Effects of different concentrations of serum on cartilage growth in an organ culture system

Summary The purpose of the present study was to examine the effects of various concentrations of serum on the behavior of neonatal condylar cartilage when cultured in an organ culture system. Mandibular condylar cartilages were obtained from newborn ICR mice, of which the zone of undifferentiated chondroprogenitor cells along with a few layers of young cartilage cells were cultivated at the medium-air interface. The incubation medium included fetal bovine serum at concentrations ranging from 0 to 10%, and the explants were kept in vitro up to 10 d. The serum-free medium maintained the chondrogenic expression, and the overall size of the cartilagenous protion of the explants increased with the decrease of the concentrations of serum in the medium. When explants were labeled with [³H]thymidine and were then processed for autoradiography, the peak of labeling was noticed at 48 h, a feature that recapitulated itself in all cultures (73, 140, 175, 201, and 129 labeled cells per chondroprogenitor zone in explants grown in 0, 1, 2.5, 5, and 10%, respectively). It can be concluded that serum-free medium maintains the chondrogenic phenotype of condylar cartilage in vitro. This study was supported in part by a research grant from the Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig-Stockheim, Federal Republic of Germany.     

Transformation of fetal secondary cartilage into embryonic bone in organ cultures of human mandibular condyles

Mandibular condyles of human fetuses, 14–21 weeks in utero, were kept in an organ culture system for up to 60 days. After 6 days in culture, the cartilage of the mandibular condyle appeared to have maintained its inherent structural characteristics, including all its various layers: chondroprogenitor, chondroblastic, and hypertrophic. After 12 days in culture, no chondroblasts could be seen; instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells in the vicinity of the chondroprogenitor zone differentiated into osteoblast-like cells that produced type I collagen. The progenitor cells were still actively incorporating ³H-thymidine. The newly formed osteoid-like tissue lacked both metachromatic reactivity and a response to antibodies against chondroitin sulfate. Instead, the tissue reacted positively for osteocalcin (bone gla-protein). The process of new bone formation further progressed and, by the 20th day in culture, the new bone reacted positively for type I collagen, osteonectin, and to a lesser extent for chondroitin sulfate. The osteoid also underwent mineralization as revealed by both the von Kossa stain and vital staining with tetracycline. The above feature appeared even more intense in 40-day-old cultures. After 60 days, the newly formed bone contained osteoblasts and osteocytes, whereas the extracellular matrix revealed a high degree of matrix polarization. The results of the present study recapitulate findings reported for organ cultures of mice mandibular condyles. However, the in vitro process of de novo bone formation in human specimens requires a 6-fold longer culture time than that needed for mice condyles.     

Histochemical study of matrical components in the mandibular condyle of the neonatal mouse

In the mandibular condyle of the newborn mouse the chondroprogenitor (CP) zone is the only layer that incorporates 3H-thymidine thus serving the source for cells of the cartilage lineage. Ultrastructurally these cells have a mesenchymal appearance surrounded by collagen fibrils as well as by additional filaments that become apparent following fixation with ferrocyanide-reduced OsO4. In addition, electron-dense particles indicative of proteoglycans are scattered throughout the matrix in the CP zone as well as in the chondroblastic and hypertrophic zones. Following labeling with 35S-sulfate the CP zone as well as the other compartments revealed a substantial number of grains following processing for autoradiography. The number of grains per cell was highest in the hypertrophic zone. Indirect immunofluorescence indicated the presence of fibronectin in the articular surface, CP zone and in the hypertrophic zone. The immunogold method localized fibronectin intracellularly in CP cells and extracellularly in the hypertrophic zone. Therefore, in the mandibular condyle the CP cells which are capable for DNA synthesis are also involved in the synthesis of macromolecules of which some are specific for the cartilage phenotype, while others are associated with other functions of connective tissue cells.     

Studies of adult amphibian heart cells in Vitro: DNA synthesis and mitosis

The ventricle of the adult newt heart was excised and cut into several pieces of approximately 0.5 – 1.0 mm. These heart pieces were then cultured for 60 days at 25 °C in a modified Leibovitz medium (L-15). Approximately 37% of the explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates which ranged 3–67 beats/min. The explants were labeled with 1 μCi/ml of ³H-thymidine for 24 hr at 7, 15, 21, 30, 45 and 60 days of culture initiation, and processed for electron microscopic autoradiography. The examination of the autoradiograms revealed that as the culture continued, the cardiac muscle cells altered their morphology, resembling embryonic cardiac muscle cells. These altered muscle cells were termed dedifferentiated cardiac muscle cells. The number of these dedifferentiated cells increased over the period of culture, showing 10.3–94% dedifferentiated cells after 7–60 days of culture respectively. DNA synthesis and mitosis were observed in the dedifferentiated cardiac muscle cells, apart from the non-muscle cells. The quantitation of the autoradiograms revealed that the number of labeled nuclei in the cardiac muscle cells gradually increased over the period of culture, and a maximum number of labeled cardiac muscle cells (30%) was observed in the third week. The peak was followed by a decline in the eighth week which exhibited 1.5 % labeled cardiac muscle cells. The trend of mitosis was similar to that of DNA synthesis. The maximum number of mitotic figures (9%) was observed in the third week of culture, which was followed by a decline and finally absent in the eighth week. The cardiac non-muscle cells, mostly fibroblasts and endothelial cells, also showed incorporation of ³H-thymidine in their nuclei. The number of labeled non-muscle cells nuclei and the mitotic index were highest (61 and 15% respectively) in the first week of culture, but then they decreased gradually over the eight-week period in culture. This study provides evidence for the first time that the adult amphibian cardiac myocytes can undergo DNA synthesis and mitosis when explanted and cultured. The significance of this cell replication is discussed.     

Differentiation of cartilage cells studied by quantitative [3H]thymidine autoradiography in vitro

The apical segments of the mandibular condylar cartilage of newborn ICR mice, containing the intact zones of progenitor cells along with a few rows of chondroblasts were initially prelabelled in vitro with [3H]thymidine and were subsequently chased and cultured for as long as eight days. Such explants underwent a process of tissue regeneration, as after three days in culture they reconstituted the original structure of the organ, thus resembling the in vivo appearance of neonatal mandibular condylar cartilage. Cellular proliferation with subsequent differentiation in the regenerating tissue was followed by means of quantitative autoradiography. Immediately after labelling, the autoradiography-positive grains were confined exclusively to progenitor cells. The latter revealed a substantial ability to proliferate in vitro, a fact that was manifested by a progressive increase in the labelling index along the course of the culture. The latter process was followed by cellular differentiation thereby obtaining hypertrophic chondrocytes. The increase in the rate of labelling index and in the total number of [3H]thymidine-labelled cells was significantly correlated with the overall growth of the regenerating explants.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

Further characterization of the chondroprogenitor zone in mandibular condyles of suckling mice. An ultrastructural and cytochemical study

In the neonatal mouse the mandibular condyle serves as an important growth center for the developing mandible. The youngest cells in this organ are the chondroprogenitor cells that are the source for new differentiating chondroblasts. The present study provides new data concerning the fine structure and cytochemical characteristics of the cartilage precursor cells as seen in suckling mice. The condylar chondroprogenitor cells normally reveal a mesenchyme-like appearance with multiple, elongated cell processes that enable close contact between neighboring cells. These cells also exhibit a variety of pinocytotic vesicles, coated pits and appear to be actively involved in the internalization of a fluid-phase marker horseradish peroxidase. Further, the progenitor cells were found to contain trimetaphosphatase reaction products within lysosomal bodies and alkaline phosphatase reactivity along their plasma membrane. Precipitates of calcium complexes in the form of calcium pyroantimonate could be demonstrated in association with the plasmalemma of the cells as well as with the extracellular collagen fibrils. Matrix granules, representing cartilage proteoglycans, became a distinct feature following staining with ruthenium red and were found to be in close contact with the extracellular collagen fibrils and with the plasmalemma. Hence, in addition to their active role in cell proliferation, the progenitor cells are also involved in the synthesis and secretion of major extracellular macromolecules such as collagen and proteoglycans.     

DNA fiber autoradiography of tobacco mesophyll protoplasts

DNA fiber autoradiography was successfully applied to protoplasts isolated from tobacco leaf mesophyll. After an incubation period of 37 h, DNA began to be labeled with ³H-thymidine. These cells, with regenerated thin cell walls, were then effectively disrupted in a lytic solution containing sodium dodecyl sulfate. DNA fiber autoradiography confirmed that the mean replicon size of this plant is approximately 60 in good agreement with the values reported previously in animal cells.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

Structural characterization of the mandibular condyle in human fetuses: light and electron microscopy studies.

Mandibular condyles from 18- to 20-week-old human fetuses were examined in the light and electron microscope with particular attention to intratissue organization and extracellular matrix. In the human fetus the condyle has been divided into five layers: (1) the most superficial, articular layer, (2) chondroprogenitor cell layer, (3) condroblast cell layer, (4) nonmineralized hypertrophic cell layer, and (5) mineralized hypertrophic cell layer. The articular layer is rich in collagen fibers (mostly of the type I collagen), but the cells seldom divide. By contrast, in the chondroprogenitor cell layer and upper part of the chondroblastic cell layer mitosis gives rise to new cells. The matrix in the latter layer is composed of thick banded 'lucent' fibrils in a loose feltwork of granules representing cartilage proteoglycans. The daughter cells in the progenitor cell layer undergo differentiation which is apparently completed along the lower border of the mineralized hypertrophic cell layer--the ossification front. The matrix in the hypertrophic cell layer reveals distinct matrix vesicles that undergo mineralization and subsequently coalesce to form larger sheets of mineralized extracellular matrix. Mineralized cartilage serves as a backbone for new bone formation as marrow-derived osteoblasts and osteoclasts attach to remnants of mineralized cartilage, which enables the turning on of the remodeling cycles involved in new bone formation. It can be concluded that the process of endochondral ossification as has been reported in lower animals is recapitulated in the human fetus, thus the dynamics associated with condylar morphogenesis is maintained through phylogeny.     

Periodic acid-Schiff reaction combined with quantitative autoradiography of 3H-thymidine- or 85S-sulfate-Labeled epithelial cells of colon

Summary The influence of periodic acid-Schiff staining on grain counts was examined using autoradiography of guinea pig colon labeled with either ³H-thymidine or ³⁵S-sulfate. Prestaining decreased the grain count, an effect due to the Schiff reagent but not to periodic acid. Poststaining altered the grains which were partly or completely lost, an effect due to periodic acid but not to the Schiff reagent. The suggested procedure is to pretreat the sections with periodic acid, process for autoradiography, and poststain with the Schiff reagent. No silver grain is then lost.     

Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

Summary The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with ³H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the lipogenic enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.     

Incorporation of Radioactive Pyrimidine Nucleosides into DNA and RNA of Eimeria tenella (Coccidia) Cultured In Vitro*

Autoradiographic methods were used to study the incorporation of tritiated cytidine, thymidine, and uridine into asexual stages of Eimeria tenella cultured in embryonic chick kidney cells. Developing parasites did not incorporate ³H-thymidine either when host cells were labeled prior to infection or when the cultures were labeled for 30 min, 48–72 hr after infection. Continuous exposure of infected cultures to ³H-thymidine for up to 18 hr resulted in light labeling of cell cytoplasm and schizonts. ³H-cytidine and ³H-uridine were incorporated into parasites developing in cultures that were labeled before infection. When the cultures were labeled for 30 min, 48–72 hr postinfection and fixed immediately, schizonts were labeled lightly with ³H-cytidine but contained dense accumulations of ³H-uridine.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

A tissue culture system supporting cartilage cell differentiation, extracellular mineralization, and subsequent bone formation, using mouse condylar progenitor cells

Undifferentiated progenitor cells of mandibular condyles of neonatal mice were kept in a tissue culture system for up to 9 days. After 2 days in culture, new chondroblasts developed within the explants, whereas the peripheries of the latter were occupied by undifferentiated cells undergoing mitosis. By 4 days in culture, many of the cartilage cells showed signs of hypertrophy, while the matrix revealed positive reactivity for type II collagen and matrix metachromasia. The process of maturation of cartilage cells appeared to have reached its final stages after 6 days in culture, at a time when the initial loci of matrix mineralization could be easily identified. Concomitantly, peripheral areas bordering the cartilaginous core, as well as portions of the cartilage, reacted positively for type I collagen and fibronectin. Light microscopy examination showed signs of new bone formation after 9 days in culture. The extracellular matrix at the upper portion of the explant, facing the medium-air interface, reacted intensely with antibodies against type I collagen and fibronectin, but not with antibodies against type II collagen. Further, the newly formed osteoid was found to have undergone mineralization, thus forming an expanded layer of new bony tissue. A close spatial association was found between mature, mineralized cartilage and new bone. Hence, we hereby introduce a new in vitro system serving as an experimental model for studies related to the differentiation of progenitor cells into chondroblasts, which in turn promote ossification.     

Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.