Osteoblast Morphology Research Articles

Establishment of human osteoblastic cells derived from periosteum in culture.

We isolated osteoblastic cells derived from human periosteum and established them in culture. Their growth depended on the presence of ascorbic acid, and the doubling time was 40 to 60 h. The requirement for ascorbic acid was used to high production of collagen. These cells produced mainly type I collagen and only small amounts of type III collagen determined by reducing sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis. The total collagen yield was about 10 mg from 2 X 10(7) cells. The cells could be continuously cultured in alpha-minimum essential medium supplemented with 10% fetal bovine serum for 18 to 40 population doubling levels, depending on the age of the donated periosteum. These cells have the ability to calcify when incubated with 2 mM alpha-glycerophosphate-Na2. Calcification as viewed by the naked eye appeared from Day 15 after treatment. Treatment with the active formed vitamin D3, 1, 25 dihydroxyvitamin D3 enhanced calcification significantly and stimulated osteocalcin production. By electron microscopy, cells with many projections on their surfaces showed well-developed rough endoplasmic reticulum and actinlike fibers, and larger numbers of lysosomes, mitochondria, and secretion granules. Many matrix vesicles, in which minerals were initially localized, and well-banded collagen fibrils were seen in the intercellular spaces. These observations demonstrate typical osteoblastic morphology. The above results indicate that cultured cells from human periostem are osteoblastic cells that have the capacity to differentiate into osteocytes and to deposit calcified minerals in response to 1,25 dihydroxyvitamin D3.

Protein adsorption at the interface between charged polymer substrata and migrating osteoblasts

The In vitro migratory morphology of neonate rat calvarial osteoblasts on positively or negatively-charged polymer substrata was observed using scanning (SEM) and transmission electron microscopy (TEM). Proteins adsorbed from the culture medium onto these charged substrata were desorbed using 2% SDS and separated using polyacrylamide gel electrophoresis (PAGE). The charge sign of the substrata affected both protein adsorption and osteoblast migratory morphology. Cells flattened and adapted so closely to positively-charged substrata that the ventral cell membrane could not be distinguished at TEM. On negatively-charged substrata the ventral cell membrane was readily visible, with only focal areas of close contact with the substratum. The depth of the ventral extracellular space varied with the surface charge carrier species on the negative substrata. Two desorbed protein fractions (MW 220 and 30 kDaltons, respectively) were correlated with osteoblast spreading on positive and negatively-charged surfaces respectively. Another protein fraction was uniquely present on PAGE profiles desorbed from negatively charged substrata. It was concluded that the migratory morphology of osteoblasts was influenced via the intermediary of specifically adsorbed proteins.

Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition

A stereologic procedure for the reconstruction of matrix and mineralized bone growth curves at formative sites in trabecular bone is presented. Iliac crest bone biopsies obtained from twenty normal individuals after tetracycline double-labeling were investigated histomorphometrically. Corresponding values for osteoid width and apparent distance between bone markers and width of mineralized bone walls were classified using a sector plotting system. Observed structure widths were converted to three-dimensional structure thicknesses by a stereologic unfolding procedure. Osteoid thickness, calcification rate, and fractional labeling of osteoid varied characteristically with increasing wall thickness and permitted the construction of curves describing the time-dependent variations in matrix and wall thicknesses. The mean thickness of completed walls was 61.9 ± 1.5 μm (SE), and the mean bone formation period (Sigma f) was 145 days (124–168; 95% confidence interval.). The initial appositional rates for bone matrix (2.1 μm 3/μm 2/day) (1.4–2.9) and bone mineral (1.1 μm 3/μm 2/day) (0.4–1.9) declined gradually toward zero at the end of Sigma f. The initial mineralization lag time was 15 days (12–24) and increased to a maximum of 27 days during the first 45% of Sigma f. Thereafter, it decreased gradually toward zero. The height of the osteoblast nuclei gradually declined from 6.7 ± 0.5 μm at the start of bone formation to 1.2 ± 0.1 μm at the end. The study demonstrates that it is possible to reconstruct growth curves for trabecular bone walls based on three-dimensional values for structure thicknesses using different sections for light and fluorescence microscopy and avoiding classification according to osteoblastic nuclear morphology. This more detailed description of the bone remodeling sequence is important for the planning of treatment of different metabolic bone diseases. The techniques described make it possible to assess bone mineral balance at the BMU level and may give further insight into the mechanisms by which different hormones and drugs influence bone remodeling.

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.

Factors influencing the effect of parathyroid hormone on endosteal cell morphology. A scanning electron microscope study

The technique of surface scanning electron microscopy (SEM) provides an opportunity to study the effect of hormones on bone at the cellular level. Parathyroid hormone (PTH) has been shown to produce characteristic changes of bone cell surface morphology (Davis et al., 1975; Krempien et al. , 1975). Using this method, we tried to analyse factors which are known to modulate the action of PTH such as uremia and immobilization. It is the purpose of the following study to describe some effects of PTH on the endosteal cells of the inner surface of rat calvaria and to report on the inhibitory effect of uremia and the stimulatory effect of immobilization on PTH-induced changes in the surface morphology of osteoblasts.

Formation of bone tissue in culture from isolated bone cells.

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.