Opioid receptor delta as a global modulator of skin differentiation and barrier function repair.

OBJECTIVES: The aims of this study were to confirm the properties of selective agonist peptide (Rubixyl) contained in the spinach towards opioid receptor delta. In fact, agonist properties of both spinach peptides (Rubiscolin-5 and Rubixyl) towards opioid receptor delta were demonstrated by Zang et al., but their effects on the other opioid receptors were not studied [1]. We also studied the expression of opioid receptor delta in epidermis under normal and stress condition (inflammatory) and its role in epidermis homeostasis under stress condition in vitro and in vivo. METHODS: Agonist properties studies were performed using functional agonist cellular model containing human opioid receptors. Opioid receptor delta expression and epidermis homeostasis were studied on human reconstructed epidermis under normal and stress conditions (inflammatory stress) using gene expression (RT-qPCR) and protein expression analysis (immunohistological analysis). Skin repair properties of opioid receptor delta agonist were based on the following parameters TEWL (trans epidermal water loss, hydration and wrinkle depth at periocular and perilabial area) on human volunteers having either intrinsic ageing (more than 40 years old and non-smoker group) and both intrinsic ageing and extrinsic ageing (more than 40 years old and smoker group). RESULTS: We have demonstrated that the Rubixyl peptide is a specific agonist of opioid receptor delta. We have demonstrated that opioid receptor delta expression is modulated under inflammatory condition. The agonist Rubixyl was able to block the depletion of opioid receptor delta seen under inflammatory condition in reconstructed human epidermis. Inflammatory conditions lead to the unbalanced gene and protein expressions of markers involved in epidermis integrity and barrier function properties. The treatment of human reconstructed epidermis with the agonist Rubixyl leads to the normalization of unbalanced gene and protein expressions. In vivo study has confirmed the efficiency of the agonist Rubixyl to repair damaged skin by decreasing TEWL, increasing hydration and decreasing wrinkle depth at the periocular and perilabial area. CONCLUSION: In this research, we have demonstrated in vitro (on inflamed reconstructed human epidermis, RHE) and in vivo (on human aged volunteers) that activation by natural agonist peptide of opioid receptor delta reduces the skin inflammation thus leading to improvement in epidermis differentiation and skin barrier properties.

Collagen-based biomaterials and cartilage engineering. Application to osteochondral defects.

Articular cartilage has a limited capacity for self-repair after trauma. Besides the conventional surgical techniques for repairing such defects, treatments involve implantation of autologous cells in suspension or within a variety of cell carrying scaffolds such as hyaluronic acid, alginate, agarose/alginate, fibrin or collagen. For the repair of full-thickness osteochondral defects, tissue engineers started to design single- or bi-phased scaffold constructs often containing hydroxyapatite-collagen composites, usually used as a bone substitute. The purpose of this study was to compare the behavior of bovine chondrocytes cultured in collagen-based scaffolds containing or not hydroxyapatite and cross-linked following two different methods. Calf chondrocytes seeded within Hemotèse and Collapat II sponges (SYMATESE biomaterials), chemically cross-linked with glutaraldehyde or EDC/NHS, were maintained up to one month in culture. The cells exhibited a similar behavior in the four scaffolds regarding proliferation level, deposition of glycosaminoglycans in the scaffolds and gene expression of types I, II and X collagens, aggrecan, MMP-1, -13 and the integrin subunits alpha10 and alpha11.

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges.

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.