How biophysical in vivo testing techniques can be used to characterize full thickness skin equivalents.

BACKGROUND: The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products. METHODS: We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time. RESULTS: High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation. CONCLUSION: Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties.

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.

Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium.

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(®)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(®)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(®)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(®)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(®)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.

Model of in vitro healing to test the influence of dedifferentiated Crithmum maritimum cells on dermal repair and epidermal regeneration.

BACKGROUND: The aim was to test the influence of dedifferentiated Crithmum maritimum cells (dCMC), totipotent vegetal stem cells, on epidermal regeneration in perfect homeostasis using a skin equivalent (SE) model. MATERIALS AND METHODS: SE are prepared by seeding fibroblasts on a collagen-glycosaminoglycan-chitosan dermal substrate (DS) epidermalized by keratinocytes 3 weeks later. The originality of this present study lies in the systemic administration of dCMC from the moment when fibroblasts are seeded in the DS right through to the reconstruction of the SE. The thickness of the epidermis as well as the number of proliferating cells expressing Ki-67 and layers expressing terminal differentiation marker (filaggrin) were compared in the dCMC-treated SE versus an untreated control group. RESULTS: dCMC accelerated the complete regeneration and differentiation of the epidermis compared to the negative control (35 days instead of 42 days). Histology showed a multilayered, thick and differentiated epithelium after 35 days of culture. The basal and suprabasal layers had increased 4.88 ± 0.41 times versus the negative control (Mann-Whitney U test: p < 0.001). This result was attributed to the greater proliferation of basal cells because the cell numbers expressing the Ki-67 proliferation marker had increased significantly compared to the negative control (Mann-Whitney U test: p < 0.001). Moreover, dCMC allowed the differentiated epithelium to recover because only treated SE expressed the terminal differentiation marker filaggrin. CONCLUSION: Our data show that dCMC enhance epidermal cell grafts by stimulating their regeneration and differentiation in perfect homeostasis. They allow the epidermis to recover its structure for protective functions faster than the negative control.

Confocal Raman microspectroscopy for evaluating the stratum corneum removal by 3 standard methods.

BACKGROUND/AIMS: Stratum corneum (SC) removal is needed in biopharmaceutical studies or in evaluating the barrier function. The most common technique is the tape stripping method. However, it results in neither a homogeneous nor a complete removal. METHODS: The removal qualities of tape stripping, cyanoacrylate skin surface biopsy and trypsinization were estimated in vitro via histological imaging and confocal Raman microspectroscopy (CRM) and compared. In addition, the potential of the noninvasive CRM as a replacement method is discussed. RESULTS: Comparison between the 3 methods showed, as expected, that the tape stripping method did not result in a uniform removal over the whole surface even after 28 strips. The trypsinization and cyanoacrylate skin surface biopsies allowed a complete and uniform removal of the SC after defining a standard protocol (2 cyanoacrylate strips with a polymerization time of 15 min). CONCLUSION: The feasibility of CRM to control SC removal was demonstrated in vitro. Tape stripping is a simple method, but it is influenced by many extrinsic factors and axial drug quantification is difficult. With trypsinization and cyanoacrylate methods, the entire SC is removed so that quantification over the whole SC is possible but not an axial drug screening.

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures.

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self-repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)-2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long-term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real-time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP-2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP-2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP-2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT.

[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.

[Human chondrocyte responsiveness to bone morphogenetic protein-2 after their in vitro dedifferentiation: potential use of bone morphogenetic protein-2 for cartilage cell therapy]. / Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage.

AIM OF THE STUDY: Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS: Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS: Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION: The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.

Contact guidance enhances the quality of a tissue engineered corneal stroma.

Corneal stroma is a very complex structure, composed of 200 lamellae of oriented collagen fibers. This highly complex nature of cornea is known to be important for its transparency and mechanical integrity. Thus, an artificial cornea design has to take into account this complex structure. In this study, behavior of human corneal keratocytes on collagen films patterned with parallel channels was investigated. Keratocytes proliferated well on films and reached confluency after 7 days in the incubation medium. Nearly all of the cells responded to the patterns and were aligned in contrast to the cells on unpatterned surfaces. Collagen type I and keratan sulfate secreted by keratocytes on patterned films appeared to be aligned in the direction of the patterns. The films showed an intermediate degradation over the course of a month. On the whole, transparency of the films increased with degradation and decreased by the presence of the cells. The decrease was, however, low and transparency level was maintained on the patterned films while on the unpatterned films a sharp decrease in transparency was followed by an improvement. This was due to the more organized distribution of cells and the oriented secretion of extracellular matrix molecules on patterned collagen films. Thus, these results suggest that application of contact guidance in cornea tissue engineering may facilitate the remodeling process, hence decrease the rehabilitation period.

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.

[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.

[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.

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.

[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.

Radioimmunofixation of human ferritin following serum isoelectric focusing.

Radioimmunofixation of human ferritin following isoelectric focusing of serum was developed to study the microheterogeneity of this protein in native serum without previous purification or concentration. This method requires only 2-10 microliter of serum and can be used with levels of ferritin as low as 10 micrograms/l. In this way, the extensive microheterogeneity of this protein was revealed, since in some cases it produced as many as 35 bands with isoelectric points in a pH range of 4.95-5.9. Very different isoelectric focusing patterns (spectrotypes) of ferritin were observed during the investigation of pathological sera. The high sensitivity of this technique makes it useful for the investigation of serum ferritin in diseases involving modifications of the metabolism of this protein.

Pathways commonly dysregulated in mouse and human obese adipose tissue: FAT/CD36 modulates differentiation and lipogenesis.

Obesity is linked to adipose tissue hypertrophy (increased adipocyte cell size) and hyperplasia (increased cell number). Comparative analyses of gene datasets allowed us to identify 1426 genes which may represent common adipose phenotype in humans and mice. Among them we identified several adipocyte-specific genes dysregulated in obese adipose tissue, involved in either fatty acid storage (acyl CoA synthase ACSL1, hormone-sensitive lipase LIPE, aquaporin 7 AQP7, perilipin PLIN) or cell adhesion (fibronectin FN1, collagens COL1A1, COL1A3, metalloprotein MMP9, or both (scavenger receptor FAT/CD36). Using real-time analysis of cell surface occupancy on xCELLigence system we developed a new method to study lipid uptake and differentiation of mouse 3T3L1 fibroblasts and human adipose stem cells. Both processes are regulated by insulin and fatty acids such as oleic acid. We showed that fatty acid addition to culture media increased the differentiation rate and was required for full differentiation into unilocular adipocytes. Significant activation of lipogenesis, i.e. lipid accumulation, by either insulin or oleic acid was monitored in times ranging from 1 to 24 h, depending on differentiation state, whereas significant effects on adipogenesis, i.e., surperimposed lipid accumulation and gene transcriptional regulations were measured after 3 to 4 d. Combination of selected times for analysis of lipid contents, cell counts, size fractionations, and gene transcriptional regulations showed that FAT/CD36 specific inhibitor AP5258 significantly increased cell survival of oleic acid-treated mouse and human adipocytes, and partially restored the transcriptional response to oleic acid in the presence of insulin through JNK pathway. Taken together, these data open new perspectives to study the molecular mechanisms commonly dysregulated in mouse and human obesity at the level of lipogenesis linked to hypertrophy and adipogenesis linked to hyperplasia.

Regulatory effects of heat on normal human melanocyte growth and melanogenesis: comparative study with UVB.

Although energy-rich ultraviolet B (UVB) is considered to be primarily responsible for most of the effects associated with solar radiation, small energy recorded as heat appears to contribute to the biologic effects of solar radiation on the skin. We compared the effects of heat and UVB on normal human melanocyte functions. In monolayer culture the following was found. (i) Heat-treated melanocytes showed an increased dendricity and exhibited a larger cell body compared with nontreated melanocytes. (ii) After multiple treatments with UVB (20 mJ per cm2, 312 nm) or heat (42 degrees C for 1 h) for 3 d, melanocytes had a lower survival than nontreated melanocytes, but they resumed proliferation within 6 d in the same manner as seen in control. (iii) The expression levels of cell cycle regulators, p53 and p21 proteins, were increased after multiple treatments with UVB or heat. (iv) The tyrosinase (dopa-oxidase) activity per cell was increased after the multiple treatments with UVB or heat. (v) The number of dopa-positive melanocytes in coculture with keratinocytes in epithelial sheets was greatly increased by UVB or heat treatments. (vi) Similarly, the increased number of tyrosinase-related protein 1 positive melanocytes was seen in skin equivalents after UVB (100 mJ per cm2) or heat (42 degrees C for 1 h) treatments for 7 d. These results suggest that heat shares significant biologic activities with UVB in melanocyte functions. These results could be considered as one of the protective or adaptive responses of the skin pigmentary system to the environment.

Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent.

Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.