Three-dimensional cell cultures (spheroids) of biopsies of human duodenum were used to develop a new noninvasive method for studying intercellular and intracellular mechanisms. Through examinations of intracellular pH regulation, high functional similarity to native tissue could be shown, as already evidenced morphologically. A special microperfusion chamber was developed to fix individual spheroids physically to a nylon net, via laminar perfusion flow through the chamber. A significant improvement over current fixation methods was shown by the increase of cell viability almost up to 100%. Viability of the spheroids was confirmed by trypan blue exclusion, by a LIVE/DEAD viability/cytotoxicity kit, and by BCECF distribution. Intracellular pH was measured by use of the pH-sensitive fluorescence dye BCECF. To investigate the intracellular pH regulation, spheroid-like vesicles were acidified by NH₄Cl prepulse technique. The subsequent active intracellular pH recovery was blocked with Na -free Krebs Henseleit (KH) solution, with amiloride KH (inhibitor of the Na -H -exchanger), or with H₂DIDS KH (inhibitor of the HCO₃⁻-Cl⁻-exchanger and Na -HCO₃⁻-cotransporter). The intracellular pH of the spheroids was 7.31 ± 0.05. pH_i-backregulation after acidification was prevented by sodium-free buffer, amiloride, and H₂DIDS. These experiments indicated the presence of a Na -H -exchanger and a Na -HCO₃⁻-cotransporter. In conclusion, the human duodenal spheroid is an excellent physiological system for in vitro studies of the human duodenum.

A trout (Oncorhynchus mykiss) epidermal skin primary explant system was evaluated over 8 d by light and electron microscopy. Three distinct regions of the explant outgrowth were identified on the basis of cell composition. The area immediately adjacent to the founder tissue contained mainly small migrating cells and mucous cells. Of the former, about 20% were mitotic and 6% apoptotic. The middle area was characterized by differentiated pavement cells and mucous cells, with fewer small migrating cells. Proliferation was approximately 30% and apoptosis 5%. Over time, total cell numbers halved as more pavement cells differentiated. The growing front contained many mucous and small migrating cells initially, with few pavement cells. About 50% of the cells were in the proliferative phase, and 5% were apoptotic. Later, there were fewer migrating and mucous cells, with a higher number of pavement cells. About 9% of the cells were apoptotic, and 70% of the cells were proliferating. As in vivo, pavement cells had apical microridges, although they were vacuolated and contained phagocytosed apoptotic bodies. The data and observations are based on the numbers of cell cultures prepared from separate trout giving the sample size n = 7. As this culture system is reproducible and closely approximates the epidermis of trout, it is a powerful tool to study the effects of pollutants, parasites, and endocrine factors on fish skin, eliminating whole-animal factors and reducing the number of experimental animals required.

A mineralizing rat dental pulp cell subclone, termed M2H4, was selected from single-cell cloning of the rat dental pulp cell line RPC-C2A by screening confluent single-cell cultures for their ability to undergo mineralization. To induce mineralization, confluent single-cell cultures were treated for 8 d with ascorbic acid followed by the addition of inorganic phosphate to a final concentration of 4 mM for an additional 3 d. Confluent M2H4 subclones were shown by immunofluorescence and electron microscopy to form collagen type I fibrils. Furthermore, using reverse transcriptase-polymerase chain reaction, this subclone was found to be capable of expressing dentin sialoprotein–phosphophoryn (DSP–PP) transcripts, an odontoblast-specific marker. Thus, this newly identified mineralizing rat M2H4 subclone possesses odontoblast-like characteristics and can serve as an in vitro model for examining the role of DSP and PP in the formation of mineralized dentin.

The disaccharide trehalose has been shown to inhibit both bone loss in ovariectomized mice and excessive osteoclastogenesis in lipopolysaccharide-injected mice. However, the mechanism of osteoclastogenesis inhibition by oral administration of trehalose is still unclear. We report here for the first time that a human intestinal epithelial cell line, FHs74Int, also produces osteoprotegerin (OPG) and that trehalose augments OPG production by this cell line. Thus, these results suggest that trehalose promotes the production of OPG by intestinal epithelial cells, which then acts on bone marrow cells, resulting in the suppression of osteoclastogenesis.

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1–induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.

Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%), compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 μg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 μg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 μg/ml TIMP-2 was greater (P < 0.05) compared with 0 μg/ml TIMP-2 (16.2 ± 4.8 versus 6.7 ± 1.4 × 10⁻³ IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.

cAMP regulatory element–binding protein (CREB)–binding protein (CBP) and its functional homolog, the adenovirus E1A–associated 300-kDa protein (p300) are nuclear coactivators and histone acetyltransferases that integrate signals from disparate pathways by bridging specific transcription factors to the basal transcription apparatus. Their role in patterning and development was suggested by studies in mice in which CBP and p300 expression was disrupted and by the human Rubinstein–Taybi syndrome, which is associated with mutations of CBP. The cAMP signal transduction pathway plays a critical role during development of the palate. The linkage between cAMP and expression of specific genes is mediated via activation of trans-acting deoxyribonucleic acid–binding proteins such as the nuclear CREB. For genes regulated by CBP- or p300-containing transcriptional complexes, rates of transcription will depend in part on cellular levels and distribution of CBP/p300. We have thus determined the temporal and spatial expression of CBP and p300 in murine embryonic palatal tissue. Both CBP and p300 proteins and messenger ribonucleic acids are expressed in palatal tissue on each d of palate development (days 12–14 of gestation), as measured by Western blotting and reverse-transcription polymerase chain reaction. Expression of both CBP and p300 was greatest on day 12 of gestation, suggesting that these transcriptional coactivators are developmentally regulated. Immunohistochemical analysis of CBP and p300 expression in the murine embryonic craniofacial region revealed a ubiquitous distribution for both proteins. These studies lay the groundwork for further investigations into the role of CBP and p300 in cellular signaling during craniofacial development.

The cyclic adenosine monophosphate (cAMP) generator choleratoxin is known to promote the growth of sebaceous epithelial cells (sebocytes) in monolayer culture in classical serum-containing media. Now that sebocytes can be grown in serum-free medium, we have examined whether choleratoxin or other cAMP generators are required for differentiation of rat preputial sebocytes in response to specific ligand activators of peroxisome proliferator–activated receptors (PPARs). Unexpectedly, choleratoxin reduced sebocyte proliferation. However, sebocyte differentiation in response to specific PPARα and PPARγ agonists required a cAMP generator such as choleratoxin, and this response was suppressed by a protein kinase A inhibitor. In contrast, the stable prostacyclin analog, carbaprostacyclin (cPGI2), a PPARα,δ agonist that also generates cAMP, stimulated differentiation independently of choleratoxin. Furthermore, unlike the selective PPARα and PPARγ agonists, cPGI2 stimulated both sebocyte DNA synthesis and proliferation. These data are compatible with the evidence that prostacyclin has the additional effect of generating cAMP. In addition, we addressed the possibility that choleratoxin may act as a surrogate for β-adrenergic catecholamines in generating cAMP. In contrast with choleratoxin, both α- and β-adrenergic catecholamines stimulated sebocyte growth and interfered with the choleratoxin effect on differentiation. These data suggest ligand-dependent, complex interactions between cAMP and the other signal transduction pathways involved in sebocyte growth and development.