatics with low baseline FEV1 (59 +/- 4% predicted) and >/= 30 % increase with beta2-agonist were recruited. Serum MMP-9 and TIMP-1 levels were measured and correlated with response to an oral CS trial (methylprenisolone 40 mg/d for 14 d). With oral CS, FEV1 changes (DeltaFEV1) ranged from -15 to +43%. The DeltaFEV1 closely correlated with the MMP-9:TIMP-1 ratios (rho = 0. 79, p = 0.0006). In conclusion, serum MMP-9: TIMP-1 ratio could predict the response of oral CS therapy in asthma. The low MMP-9:TIMP-1 ratio observed in subjects with little or no FEV1 improvement with CS supports the hypothesis that, in these asthmatic subjects, bronchial fibrogenesis predominates over inflammation.
  American Journal of Respiratory and Critical Care Medicine 03/1999; 159(2):596-602. · 11.08 Impact Factor
• Article: The multilayered organization of engineered human skin does not influence the formation of sunlight-induced cyclobutane pyrimidine dimers in cellular DNA.

  J P Therrien, M Rouabhia, E A Drobetsky, R Drouin
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  ABSTRACT: Solar UVB initiates skin cancer mainly by generating highly premutagenic cyclobutane pyrimidine dimers (CPDs) and subsequent mutations in critical growth control genes. It is universally presumed that the upper epidermis in human skin blocks a significant portion of the incident UVB, thereby protecting the cancer-prone basal layer from CPD formation. Using two sensitive techniques for measuring CPD in cellular DNA, we confirmed that the multilayered organization of engineered human skin efficiently shields the basal layer against 254-nm UVC (which is not present in terrestrial sunlight) but, very unexpectedly, provides virtually no protection against environmentally relevant UVB. This underscores the importance of regular sunscreen use, which, in light of our data, may constitute a considerably more important first line of defense against photocarcinogenesis than previously believed.
  Cancer Research 02/1999; 59(2):285-9. · 7.86 Impact Factor
• Article: Tissue-engineered human skin substitutes developed from collagen-populated hydrated gels: clinical and fundamental applications.

  F A Auger, M Rouabhia, F Goulet, F Berthod, V Moulin, L Germain
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  ABSTRACT: 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.
  Medical & Biological Engineering & Computing 12/1998; 36(6):801-12. · 1.88 Impact Factor
• Article: Permanent skin replacement using engineered epidermis containing fewer than 5% syngeneic keratinocytes.

  F Larochelle, G Ross, M Rouabhia
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  ABSTRACT: This study was conducted to investigate permanent skin replacement using heterologous (syngeneic-allogeneic) epidermal substitutes containing fewer than 5% syngeneic keratinocytes. Keratinocytes were isolated from the skin of new-born Balb/c (B) and C3H/HeN (C) mice and cocultured in different ratios: 20%B-80%C, 10%B-90%C, 5%B-95%C, and 2%B-98%C (and vice versa). After 4 to 5 days, graftable epidermal substitutes were obtained and transplanted onto adult Balb/c or C3H/HeN male mice. Recipients always received the heterografts containing the lower percentage of their own keratinocytes. On Days 15 and 30 postgrafting, all heterografts showed significant graft take (> 75%) and skin replacement compared with allografts. Regenerated tissues were well structured and well vascularized. These tissues contained only syngeneic keratinocytes. These results led us to question whether this was an active immune situation. Structural analyses revealed the presence of leukocyte infiltration that was dependent on the percentage of allogeneic keratinocytes present in the heterologous implant. However, even with the 2% syngeneic-98% allogeneic implant, infiltration was lower than with the allograft. Leukocyte phenotyping confirmed the presence of immune cells infiltrating the heterologous implants and revealed the involvement of CD8+ and CD4+ lymphocytes in this immune activation. The percentages of these two cell populations were lower than those obtained with the allografts, suggesting moderate cellular activation after each heterograft compared with the allografts. In conclusion, it is possible to generate functional skin even after 2% syngeneic-98% allogeneic heterografts; there was moderate cellular immune activation compared with the allografts.
  Laboratory Investigation 10/1998; 78(9):1089-99. · 3.64 Impact Factor
• Article: In vitro production and transplantation of immunologically active skin equivalents.

  M Rouabhia
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  ABSTRACT: In this study, we produced in vitro epidermal equivalents (EE) and skin equivalents (SE) with and without spleen lymphocytes. These skin substitutes were used for in vitro and in vivo (after isograft) histologic studies of cell and extracellular matrix organization and for protein synthesis. Then, using spleen lymphocytes in syngeneic and allogeneic SE, we assessed the immunogenicity of these skin substitutes after transplantation. In vitro histologic analyses showed that the epidermal organization of EE was comparable to that of SE. Fibroblasts and spleen lymphocytes were present in the extracellular matrix, as is the case in normal skin. Comparative immunohistologic studies after EE and SE isografting showed that the newly generated cutaneous tissues were well structured and vascularized. There were indications of physiologically active skin. The dermal component in these regenerated skins was, however, more organized after SE than after EE isografting, which indicates the importance of the dermis. Lastly, allografting of SE with and without spleen lymphocytes showed interesting results. Indeed, 10 days after allografting, all SE allowed skin regeneration comparable to isografts. Moreover, leukocyte infiltration in allografts was observed as early as 10 days and increased during the postgrafting period. Also, the presence of allogeneic spleen lymphocytes alone in syngeneic SE initiated recipient immune activation and induced leukocyte infiltration and graft rejection. The density of infiltrating leukocytes was higher in the complete allograft (allogeneic keratinocytes, fibroblasts, and spleen lymphocytes) compared with the partial allograft (only spleen lymphocytes were allogeneic), with the allograft (allogeneic keratinocytes and fibroblasts), and with the partial isograft (presence of syngeneic lymphocytes with allogeneic keratinocytes and fibroblasts). Mac-1+ and CD8+ cells were present in these leukocyte infiltrations, which indicates recipient immune system activation and allograft rejection. CD4-positive cells were not, however, seen in these leukocyte infiltrations. These results suggest that the incorporation of spleen lymphocytes in SE enhanced their immunogenicity as judged by leukocyte infiltration and the presence of CD8+ cells in the implants.
  Laboratory Investigation 11/1996; 75(4):503-17. · 3.64 Impact Factor
• Article: Grafting on nude mice of living skin equivalents produced using human collagens.

  C A López Valle, L Germain, M Rouabhia, W Xu, R Guignard, F Goulet, F A Auger
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  ABSTRACT: 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.
  Transplantation 09/1996; 62(3):317-23. · 4.00 Impact Factor
• Article: Permanent skin replacement using chimeric epithelial cultured sheets comprising xenogeneic and syngeneic keratinocytes.

  M Rouabhia
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  ABSTRACT: The present study was undertaken to evaluate the possibility of permanent skin replacement using chimeric xenogeneic-syngeneic graftable sheets previously obtained in vitro. Newborn (<3 days old) BALB/c and human keratinocytes were isolated and cocultured in different ratios as follows: 50% BALB/c to 50% human and 25% BALB/c to 75% human keratinocytes. Four to 5 days after culture and prior to their grafting, all chimeric sheets contained both cell types in ratios similar to those used to seed the initial chimeric cultures. Fourteen and 30 days after chimeric sheet grafting onto BALB/c mice dorsum, the newly generated cutaneous tissue showed a histologically well-organized epidermis presenting basal and suprabasal cell layers. Cutaneous cells in these structures secreted laminin and type IV collagen in blood vessels, and at ground level of the dermoepidermal junction there were signs of physiologically active skin. Cell phenotyping revealed the presence of only syngeneic keratinocytes, whereas xenogeneic cells were passively eliminated without a total rejection of the chimeric implant. This selective and passive elimination of xenogeneic keratinocytes went through cellular and humoral immunity activation. Data suggest that this chimeric culture method can be used for cutaneous therapies such as large congenital nevi, skin ulcers, and extensively burned skin. Indeed, for large third-degree wounded skin treatment, this culture method may shorten the time (4-5 weeks) needed for cell growth and graftable sheet production. Moreover, since the ultimate aim in allogeneic and xenogeneic transplantation is to achieve an immunological acceptance and tolerance to these foreign tissues, the chimeric culture approach may provide ways to lighten tolerance phenomena on cutaneous tissue.
  Transplantation 05/1996; 61(9):1290-300. · 4.00 Impact Factor
• Article: Cutaneous cell and extracellular matrix responses to ultraviolet-B irradiation.

  I Pâquet, N Chouinard, M Rouabhia
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  ABSTRACT: The present study examined fibroblasts and keratinocytes in monolayers and cultured within dermal and skin substitutes and their use in assessing the effect of UVB irradiation on cutaneous cells and extracellular matrix organization. Dermal substitutes (DS) were produced by incorporating normal fibroblasts into a collagen lattice and skin substitutes (SS) were obtained by seeding normal keratinocytes onto the DS. Keratinocyte monolayers, fibroblast monolayers, DS, and SS were exposed once a day to a UVB source (10 mJ/cm2). The irradiation protocol was stopped when the keratinocytes of the non-irradiated cultures (control groups) had reached confluence. Microscopic observations revealed that UVB radiation decreased both fibroblast and keratinocyte growth and enhanced their differentiation resulting in (1) less fibroblasts in the DS and SS, and (2) incomplete coverage of the DS by keratinocytes. Microscopic observations and histological analyses revealed major morphological changes. Both cell types became bigger and presented wide nuclei and vacuoles in the cytoplasm. No organized deep epidermal layer was observed in irradiated compared to non-irradiated SS. Irradiated DS and SS extracellular matrices showed an irregular aggregating collagen fiber organization with serious discrepancies suggesting large defects in the structural properties of the extracellular matrix. The present study demonstrated that exposure to a UVB source led to profound morphological and functional disturbances in both cutaneous cells and in the extracellular matrices of the DS and SS. The present technology would be of great interest for step-by-step studies of UVR effects on cutaneous cell morphology and functional properties, and could be an alternative to using animals for pharmacological and toxicological evaluations.
  Journal of Cellular Physiology 03/1996; 166(2):296-304. · 3.87 Impact Factor