Impact of 3D-culture on the expression of differentiation markers and hormone receptors in growth plate chondrocytes as compared to articular chondrocytes.

In this study we compared the expression of hormone receptors and markers of differentiation in growth plate chondrocytes (GPC) and articular chondrocytes (AC) under different culture conditions by real-time PCR and immunofluorescence. After one week of monolayer culture, porcine GPC and AC of 6-8-week-old piglets were either seeded in alginate beads or further grown in monolayer culture up to 5 weeks. During monolayer culture the expression of Col2, Col1, Col10, aggrecan, ERalpha and ERbeta decreased dramatically, whereas culture in alginate beads restored an expression pattern similar to native tissue. The receptors IGF-1R, IGF-2R and GHR were, however, increased in monolayer culture compared to alginate culture or native tissue. The relative proportion of ERalpha and ERbeta expression was different in GPC when compared to AC. All of our results on mRNA expression during culture in alginate beads were also verified to be translated to the protein level. Thus, the expression of hormone rereptors and differentiation markers were found to be highly influenced by culture conditions. Moreover, the porcine model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research.

Macroscopic and histological investigation of guanaco footpads (Lama guanicoe, Müller 1776).

The surface of guanaco footpads is characterized by hairless skin with up to 4-mm-thick stratum corneum that protects from abrasion. The horny layer is pliable and elastic, and ensures firm contact with irregular ground. It is padded with a particular structure of the subcutaneous layer, the digital cushion. The flat cushions of each of the two digits are of elongated ovate shape, each about 45-mm long, up to 20-mm wide, and 8-mm thick. The cushions are lined by a 1-2-mm-thick capsule that resembles a tunica albuginea. The capsule consists of coarse collagen fibers, with elastic fibers absent. The cushion capsule and dermis approach each other, and fuse along a line that runs parallel to the longitudinal axes of cushion and digit. Loose connective tissue rich in elastic fibers and acidic glycosaminoglycans separates dermis and cushion capsule lateral to the narrow interconnecting zone. The cushion capsule encloses cloudy yellowish, gelatinous material. Microscopy shows bundles of elastic fibers in abundant mucinous matrix. Tightly gathered elastic bundles adjoin the inner surface of the capsule. Rough cords of elastic fibers branch out from there and traverse to the opposite side. The cushion is pressed flat, and elastic fibers are stretched when bearing weight. With relief of load, elastic fibers contract and reset the cushion's shape. Contractile cells are absent. A resistant capsule and easily malleable mucinous contents establish the functioning as a gel pad. Mucinous connective tissue between elastic fiber bundles contains abundant basophilic matrix. Hyaluronan, chondroitin sulfate, and dermatan sulfate are main matrix constituents. Spindle-shaped or stellate fibroblasts contain vimentin, S100 protein, and neuron specific enolase. Moprhology, staining characteristics and synthesis activities of these cells meet the criteria to be classified as myxoid cells. The connective tissue in guanaco digital cushions represents myxoid tissue.

Distribution of actin and myosin in the cochlear microvascular bed.

OBJECTIVE: Previous studies of the microvascular bed of the rat inner ear showed vascular constriction after i.v. application of endothelin-1 (ET-1). Luminal narrowing together with multiple circumscribed constrictions were observed on vascular corrosion casts of initial and small calibre veins. These constrictions were interpreted as being caused by contractile cytoplasmic fibrils, most probably of pericytes; pericytes reportedly respond to ET-1 and the frequency, distribution and dimension of pericytes and their cytoplasmic processes closely corresponded to the constrictions observed. In the present study we analysed the distribution of actin and myosin in order to directly show the presence of contractile cytoplasmic fibrils. MATERIAL AND METHODS: We performed standard immunostaining for actin, smooth muscle actin, smooth muscle myosin and tropomyosin in the cochlea. We used different fixation protocols (methacarn, neutral formalin, Bouin's fluid) and compared observations in two species (rat and guinea pig). RESULTS: Immunohistochemistry confirmed the presence of contractile cytoplasmic fibrils in cochlear pericytes and vascular smooth muscle cells. Microvessels in the cochlea were much better provided with contractile fibrils in rats compared to guinea pigs. The distribution of contractile fibrils in rats corresponded well to the luminal constrictions observed on vascular corrosion casts. CONCLUSIONS: Our results support the assumption that active myofibrillar contraction (in response to ET-1 stimulation of pericytes) causes luminal constriction in cochlear microvessels. Contraction of myofibrils can be influenced by intrinsic or extrinsic agents, which offers new therapeutic regimens to govern cochlear blood flow. As the frequency of contractile cells on cochlear microvessels varied with the species studied, evaluation of human material will be the next step.

IGF expression patterns and regulation in growth plate chondrocytes.

IGF-I and IGF-II are key regulators of growth and metabolism. Still, data about their expression and distribution within the growth plate in different animal models remain contradictory. Inferences drawn from rodent animal models can only be applied to human conditions to a limited extent as the rodent's growth plate never fuses. In this study, we compared the expression of IGF-I and IGF-II in native growth plates of prepubertal piglets and under different cell culture conditions. We detected IGF-I mRNA expression and abundantly expressed IGF-II within the growth plate. IGF-I expression increased during monolayer cell culture while IGF-II expression dramatically decreased. Our studies revealed that these expression patterns remained unaffected by growth hormone stimulation in vitro. The abundant expression of IGF-II in porcine growth plate tissue, both on the mRNA and on the protein level, suggests that IGF-II also has a role in growth regulation at the early postnatal stage.