Growth and proteoglycan metabolism of chick embryonic cartilaginous long bone rudiments and of isolated epiphyses

Summary Turnover of extracellular matrix (ECM) proteoglycans was studied in chick cartilaginous femur rudiments grown in organ culture. Femora from six-day-old embryos showed nearly normal growth rates during the first few days in culture. By labeling the rudiment with ³⁵S-sulfate or ¹⁴C-glucosamine, it was demonstrated that the cartilaginous ECM undergoes rapid turnover. It was also found that the metabolic fate of the proteoglycans is to be released as macromolecules into the culture medium. When a rudiment was cut to obtain two epiphyses it was observed that each part grows and synthesizes proteoglycans at nearly normal rates, which indicates that the isolated epiphyses, like the whole rudiment, behave as autonomous systems. We suggest that the turnover of ECM components is part of the continuous remodelling process rudiments undergo during their growth and development. In order to study cell-ECM interaction in morphogenesis, we made an attempt to prepare an intact cell-free ECM. Epiphyses were heated at 45.2° C for 1 h. The treatment caused complete cessation of growth and biosynthesis. When the cut surface of a live epiphysis was brought into apposition to a heat-treated epiphysis and the attached pair placed in organ culture, it was found that the heat-treated epiphysis begins to grow and reaches almost the same size as its live counterpart. We discuss the possible advantage of this new experimental system for studies on the role of ECM in morphogenesis.     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures

Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line. ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index. Cells on ECM were heterogeneous in size and insulin content. They showed extended areas of confluence. Cultures on plastic demonstrated an organisation in clusters and low mitotic activity. However, ECM did not allow for reconstitution of an islet-like structure. When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture. Glucagon and somatostatin secretion were similar on both substrates. These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.     

The anomalous archaic Homo femur from Berg Aukas, Namibia: a biomechanical assessment.

The probably Middle Pleistocene human femur from Berg Aukas, Namibia, when oriented anatomically and analyzed biomechanically, presents an unusual combination of morphological features compared to other Pleistocene Homo femora. Its midshaft diaphyseal shape is similar to most other archaic Homo, but its subtrochanteric shape aligns it most closely with earlier equatorial Homo femora. It has an unusually low neck shaft angle. Its relative femoral head size is matched only by Neandertals with stocky hyperarctic body proportions. Its diaphyseal robusticity is modest for a Neandertal, but reasonable compared to equatorial archaic Homo femora. Its gluteal tuberosity is relatively small. Given its derivation from a warm climatic region, it is best interpreted as having had relatively linear body proportions (affecting proximal diaphyseal proportions, shaft robusticity, and gluteal tuberosity size) combined with an elevated level of lower limb loading during development (affecting femoral head size and neck shaft angle).     

Behavioural evidence for visual recognition of predators by the mangrove climbing crab Sesarma leptosoma

The climbing crab Sesarma leptosoma colonizes the mangrove roots and canopy of East African mangrove swamps, an intricate three-dimensional habitat in which it orients itself visually. To ascertain if vision helps this tree crab to detect dangers such as predators, we used dummy objects: (1) a preserved specimen of its predator, the crab Epixanthus dentatus in its typical ambush posture; (2) a piece of wood with real E. dentatus claws attached to it, the same size as, and painted to resemble (to the human eye), this predator; and (3) a piece of wood the same size and colour as a live crab but without claws. When these dummies were presented to migrating S. leptosoma in the field, they stopped their normal migratory flow only when they were able to see the open claws of the predator. Thus S. leptosoma showed a considerable ability to perceive shape, being able to distinguish motionless objects of different shapes but similar size and to associate the detected shapes with the presence of danger.     

Blood vessels of the epiphysis in comparative-anatomical aspect]

The structure of the epiphysis and its inner blood vessels were studied in the representatives of nine orders of placental mammals and in man by means of injection of stained masses into the arteries and veins and subsequent preparation of histological sections. Not only form and topography of the organ differ in the representatives of different orders, but histological picture of the epiphysis is specific for each of them. In insectivores and chiroptera the loops of the inner three-dimensional capillary network are stretched along the longitudinal axis of the organ. In the epiphysis of carnivores, ungulata and monkey, the intraorganic vessels are situated in stromal trabeculae and the loops of the capillary network have polygonal shape. The intraepiphyseal vessels in man are arranged in peculiar baskets which envelope parenchymal lobules. The intraorganic veins beginning from the loops of the capillary network do not follow the arteries penetrating into the organ, but independently go to different surface parts of the organ where they flow into extraorganic veins.     

Effects of Feed Supplementation on Mineral Composition,Mechanical Properties and Structure in Femurs of Iberian Red Deer Hinds (Cervus elaphus hispanicus)

Few studies in wild animals have assessed changes in mineral profile in long bones and their implications for mechanical properties. We examined the effect of two diets differing in mineral content on the composition and mechanical properties of femora from two groups each with 13 free-ranging red deer hinds. Contents of Ca, P, Mg, K, Na, S, Cu, Fe, Mn, Se, Zn, B and Sr, Young’s modulus of elasticity (E), bending strength and work of fracture were assessed in the proximal part of the diaphysis (PD) and the mid-diaphysis (MD). Whole body measures were also recorded on the hinds. Compared to animals on control diets, those on supplemented diets increased live weight by 6.5 kg and their kidney fat index (KFI), but not carcass weight, body or organ size, femur size or cortical thickness. Supplemental feeding increased Mn content of bone by 23%, Cu by 9% and Zn by 6%. These differences showed a mean fourfold greater content of these minerals in supplemental diet, whereas femora did not reflect a 5.4 times greater content of major minerals (Na and P) in the diet. Lower content of B and Sr in supplemented diet also reduced femur B by 14% and Sr by 5%. There was a subtle effect of diet only on E and none on other mechanical properties. Thus, greater availability of microminerals but not major minerals in the diet is reflected in bone composition even before marked body effects, bone macro-structure or its mechanical properties are affected.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Tissue elongation requires oscillating contractions of a basal actomyosin network

Understanding how molecular dynamics leads to cellular behaviours that ultimately sculpt organs and tissues is a major challenge not only in basic developmental biology but also in tissue engineering and regenerative medicine. Here we use live imaging to show that the basal surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or their coupling to extracellular matrix (ECM) blocked elongation of the whole tissue, whereas enhancement of the contractions exaggerated it. Myosin accumulated in a periodic manner before each contraction and was regulated by the small GTPase Rho, its downstream kinase, ROCK, and cytosolic calcium. Disrupting the link between the actin cytoskeleton and the ECM decreased the amplitude and period of the contractions, whereas enhancing cell-ECM adhesion increased them. In contrast, disrupting cell-cell adhesions resulted in loss of the actin network. Our findings reveal a mechanism controlling organ shape and an experimental model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet.     

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.     

In vitro effect of KCA-098, a derivative of coumestrol,on bone resorption of fetal rat femurs

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoline-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of ⁴⁵Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of ⁴⁵Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.     

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

Development of rat neuromuscular junctions in organ culture

Pieces of thoracic body wall, including intercostal muscles, ribs, and the spinal cord were explanted from 15 to 18-day embryonic rats and maintained in organ culture for up to 6 days. During the time in culture muscle fibers continued to increase in size, and nerve sprouts extended along the center of the muscle. When muscle-spinal cord explants were cultured at 15 days gestation, the number of synaptic inputs per fiber increased with time in culture. Subsequently synapse elimination began with a time course similar to that recorded in vivo. In 15-day explants acetylcholine receptors were uniformly distributed along the fibers and focal cholinesterase (ChE) was not detected. The cholinergic receptors started to cluster at the midregion of the fibers after 1 day explantation, and ChE was detected in the fibers after 2 days in culture. The central receptor clusters were associated with ChE and their formation was dependent on the presence of nerve terminals. We conclude that neuromuscular contacts develop in organ culture with a pattern and time course similar to that of synapes developing in utero.     

Biomimetic bone mechanotransduction modeling in neonatal rat femur organ cultures: structural verification of proof of concept

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest then maintained in culture for 1 week at which time structural tests were conducted. Comparing 1 and 8 days in culture, bones grew significantly in size over the 7-day culture period. In addition, histology supported adequate diffusion and organ viability at 2 weeks in culture. For stimulation studies, 350 cycles of physiologic loading 24 h post-harvest resulted in increased bone strength over the 7-day culture period. In this work, structural proof of concept was established for the use of whole bone organ cultures as mechanotransduction models. Specifically, this work established that these cultures grow and remain viable in culture, are adequately nourished via diffusion and are capable of responding to a brief bout of mechanical stimulation with an increase in strength.     

Growth and mating of southern African Lycoteuthis lorigera (Steenstrup, 1875) (Cephalopoda; Lycoteuthidae)

Lycoteuthis lorigera is an oceanic squid that is abundant in the Benguela system. Little is known about the biology of this squid except that it is eaten in large numbers by numerous oceanic predators and that males grow to larger size than females, which is unique for oegopsid squid. The aim of this study was to better understand the biology of this species by investigating its age and growth, as well as its mating system. Toward this end, the age of 110 individuals, ranging from 35 to 110 mm, was estimated by counting statolith growth increments. Estimates of age ranged from 131 to 315 days and varied with mantle length. No significant differences were found in the size of males and females of equivalent ages. The relationship between ML and age for both sexes was best described by an exponential growth curve, probably because no early life stages were aged in this study. Only one mature male (ML 160 mm) was aged, and preliminary estimates suggest it was 386 days old. Instantaneous growth rates were low (0.54% ML/day and 1.4% BM/day) but consistent with enoploteuthid growth rates. When the growth rate of L. lorigera was corrected for temperature encountered during the animal’s life, the growth rate was fast (0.47% BM/degree-days) and consistent with the hypothesis that small cephalopods grow fast and that large cephalopods grow older, rather than fast. Mature females were often mated and had spermatangia in a seminal receptacle on the dorsal pouch behind the nuchal cartilage. Males probably transfer spermatangia to the females using their long second and/or third arm pair since the paired terminal organs open far from the mantle opening. M. A. C. Roeleveld deceased.     

Tissue culture of human neurocytomas induces the expression of glial fibrilary acidic protein

Summary. Cell cultures were established from three human neurocytoma specimens (primary and recurrent). The phenotypic evolution was analyzed by immunocytology in different culture conditions in the presence and absence of serum including the addition of epidermal growth factor, rat caudate extract, retinoic acid, and N-acetyl cystein. The cells were grown on glass cover slides or an extracellular matrix (ECM) from bovine corneal endothelial cells. Immunostainings were performed after overnight incubation and were repeated after 5 and 10 days of culture. The cultures were compared to an oligoastrocytoma also arising at the foramen of Monro and an ependymoma of the frontal lateral ventricle, two tumors supposedly originating from the same tissue matrix as the neurocytoma. After overnight incubation, 90% of the neurocytoma cells were positive for A2B5 and synaptophysin. GFAP reactivity appeared in the periphery of cell processes in less than 1% of the cells. The staining patterns and morphology were nearly identical under the different culture conditions. After 5 days, almost all cells were strongly positive for GFAP, while the number of cells remaining positive for synaptophysin and A2B5 was unchanged from the earlier time point. Again, there were no fundamental differences between the incubation conditions. At this point, cultures maintained on ECM were compared to their counterparts on untreated glass cover slides with identical staining results, although many fewer cells had attached. An identical immuno-reactive pattern was found on day 10. In contrast to the neurocytoma cultures, there was an immediate strong GFAP signal in both the mixed glioma and the ependymoma. A2B5 was also positive, but synaptophysin was absent. Because the neurocytoma specimens were synaptophysin positive but GFAP negative by immunohistochemistry, it is concluded that neurocytomas may represent a human neuronoglial precursor tumor that switches its phenotype in culture to astroglial differentiation despite very diverse culture conditions.     

Pancreas acinar cell differentiation VIII. Effect of methionine on DNA synthesis of pancreas anlage in organ culture

Rat pancreas embryonic rudiments incubated in a control chemically-defined (CD) medium grew and differentiated during 9 days of organ culture to adult acinar cells characterized by zymogen granules and relatively high levels of enzyme activity. Incubation of the anlagen in a chemically-defined medium with a lower methionine concentration (CD-MD) gave growth to a comparable size but no acinar cell formation, no zymogen granules and no increases of enzyme activity. Anlagen incubated in the CD medium showed a marked decrease of the autoradio-graphic labeling index of acinar cell nuclei after exposure to ³H-thymidine during the period of organ culture, whereas culture of anlagen in the CD-MD medium led to far less decrease of the acinar cell labeling index. When anlagen which had been grown in the CD-MD medium for 9 days were incubated for 3 additional days in the CD medium there was a highly significant drop in the previously high labeling index. The higher level of methionine in the CD medium may have been involved in the synthesis of, or the methylation of, an inhibitor of DNA synthesis.     

The effect of phosphorus and nitrogen deficiency on growth of seedlings of spring barley in dependence on irradiance: Growth analysis

Seedlings of spring barley,Hordeum vulgare L. cv. Mirena, were grown in a controlled environment chamber at high (HI: 122 Wm^?2) and low (LI: 28 Wm^?2) irradiance in the complete Richter’s nutrient solution (R) or in solution lacking either phosphorus (R -P) or nitrogen (R -N). The experiment was terminated 15 days after sowing when plants (R-N) at HI ceased to grow. At that time the dry mass of one plant was 449.8 mg, 145.7 mg and 116.8mg at HI and 203.4 mg, 110.1 mg and 91.0 at LI for R, (R-P) and (R-N), respectively. Deficiency of P and especially N reduced the size of loaf area more under HI than under LI conditions. Specific dry mass of leaves was the highest in R-N plants. The values of relative growth rate and assimilation rate are presented. Interaction of the effects of deficiency of mineral nutrients and irradiance during cultivation should be analyzed in further experiments for determination of optimum conditions for utilization of mineral nutrients.     

The rate of linear deposition of bone tissue in the bone spongiosum of lactating rats on a low calcium diet

In the epiphysis and metaphysis of lactating rats, submitted to a Ca++ depletion for 10 and 30 days and a Ca++ repletion diet for 10 days, the density of spongiosa framework and the bone tissue linear accretion rate were compared with those of control rats. The distal metaphyses of femora of the rats fed a calcium free diet for 10 and 30 days lose 50% and 90% of the trabecular framework respectively, while the epiphysis of the same bone lose only 45% and 56%. The linear accretion rate in these regions increases by 7.9 and 24.7% in the epiphysis and by 11.3% and 75.6% in the metaphysis of rats fed a calcium-free diet for 10 and 30 days respectively. Our data indicate that the bone tissue linear accretion rate changes not only between the corresponding regions of control and experimental rats but, in the latter, also in different regions of the same bone. Moreover, the higher the bone loss is, the higher bone accretion rate will be. The correlation between the bone tissue linear accretion rate and the bone loss indicates that the same local factor - probably mechanical - controls the activity and distribution of osteoblasts and osteoclasts.