Céline Auxenfans

Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering

Since the culture of keratinocytes on feeder layers, research to produce skin equivalents has been motivated by the challenge of treating large burns and chronic wounds and by European regulations which both require proof of the innocuousness and the effectiveness of cosmetic products, and which forbid animal testing. The dynamism in fundamental research, dermocosmetology and the pharmaceutical industry has led to the evolution and complexification of reconstructed skin. The Collagen-GAG-Chitosan sponge, as well as the self-assembly model, allow dermal reconstruction in which the neosynthesized extracellular matrix contains all of the desired macromolecules. It is deposited forming an ultrastructurally organised architecture. The quality of the dermis obtained allows the development and regeneration of a pluristratified and differentiated epidermis firmly anchored by an organised dermal-epidermal junction. Evolution of reconstructed skin into models which are more and more similar to the physiological skin results in higher graft take rates in the treatment of burns and chronic wounds, and brings to research, to dermocosmetology and to the pharmaceutical industry, a wide range of products such as pigmented, endothelialized, immunocompetent, and now adipose reconstructed skins. The present review will mainly concentrate on the latest developments in skin engineering and will mostly concern the studies carried out by our groups.

Reconstruction of a full-thickness collagen-based human oral mucosal equivalent

Tissue engineered human oral mucosa has the potential to be applied to the closure of surgical wounds after tissue deficits due to facial trauma, malignant lesion surgery or preposthetic procedure. It can also be used to elucidate the biology and pathology of oral mucosa and as a model alternative to animals for safety testing of oral care products. Using the technology previously developed in our laboratory for the production of a skin equivalent, we were able to reconstruct a nonkeratinized full-thickness human oral mucosal equivalent closely mimicking human native oral mucosa. The successive coculture of human lamina propria fibroblasts and human oral epithelial cells isolated from the nonkeratinized region of oral cavity in a porous collagen-glycosaminoglycan (GAG)-chitosan scaffold gave rise to a lamina propria equivalent (LPE) and then to an oral mucosa equivalent (OME). The results of the histology, immunohistology and transmission electron microscopy of this OME demonstrated the presence of a nonkeratinized pluristratified and differentiated epithelium as in native nonkeratinized human oral mucosa expressing both K13 and K3/76. This epithelium was firmly anchored to the LPE by a continuous and ultrastructurally well-organized basement membrane. In the LPE, fibroblasts synthesized new extracellular matrix where the average collagen fibre diameter was 28.4 nm, close to that of native oral mucosa. The proliferative capacity of the basal cells was demonstrated by the expression of Ki67.

Influence of negative pressure when harvesting adipose tissue on cell yield of the stromal–vascular fraction

Adipose tissue is the standard autologous filling material used in plastic surgery today. At the same time it is also a source of mesenchymal stem cells, situated in the Stromal-Vascular Fraction (SVF) and easy to obtain in large quantities. The method of harvesting adipose tissue is an important stage for cell survival. So far, comparative studies on harvesting techniques have only concerned MTT cell viability of mature adipocytes. The aim of our study was to determine the influence of pressure on the yield of SVF cells in relation to the syringe aspiration technique which is the standard technique in plastic surgery. For this, six different harvesting conditions were tested on 3 patients. For each condition, a sample was taken from the trochanter region with the help of a 3 mm cannula, manual aspiration by a 10 ml syringe; wall suction; the traditional pump suction at -350 and -700 mmHg; the power assisted liposuction at -350 and -700 mmHg. Cell yield with a pressure of -350 mmHg, assisted or not, was greater than that obtained at -700 mmHg and significantly superior to aspiration with a syringe (p<0.05). At -350 mmHg, the use of power-assisted liposuction gave better results for two out of three patients when compared to non-power-assisted liposuction. Negative pressure is a factor influencing the number of SVF cells harvested.

Cultured allogenic keratinocytes for extensive burns: A retrospective study over 15 years §

UNLABELLED The aim was to review the use and indications of cultured allogenic keratinocytes (CAlloK) in extensive burns and their efficiency. MATERIALS AND METHODS This retrospective study comprised 15 years (1997-2012). INCLUSION CRITERIA all patients who received CAlloK. EXCLUSION CRITERIA patients who died before complete healing. Evaluation criteria were clinical. Time and success of wound healing after CAlloK use were evaluated. RESULTS The CAlloK were used for 2 indications - STSG donor sites and deep 2nd degree burns in extensively burned patients. A total of 70 patients were included with severity Baux score of 99.2 (from 51 to 144) and mean percentage of TBSA of 63.49% (from 21 to 96%). Fifty nine patients received CAlloK for STSG donor sites with a mean number of applications of 4 and mean surface of 3800 cm(2) per patient. Treated donor sites were re-harvested 2.5 times. The mean time of complete epithelialization was 7 days. In 11 patients, CAlloK were used for deep 2nd degree burns. The mean percentage of burned surface was 73.7%. The mean surface of CAlloK per patient was 2545 cm(2). Complete healing was achieved in 6.4 days. CONCLUSION The CAlloK allow rapid healing of STSG donor-sites and deep 2nd second degree burns in extensively burned patients.

Cultured autologous keratinocytes in the treatment of large and deep burns: a retrospective study over 15 years.

AIM The aim was to review the use and indications of cultured autologous epidermis (CAE) in extensive burns and to evaluate the efficiency of our strategy of burn treatment. MATERIALS AND METHODS This retrospective study comprised 15 years (1997-2012). INCLUSION CRITERIA all patients who received CAE. EXCLUSION CRITERIA patients who died before complete healing and patients who received exclusively cultured allogeneic keratinocytes. Evaluation criteria were clinical. Time and success of wound healing after CAE graft were evaluated. RESULTS A total of 63 patients were included with severity Baux score of 107 (from 70 to 140) and mean percentage of TBSA of 71% (from 40% to 97%). The CAE were used as Cuono method, in STSG donor sites and deep 2nd degree burns and in combination with large-meshed STSG (1:6-1:12) in extensively burned patients. Cuono method was used in 6 patients. The final take was 16% (0-30) because of the great fragility of the obtained epidermis. Nine patients with deep 2nd degree burns (mean TBSA 81%, from 60 to 97%) were successfully treated with only CAE without skin grafting. Combined technique (STSG meshed at 1:6-1:12 covered with CAE) was used in 27 patients (mean TBSA 69%, from 49% to 96%) with 85% success rate. Finally, donor sites treated with CAE in 49 patients could be harvested several times thanks to rapid epithelialization (time of wound healing was 7 days (from 5 to 10 days)). CONCLUSION The CAE allow rapid healing of STSG donor sites and deep 2nd second degree burns in extensively burned patients.

Keratin 13 immunostaining in corneal impression cytology for the diagnosis of limbal stem cell deficiency.

PURPOSE The aim of this study was to develop a validated, reliable, and minimally invasive technique for diagnosing limbal stem cell deficiency (LSCD) by immunocytochemical detection of conjunctival and corneal keratins on epithelial cells collected by impression cytology (IC). METHODS After validation of labeling techniques on a cohort of 10 healthy control patients, keratins K12, K13, and K19 were labeled on corneal IC of 10 eyes suspected of LSCD. Positive scores for the conjunctival markers K13/K19, coupled with the rarity of the corneal marker K12, were diagnostic proof of LSCD. RESULTS IC is a reliable and noninvasive technique for collecting epithelial cells. The labeling validation phase has permitted K3 labeling to be eliminated due to lack of corneal specificity. Among patients with LSCD, nine samples were diagnosed with LSCD (K13+/K19+), which was severe (K12-) in eight cases and mild (K12+) in one case. One sample could not be analyzed due to lack of cells. CONCLUSIONS K13 has shown to be a new marker of conjunctival differentiation. The immunocytochemical search for the K13/K19 couple by corneal IC provides a simple and reliable method for diagnosing LSCD, whereas the level of K12 could provide a score of disease severity. On the other hand, the authors question the corneal specificity of K3 as conventionally established.

Corneal Collagen Cross-linking for the Treatment of Progressive Corneal Ectasia: 6-Year Prospective Outcome in a French Population

PURPOSE To evaluate 6-year results of standardized epithelium-off corneal collagen cross-linking (CXL) for treatment of progressive corneal ectasia. DESIGN Prospective, consecutive, interventional case series. METHODS Thirty-six eyes of 25 consecutive patients with documented progressive primary or iatrogenic corneal ectasia underwent CXL following the Siena protocol. The main outcome measures included uncorrected (UDVA) and corrected (CDVA) distance visual acuities, biomicroscopy and fundus appearance, topography-derived steep and flat keratometry (Kmax, Kmin), central corneal thickness (CCT), intraocular pressure with Goldmann applanation tonometer (GAT-IOP), and endothelial cell density (ECD), recorded at baseline and months 1, 3, 6, 12, 24, 36, and 72. Bilateral macular optical coherence tomography was performed at the endpoint visit. The mean follow-up was 66 ± 6 months (range, 60-78 months). RESULTS At 6 years, CXL stabilized primary and iatrogenic corneal ectasia in 89% of the patients. In bilateral CXL, the progression of the first eye was highly predictive of the fellow eyes outcome. At the endpoint follow-up, the mean outcome variations were: UDVA: -0.08 ± 0.36 logMAR (P = .2); CDVA: -0.14 ± 0.28 logMAR (P = .004); Kmax: +0.11 ± 1.70 diopters (D) (P = .7); Kmin: -0.25 ± 1.25 D (P = .2); CCT: -16.38 ± 37 μm (P = .01); GAT-IOP: +1.0 ± 2.3 mm Hg (P = .01); ECD: +31 ± 400 cells/mm(2) (P = .6); no cases of macular toxicity or severe adverse events were reported. CONCLUSIONS At 6 years, CXL maintains long-term results in halting the progression of corneal ectasia, with significant improvement in CDVA and long-term stability of keratometry. Further clinical studies with longer follow-up and larger series would be necessary to definitely confirm these results.

Optimization of a culture medium for the differentiation of preadipocytes into adipocytes in a monolayer

UNLABELLED Our objective was to optimize a medium for preadipocyte differentiation into adipocytes. METHODS The differentiation medium contains fixed components as well as 7 variable ones. To perform this study, different experiments were designed and the study was carried out in 4 stages. The first two stages tested the influence of serum, dexamethasone, hydrocortisone and an cAMP activator. In the third stage, two new variables were added: rosiglitazone and insulin. In the final stage, the medium selected in stage 3 was validated. The differentiation selection criteria consisted of the number of mature adipocytes and adiponectin secretion. RESULTS We have shown that each variable was indispensable and that positive interactions occurred between some variables. No negative interactions were found and it was possible to optimize the concentration of each variable. CONCLUSIONS We selected the following medium, which provides optimal adipocyte size and adiponectin secretion: DMEM/HAMF12+10% Foetal Clone Serum (FCS)+2 nM triiodothyronine+10 nM hydrocortisone +0.5 mM IsoButyl Methyl Xanthine (IBMX)+500 nM dexamethasone+1 microM rosiglitazone+0.15 UI/ml insulin+antibiotics.

Does adipose tissue cultured with collagen matrix and preadipocytes give comparable results to the standard technique in plastic surgery

INTRODUCTION Repairing contour defects is a challenge in plastic surgery. Different filling materials have been used with inadequate results and complications. The autologous fat transfer is the standard technique at the moment, but adipose tissue reserves are limited. The aim of our study was to compare in vivo on an animal model, preadipocytes cultured in a collagen scaffold versus adipose tissue transferred by the usual surgical technique. MATERIALS AND METHODS In order to compare adipocytes resulting from the differentiation of preadipocytes with those of purified adipose tissue, we implanted them in 10 nude mice. The preadipocytes were implanted using a collagen scaffold as intermediary and the adipose tissue following the plastic surgery protocol described by SR Coleman. After 8 weeks, tissue fragments were explanted and analysed after staining with HPS, Oil Red O and labelling with human anti-vimentin antibodies. RESULTS The scaffold seeded with preadipocytes had the macroscopic appearance of adipose tissue with peripheral neovascularisation. The preadipocytes had been transformed into mature adipocytes. Purified adipose tissue also presented peripheral neovascularisation. Numerous mature adipocytes were found. There was an abundant murine extracellular matrix since anti-vimentin labelling was negative. CONCLUSION This experimental study showed that adipose tissue engineering is feasible and gives comparable results to fat grafting. It allows a better understanding of the sequence of events following the transfer of adipose tissue. It provides not only volume but also undeniable stimulation, leading to significant thickening of the extracellular matrix.

Immunocytochemical Diagnosis of Limbal Stem Cell Deficiency: Comparative Analysis of Current Corneal and Conjunctival Biomarkers.

Purpose: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD). Methods: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD. Results: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed. Conclusions: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction.