Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

Although the trace elements zinc, copper and manganese are used in vivo for their healing properties, their mechanism of action is still only partially known. Some integrins expressed by basal layer keratinocytes play an essential part in healing, notably alpha2beta1, alpha3beta1, alpha6beta4 and alphaVbeta5, whose expression and distribution in epidermis are modified during the re-epithelialization phase. This study demonstrates how the expression of these integrins are modulated in vitro by trace elements. Integrin expression was studied in proliferating keratinocytes in monolayer cultures and in reconstituted skin that included a differentiation state. After 48 h incubation with zinc gluconate (0.9, 1.8 and 3.6 microg/mL), copper gluconate (1, 2 and 4 microg/mL), manganese gluconate (0.5, 1 and 2 microg/mL) and control medium, integrin expression was evaluated by FACScan and immunohistochemistry. Induction of alpha2, alpha3, alphaV and alpha6 was produced by zinc gluconate 1.8 microg/mL in monolayers, of alpha2, alpha6 and beta1 by copper gluconate 2 and 4 microg/mL and of all the integrins studied except alpha3 by manganese gluconate 1 microg/mL. Thus, alpha6 expression was induced by all three trace elements. The inductive effect of zinc was particularly notable on integrins affecting cellular mobility in the proliferation phase of wound healing (alpha3, alpha6, alphaV) and that of copper on integrins expressed by suprabasally differentiated keratinocytes during the final healing phase (alpha2, beta1 and alpha6), while manganese had a mixed effect.

In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese.

Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. "Air-liquid interface" cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a "permissive switch" that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.

/pdf/engineering-of-three-dimensional-microenvironments-to-1et9vdnsya.pdf

Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Prunieras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm2 x 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.

Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

Recently a change to a low-salt mineral water has been associated with an improvement in atopic dermatitis [2]. In a similar manner, balneotherapy using spa water has long been known as an effective approach to the management of inflammatory skin diseases such as psoriasis and atopic dermatitis. However, only a limited number of studies have addressed the mechanism(s) responsible for the overall satisfactory clinical results which so far have been only empirical in nature [ 11 ]. Trace elements such as selenium and strontium, which are sometimes found in high concentrations in spa waters, may play a significant role in the observed effects. Using a reconstituted skin model, we studied the in vitro modulatory effect of these two trace elements on the production of cutaneous inflammatory cytokines (IL-lo~, TNFcz, IL6) and compared the results with those obtained with a seleniumand strontiumrich spa water known to be particularly effective in the management of inflammatory skin diseases. Samples of inflammatory skin and normal skin were obtained from biopsies of atopic dermatitis lesions and during plastic surgery, respectively. Skin was reconstituted according to the method of Prunieras [8] as modified by Basset-Seguin et al. [3]. After deepidermized dermis (DED) had been obtained, a 2-mm punch biopsy specimen of atopic dermatitis or normal skin was placed on top of each specimen of DED, which was then maintained at the air-liquid interphase with a metallic support in a 60-

Modulatory effects of selenium and strontium salts on keratinocyte-derived inflammatory cytokines.

Reconstituted skin in culture: a simple method with optimal differentiation.

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Reconstituted skin in culture: a simple method with optimal differentiation.