[Corneal endothelial cell therapy, a review]. / Thérapie cellulaire cornéenne endothéliale. Revue de la littérature.

In France, endothelial dysfunction represents approximately one half of the indications for corneal transplants performed each year. However, the use of endothelial keratoplasty is limited by the technical difficulty of the procedure, a shortage of available grafts, and the potential for graft failure or rejection. These limitations are driving researchers to develop new, less invasive, and more effective therapies. Corneal endothelial cell therapy is being explored as a potential therapeutic measure, to avoid the uncertainty associated with grafting. The human cornea is an ideal tissue for cell therapy. Due to its avascular and immunologically privileged characteristics, transplanted cells are better tolerated compared with other vascularized tissues and organs. Advances in the field of stem cell engineering, particularly the development of corneal epithelial stem cell therapy for the treatment of severe ocular surface disease, have aroused a massive interest in adapting cell therapy techniques to corneal endothelial cells. This chapter, based on a review of the literature, aims at educating the reader on the latest research in the field of corneal endothelial cell therapy.

[Descemet's Membrane Endothelial Keratoplasty. Indication, surgical technic, postoperative management and review of literature]. / Descemet's Membrane Endothelial Keratoplasty. Indication, technique chirurgicale, gestion postopératoire et revue de la littérature.

Endothelial keratoplasty is currently the preferred method for the treatment of endothelial dysfunctions and dystrophies. Descemet Membrane Endothelial Keratoplasty (DMEK), described by Gerrit Melles in 2006, is performed by selectively replacing the damaged endothelium with a healthy counterpart. It leads to a faster visual recovery and better refractive outcomes with a limited risk of rejection compared to Descemet's Stripping Automated Endothelial Keratosplasty (DSAEK), which includes a thin stromal layer. Open debate still exists between DMEK and DSAEK. This article aims to provide a literature review and enlighten the reader on the DMEK technique, its results and complications.

[Limbal stem cell deficiency management. A review]. / Traitement du déficit en cellules souches limbiques. Revue de la littérature.

Limbal stem cell deficiency is predominantly caused by severe eye burns resulting in a decreased or a complete ablation of the regenerative potential of these stem cells. The inability to reconstruct the corneal epithelium further leads conjunctivalization of the gimbal-epithelial barrier. These abnormalities collectively result in the progressive opacification of the cornea responsible for blindness that is driven by chronic corneal ulceration and neovascularization. The underlying pathology of the cornea affects the homeostasis of the neighboring conjunctiva, eyelids, and tear film. Therefore, the ocular reconstruction to treat limbal stem cell deficiency is quite prolonged and involves a continued treatment plan. The management of limbal stem cell deficiency has undergone a multitude of changes over the past several decades. The understanding of limbal anatomy and physiology, as well as therapeutic advances in the stem cell field have propelled the development of new treatments offering new hope to severely disabled patients. Cultivated limbal epithelial and oral mucosal epithelial transplantations are therefore viable alternatives that could be utilized for the treatment of limbal stem cell deficiency.

A simple way to reconstruct a human 3-d hypodermis: a useful tool for pharmacological functionality.

BACKGROUND: Adipose tissue engineering has been hampered by the inability to culture mature adipocytes. Adipose-derived stem cell (ASC) culture opens the way for the preparation of human 3-D hypodermis in large quantities. These models play a role in obesity-related active molecules and slimming agent screening. Moreover, they contribute to a better understanding of the mechanisms underpinning obesity. MATERIALS AND METHODS: Freshly extracted ASC from fat tissue were characterized by flow cytometry for CD73, CD90, CD105, HLA-ABC, CD14 and CD45 markers and by Western blot for pref-1. Their differentiation in mature adipocytes was followed by lipid and adiponectin secretion or by oil red O staining and radioimmunoassay. Neosynthesized extracellular matrix (ECM) of 3-D hypodermis was investigated by immunohistochemistry (collagen type I, V and VI) and transmission electron microscopy. RESULTS: Our results demonstrate that the culture of preadipocytes in proliferation medium for 15 days followed by 16 days of culture in differentiation medium allowed production of the thickest single-layer hypodermis in which preadipocytes and mature adipocytes coexist and synthesize adiponectin and ECM components. Functionality of our 3-D single-layer hypodermis was demonstrated both by a 3.5-fold glycerol production after its stimulation with norepinephrine (adrenergic agonist) and by its slimming after caffeine treatment versus the nontreated 3-D hypodermis. CONCLUSION: This economic 3-D model, easy to prepare and giving reproducible results after the treatment of actives, is useful for pharmacotoxicological trials as an alternative to animal experimentation.

Adipose-derived stem cells (ASCs) as a source of endothelial cells in the reconstruction of endothelialized skin equivalents.

Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models.

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.

[Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering]. / Matrice poreuse et culture de cellules primaires : un même concept pour la reconstruction cutanée et cornéenne.

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.

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.

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.

Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing.

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

Use of allogenic epidermal sheets for difficult wound healing: selection and testing of relevant growth factors.

The clinical interest of using allogenic epidermal sheets (AES) has largely been shown [1,2,3]. As well as covering, they also stimulate healing, by simultaneously secreting numerous growth factors (GFs), although little is known on their mechanism of action. Our objectives were to: (a) devise a test for the efficacy of AES release, (b) select keratinocyte-secreting strains and optimal culture conditions. Three GFs were selected: IL-1alpha, IL-8 and VEGF. Three different keratinocyte strains were cultured for 3 and 6 days after confluence for 3 passages. Assays were performed after 3 h and 24 h+3 h after dispase treatment (AES conservation for 24 h then change of medium and sampling after 3 h). AES were found to secrete GFs in DMEM and the amounts were greater when cultured for 6 rather than 3 days after confluence. Each strain had different secretory patterns depending on passage and time in culture, this variability being explained by inter-individual heterogeneity.

[Legal framework relating to human tissues used for research ends]. / Droit concernant les tissus humains à finalité de recherche.

Development in cell and tissue engineering needs human tissue samples. If French jurisdiction concerning the human tissue sample collected in a therapeutic goal is well established, the French and European legal context concerning the scientific research is not clear and controversial. In our lab, we aim to conjugate the professional and the moral duty and to impose on our researchers the respect of strictly defined procedures. In order to organize the management of these biological resources, we chose not only to take into account the present legal context concerning the collection of tissues for research purposes, but also to precede the French legal framework by inspiring from good practice, concerning on one hand the conservation, the transformation and the transport of human tissues used to therapeutic ends (decree of December 29, 1998) and on the other hand, from the ethical recommendations of the european directives. It is why, we put some procedures in place to guarantee the donor's information, the staff's security, the confidentiality as well as the tracability.

Lipid peroxidation assessment by malondialdehyde measurement in parenteral nutrition solutions for newborn infants: a pilot study.

AIM: To determine malondialdehyde (MDA) concentrations in parenteral nutrition admixtures exposed to ambient room light, and in the serum of neonates. METHODS: Using a new method to measure MDA specifically, this study analysed MDA of lipid-containing all-in-one admixtures provided by the pharmacy, with a composition identical to that used in routine clinical conditions. First, 12 admixtures were exposed to ambient light for 24 h, in the neonatal intensive care unit. Secondly, 18 solutions were either exposed to (n = 9) or protected from ambient light (n = 9) during the same period. Samples of admixtures were collected at baseline and 24 h later, for MDA measurement. Serum MDA was also randomly measured in orally fed healthy neonates. RESULTS: After 24 h exposure to ambient room light, MDA concentrations in parenteral nutrition admixtures increased from 179 (129, 348) nmol l(-1) to 5800 (1632, 14679) nmol l(-1) (p = 0.0002) [50th (10th, 90th) centiles]. When admixtures were protected from light, the increase in MDA was significantly lower than without protection: 187 (60, 429)nmol l(-1) versus 13 696 (3472, 26 049)nmol l(-1) (p = 0.0003). In 54 infants with a gestational age of 33 (28, 39) wk and a birthweight of 1750 (960, 3388) g, plasma MDA concentrations were 173 (98, 315) nmol l(-1). CONCLUSION: In solutions protected from light, MDA concentrations were low and were close to the serum MDA concentrations observed in orally fed neonates. Administration of all-in-one admixtures containing lipids in ambient lighting results in intravenous infusion of high levels of MDA which may present an additional source of morbidity in immature infants. This study confirms the need to protect parenteral admixtures from light.