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The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silbermann, M., D. Lewinson, H. Gonen, M. A. Lizarbe, and K. von der Mark. 1983. Anat. Rec. 206:373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.
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PMID:Gene expression during osteogenic differentiation in mandibular condyles in vitro. 169 Nov 90

Intrinsic differences in bone formation rate, cell numbers, and the percentages of cells expressing alkaline phosphatase activity were studied in explants of chick calvaria periosteum cultured for 4 days and 6 days. Proliferation, differentiation, and bone production were examined in radioautographs of plastic sections and by using whole-culture biochemical assays of protein and alkaline phosphatase. Ectocranial explants at both 4 days and 6 days exhibited more alkaline phosphatase-positive cells and significantly more bone formation than endocranial cultures. There were no detectable differences in cell numbers or 3H-thymidine labeling indices. The volume of bone synthesized per osteoblast was significantly higher in the ectocranial group. Examination of bone stripped of periostea and then cultured for 4 days revealed that large areas of bone were covered by osteoblasts, indicating that the periosteal explant cultures were composed almost exclusively of osteoprogenitor cells and fibroblasts. The data suggest that the level of expression of predetermined osteogenic phenotypes can be maintained in vitro for 6 days following explantation and that variations in the rate of osteogenesis are programmed into progenitor cells prior to their differentiation into osteoblasts.
Anat Rec 1989 Jan
PMID:Site-specific regulation of osteogenesis: maintenance of discrete levels of phenotypic expression in vitro. 291 54

The anterior part of the mammalian nasal septum (NS) persists throughout the life span as hyaline cartilage, in contrast to cartilage in most parts of the body, which is gradually replaced by bone during development. In this study, we have cultured differentiating rat NS under various experimental conditions in an attempt to gain some insight into the osteogenic potential, if any, of the NS and its surrounding connective tissue. Differentiating NS from E15 and E19 rat embryos were dissected and grown under the following conditions: 1) organ cultured in Waymouth's medium or modified Eagle's medium, with or without serum; 2) cultured on chick chorioallantoic membrane (CAM); 3) implanted under rat kidney capsule (KC). Bone-like substance (BLS) never developed in organ cultures, but was observed in CAM cultures and KC implants after 7 days. The BLS was located external to the perichondrium of the NS and was stained red by the van Gieson's technique, indicating the presence of mature collagen. Further evidence of its bone-like characteristics was demonstrated by the presence of alkaline phosphatase and type I collagen. The CAM and KC represent two experimental conditions under which progenitor cells in the nasal septum area may be induced to synthesize BLS.
Anat Rec 1988 Aug
PMID:Development of bone-like substance in cartilaginous rat nasal septum under experimental conditions. 318 74

We divided 116 rats weighing 50 gm into four groups with tails either left in situ or transplanted as follows: straight in situ: untreated controls; bent in situ: five caudal vertebrae (CV) in the loop; straight transplants: three CV skinned and transplanted autologously; and bent transplants: five CV skinned, bent to form a loop, and transplanted autologously. Tails were radiographed weekly up to 6 weeks and at 12 weeks, and microradiographic and histological studies were undertaken on selected specimens. At 12 weeks the bones in the apex of the loop of tails left in situ appeared bent with a straight-to-convex shaft on the outer side and a thicker, more concave one on the inner side. In the transplanted bent segments the bone shaft died and initially the reverse occurred: the outer shaft thickened and the inner resorbed completely. A new concave inner diaphysis then formed so that the bones in both instances were essentially similar in final shape. In the bent transplants the surviving osteogenic tissues regenerated and, adapting to the altered forces, formed a new bone shaft. This involved a change in the direction, amount, and nature of endochondral, periosteal, and regenerative growth and subsequent remodeling of bone. The results support previous observations that, within limits, the strain in the osteogenic envelope is an important factor in adaptation of bones to changing stress and that, where the envelope is deficient, the surviving tissues have the capacity to regenerate and repair defects in the bone so that it best resists the changing stresses applied to it.
Anat Rec 1986 Jun
PMID:Remodeling of bone and bones: effects of altered mechanical stress on the regeneration of transplanted bones. 352 16

Dexamethasone is an important regulator of cellular proliferation and differentiation, but paradoxical effects have been noted in a variety of culture systems. The purpose of this study was to determine whether dexamethasone induces proliferation and differentiation of osteogenic precursor cells. Periosteal explants from embryonic chicks were grown in culture for 3 or 4 days, treated continuously with dexamethasone or ethanol vehicle, and then either pulse-labeled with 3H-thymidine at 3 days or labeled for 24 hr between day 3 and day 4. Histochemical and autoradiographic procedures were used to assess the proliferation and differentiation of osteogenic cells. At 3 days, the area of bone, the percentage of alkaline phosphatase-positive cells, the percentage of 3H-thymidine-labeled cells, and the percentage of cells labeled with both markers were significantly higher in dexamethasone-treated cultures. Between day 3 and day 4 no significant changes in these parameters were observed in the dexamethasone-treated cultures. In comparison, control cultures exhibited significant increases in the percentage of 3H-thymidine-labeled cells after 24 hr of continuous labeling. The data show that dexamethasone induces a burst of proliferation in a cohort of cells that undergo differentiation. Once these cells have divided, further proliferation within the culture is limited. Finally, it is apparent that the timing of experiments may be critical in determining whether dexamethasone will inhibit or stimulate proliferation.
Anat Rec 1986 Aug
PMID:Dexamethasone induces proliferation and terminal differentiation of osteogenic cells in tissue culture. 374 Apr 74

Morphogenesis and maturation of the sagittal suture in newborn C57B1/6J strain mice were studied using light and electron microscopy. Morphodifferentiation of the murine parietal bones progresses radially with the interposed sagittal suture, assuming a greater level of maturity at birth at a midpoint along its length. The presumptive suture develops in a sulcus, deeper posteriorly, more shallow anteriorly. Cells at the osteogenic front (OF) are distinguished from the surrounding fibrocytic cells by a number of distinctive characteristics: 1. increased cytoplasmic density; 2. extensive endoplasmic reticulum; 3. dispersed nuclear chromatin aggregates; 4. extensive surface projections; 5. close approximation. Mineralization of the developing parietal bone occurs extracellularly with the initial deposits of apatite crystals exhibiting no oriented relationship to either cellular or extracellular fibrillar elements. The majority of collagen fibers lie superior and inferior to the presumptive suture, oriented anteroposteriorly with their long axes parallel to the ectocranial surface. Other fiber bundles more intimately associated with the developing suture display a more random orientation.
Anat Rec 1985 May
PMID:Light and electron microscopy of the new born sagittal suture. 407 46

The frontal region of the embryonic chick was studied to determine whether epithelial influences are necessary for frontal bone development. The frontal bone is a membrane bone, of neural crest and head mesodermal origin, which develops within mesenchyme sandwiched between two epithelia, neural ectoderm and epidermis. Rudiments were treated enzymatically to separate epithelial and mesenchymal tissues. Frontal mesenchyme then was grown as chorioallantoic membrane grafts either in the presence or absence of neural ectoderm and/or epidermis. The results indicate that neural ectoderm, though required during early stages of development to induce frontal bone development (Schowing, 1968), is not required during later stages (HH 22-30, the stages tested in this study) for osteogenesis. Epidermis, however, was shown to be required for frontal bone development during the stages tested. Frontal mesenchyme formed bone when epidermis was present on the outer aspect of the mesenchyme, and did not form bone when the epidermis had been removed prior to grafting, whether or not neural ectoderm was present. This dependence upon epidermis continues beyond the onset of meningeal differentiation. Once the outer ectomeninx-dermis is distinguishable from the inner endomeninx, osteogenic capabilities are confined to the ectomeninx-dermis layer. Furthermore, the ectomeninx-dermis layer attached to epidermis is able to form membrane bone in the absence of the endomeninx and neural ectoderm. The endomeninx, though normally nonchondrogenic, was shown to be capable of forming cartilage when the neural ectoderm is removed. Neural ectoderm, therefore, may have an inhibitory effect on chondrogenesis in the endomeninx.
Anat Rec 1983 May
PMID:Development of the frontal bone and cranial meninges in the embryonic chick: an experimental study of tissue interactions. 688 51

We describe a method for clearly separating the cell layers at the bone-marrow interface, which reveals that the myeloid tissue is invested by an epithelial-like layer of specialized squamous cells we call the marrow sac. The scanning electron microscope showed that the sac was fenestrated and that some of its cells pass as perivascular elements with the marrow capillaries that penetrate the bony cortex. The transmission electron microscope (TEM) showed that the cells comprising the marrow sac are less than 0.1 micrometer thick, overlap at their margins without specialized cell junctions, and are more electron dense than the reticular or fibroblastic cells of the marrow stroma. The fenestrations in the sac were intercellular and were usually occupied by cells having an ultrastructure compatible with an osteoprogenitor cell (OPC) lineage. The observation of a close proximity between the cells of the marrow sac and the osteogenic cells that line the endosteal surfaces of bone suggest that the sac cells, along with the OPCs of the superficial marrow stroma, should be included in any morphological or functional definition of an endosteum.
Anat Rec 1982 May
PMID:A cellular investment of bone marrow. 704 2

Osteogenesis was inhibited when mandibular processes from 3 1/2-day-old embryos were cultured in BUdR, LACA, alpha, alpha'-Dipyridyl, 4-Methylumbelliferone, and 4-Methylumbelliferyl-beta-D-glucoside or beta-D-xyloside. Mandibular processes were then cultured in the test substances for 3 days, enzymatically separated into their epithelial and ectomesenchymal components, combined with mandibular components from untreated embros, and either organ-cultured or grafted to chorioallantoic membranes of host embryos. Osteogenesis was inhibited when treated epithelium, but not when treated ectomesenchyme, was present in the tissue recombinations. Analysis of the known action of these inhibitors indicates that proliferation, hydroxylation of collagen, and synthesis of proteoglycans by epithelial cells are all necessary components of this osteogenic epithelial-ectomesenchymal interaction.
Anat Rec 1980 Jul
PMID:The role of epithelial collagen and proteoglycan in the initiation of osteogenesis by avian neural crest cells. 743 7

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.
Anat Rec 1998 12
PMID:Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue. 984 6


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