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Notes: Skin firmness, elasticity and tone are gradually lost with age. These changes originate in the dermis and correspond to a decrease in the ability of cells, particularly the fibroblasts, to regenerate the molecules which make up the extracellular matrix. Skin ageing is also characterized by a reduction of the epidermal thickness and by a flattening of the basal membrane. The recent development of two 3-dimensional culture systems, in which the cells develop within a porous structure reproducing the extracellular matrix of the human dermis, is a way of reproducing in vivo conditions and demonstrating the biological effects of anti-ageing compounds. The dermal equivalent model used in this study is composed of a dermal matrix made of collagen–chitosan–glycosaminoglycans populated by normal human fibroblasts which synthesized their own extracellular matrix. A skin equivalent model is obtained by the cell culture of normal human keratinocytes onto a dermal equivalent elevated at the air–liquid interface. Such models were used to prove anti-ageing activity of promising compounds. Cosmetic Science has used many protein hydrolysates in order to fight skin ageing, but up to now, these natural peptides were poorly studied, and their efficacy poorly demonstrated. Eight protein hydrolysates were screened in a proliferation study in monolayered cultures giving two selected polypeptides. A soya derived peptide was used for an efficiency study in 3-dimensional models. In the dermal equivalent model, this peptide increased fibroblast proliferation by 40% and led to a stimulation of collagen formation (165%) and elastin (116%) synthesis. The effect of this soya peptide on glycosaminoglycan synthesis was also significant, with increases of 36% for chondroitin-4-sulfate and 68% for hyaluronic acid. These results were confirmed using a skin equivalent model. In this model, the soya peptide increased the thickness of the epidermis.〈section xml:id="abs1-2"〉〈title type="main"〉RésuméLa fermeté, l'élasticité et le tonus de la peau diminuent progressivement avec l'âge. Ces transformations visibles ont leur origine dans le derme et correspondent à une diminution du renouvellement, par les fibroblastes, des molécules qui composent la matrice extracellulaire. Le vieillissement se manifeste également par une diminution de l'épaisseur de l'épiderme ainsi que par un affaissement de la membrane basale. Le développement récent des systèmes de culture tridimensionnels, dans lesquels les cellules se développent à l'intérieur d'une structure poreuse reproduisant la matrice extracellulaire du derme humain, est un moyen de mimer les conditions naturelles et de démontrer les effets biologiques des actifs anti-âge. Le modèle de derme équivalent utilisé ici est composé d'un substrat dermique collagène–chitosane–glycosaminoglycannes au sein duquel se développent des fibroblastes humains normaux, néosynthétisant leur matrice extracellulaire. Le modèle de peau reconstruite est obtenu en cultivant des kératinocytes sur un derme équivalent élevéà l'interface air–liquide. Ces deux modèles ont été utilisés pour prouver l'efficacité anti-âge d'actifs prometteurs. En effet, la cosmétique utilise de nombreux hydrolysats protéiques pour lutter contre le vieillissement de la peau, mais jusqu'à présent, ces peptides naturels étaient peu étudiés et leur efficacité rarement démontrée. Huit hydrolysats protéiques ont étéévalués dans un test de prolifération en monocouche et deux actifs prometteurs ont été sélectionnés. L'efficacité de l'un de ces actifs, dérivé du soja, a ensuite étéévaluée en modèles tridimensionnels. Dans le modèle de derme équivalent, ce peptide augmente la prolifération fibroblastique de 40%, stimule la synthèse des collagènes et d'élastine respectivement de 65% et 16% mais également celle des glycosaminoglycannes, respectivement 36% pour les chondroitines sulfate et 68% pour l'acide hyaluronique. Ces résultats ont été confirmés dans le modèle de peau reconstruite oò le peptide de soja accroît également l'épaisseur de l'épiderme. © Rapid Science Ltd. 1998

Notes: Calcifications found in the coronal pulps of primary teeth extracted in an 8-year-old child were studied by TEM. Different types of relationship were observed between fibroblasts and pulp calcifications: extension of cell processes towards calcifications, modelling of the cells upon calcifications, internalizing process of calcifications. Fibroblasts proved to be able to enclose small pulp calcifications within intracytoplasmic vesicles. There was no evidence of any active role played by fibroblasts in the genesis of pulp calcifications. It was shown that collagen fibres could be involved or not in the mineralizing process. It is suggested that mitochondria might provide an adequate environment for initial mineralization. It is likely that the role played by the cytoskeleton in the internalizing process of calcifications is important.

Notes: Abstract. Rapid developments in tissue engineering have renewed interest in biodegradable three-dimensional structures such as collagen-based biomaterials. Collagen matrices seeded in vitro with fibroblasts, osteoblasts, and chondrocytes can form tissues resembling skin, bone, and cartilage that could be used as functional substitutes for damaged tissues. Collagen is associated with both dystrophic calcification of collagenous implants and bone mineralization. We report here the calcification properties of collagen sponges incubated in cell-free media. Mineral deposited in sponges was identified by X-ray and electron diffraction, Fourier transform infrared spectroscopy, and the molar ratio of calcium:phosphorus (Ca:P) as a poorly crystalline apatite similar to bone. The degree of calcification increased with length of incubation and the Ca and P content of the media, with 10–15% Ca (dry weight) after 21 days' incubation in media containing 1.6–3 mM Ca and a Ca × P molar product of 2–3 mM2, but only 2% Ca after incubation in medium with 1.33 mM Ca and a 1.7 mM2 Ca × P molar product. Mineral deposition was completely inhibited in sponges that were washed extensively and initially contained less than 0.01% P. Readdition of phosphate in these sponges and subsequent freeze drying and sterilization restore their mineralization capacity, suggesting that collagen per se cannot initiate calcification and that the inorganic phosphate content associated with the collagen preparation process is in the solid state a potential nucleator. Addition of chondroitin 4-sulfate to the sponges partially or totally inhibited mineral deposition, even though 80–90% of the compound was released within 24 hours. These results indicate that acellular calcification of collagen-based biomaterials can occur under the culture conditions currently used in tissue engineering.

Notes: Gingival fibroblasts were cultured with four different calcium phosphate minerals (hydroxyapatite, whitlockite, b̃-tricalcium phosphate, and octocalcium phosphate). 3H-thymidine incorporation into DNA and alkaline phosphatase specific activity were determined after different incubation periods. As a consequence of the phagocytosis of calcium phosphates crystals, we pointed out, compared to control, a stimulation of the rate of 3H-thymidine incorporation and sharp decreases in alkaline phosphatase activity. The magnitude of the alkaline phosphatase activity inhibition was observed to be increased with the solubility of the materials. We propose that the effects of calcium phosphates on alkaline phosphatase and 3H-thymidine incorporation could be calcium-mediated events, resulting from intracellular dissolution of phagocytized materials. We suggest that in vitro determination of 3H-thymidine incorporation and alkaline phosphatase activity, which are highly sensitive tests, could be involved in evaluation procedures of calcium phosphates biomaterials.

Notes: An in vitro method is described to assess the influence of synthetic calcium phosphate powders on osteoblast activities. Human osteoblast cell cultures were established from iliac crest. MC3T3-E1, an established osteogenic cell line, was employed as a control. Scanning and transmission electron microscopic observations clearly demonstrated the internalization of particles of calcium phosphate by the two osteoblast cell populations. As a consequence to the phagocytotic process, RNA transcription and protein synthesis were stimulated, as indicated by the measurements of labeled uridine, leucine and proline uptakes. From these data, it is proposed that such an in vitro model, using one of the specific cell types involved in the tissue responses to implants, could be useful to assess the biological response at the cell-biomaterial interaction.