Periosteal Fibroblasts Research Articles

Periosteal Fibroblasts Synthesize a Cytokine That Stimulates Bone Resorption and Demonstrates Interleukin-1 Activity After Partial Purification

Culture supcrnatants from rabbit periostea] fibroblasts stimulate 45Ca2+ release from prclabcllcd mouse bones in viiro and the production ofcollagcnase by rabbit articular chondrocytes and mouse calvanalosteoblasts. Bonc-resorptivc and chondrocytc-stimulating activities arc found in gel-filtration fractions corresponding to an Mr in the range 15000-25000 When tested in the standard mouse thymocyte assay for interlcukin-1 (1L-1). crude and fractionated culture media are inactive. After isoelectnc focusing, however, chondrocytc-stimulating activity and IL-1 activity arc both focused at pH 4.8 These results suggest that an inhibitor of the response of mouse thymocytcs to IL-1 was present in the crude and fractionated material In view of the recent report that pure pig IL-1 (Mr 21 (KK). pi 4 7) has diverse effects on connective tissues and is a potent inducer of bone rcsorption in vitro, it is probable that the fibroblast-denved rcsorptivc activities reside in a molecule closely related to pig IL-1

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an M r in the range 15 000–25 000 and a p I corresponding to approx. pH 4.7. These biological and physicochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.

The use of in vitro models for investigating the response of fibrous joints to tensile mechanical stress

To clarify the role of mechanical deformation in the remodeling of fibrous joints, organ culture systems have been developed to apply mechanical stress to cranial sutures under controlled experimental conditions. Tensile mechanical stress applied to cranial sutures from newborn rabbits produces a two- to threefold increase in protein synthesis and a twofold increase in collagen synthesis that can be detected within 6 hours. There is also a threefold increase in the DNA content of the sutures after 48 hours. Under normal conditions sutural fibroblasts synthesize type I collagen but respond to tensile deformation by synthesizing significant amounts of type III collagen. This suggests that the biomechanical environment of a connective tissue cell is an important determinant of the collagen type synthesized. However, the effect is likely to be an indirect one by virtue of its influence on the metabolic activity of the cells. Mechanically activated cells do not preferentially synthesize structural proteins, since mechanical stress stimulates the synthesis not only of structural macromolecules but also of the enzymes responsible for their specific hydrolysis. This is not accompanied by increased degradation, however, perhaps because the metalloproteinase inhibitor TIMP synthesized by the tissues is also increased. Confluent rabbit and mouse periosteal fibroblasts synthesize and release into the culture medium factors that can inhibit bone cell proliferation and stimulate bone resorption in vitro. It seems likely that further investigation of the interaction between fibroblasts and osteoblasts at the bone—fibrous tissue interface will require a reassessment of current thinking concerning the mechanisms regulating sutural osteogenesis.

Biochemical and histological studies on various bone cell preparations.

Four different cell populations--designated PF, OB, OC, and PC--were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the "transition state" between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria from removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E1 (PGE1) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.

Nuclear uptake of sex steroids in gingiva of the baboon.

Influences of sex steroid hormones on the periodontium are documented. Confirmation of the presence and location of receptors for these hormones is important in achieving a more complete understanding of their role in periodontal disease. Biochemical studies have provided evidence of sex steroid receptors in cytosols prepared from gingival tissues, however, no morphologic data are available on the location or specific cell types with receptors. For this study, three mature male and three female baboons received tritiated 5-alpha-dihydrotestosterone (3H-DHT) while three similar males and three females received tritiated estradiol-17 beta (3H-E2). A control animal from each group of three was given an excess of the corresponding unlabeled hormone in addition to the tritiated dose. Gingival specimens were obtained at necropsy as part of a total body study of receptor distribution, and autoradiographs were prepared on emulsion-coated slides. Heavy nuclear uptake was observed in periosteal fibroblasts and scattered fibroblasts of the lamina propria in male given 3H-E2. Females had moderate uptake of E2 in similar but fewer sites than males. There was minimal uptake of 3H-DHT in one male and two females. These data provide additional evidence of steroid receptors in the periodontium and contribute to our understanding of responses of the periodontium to sex steroids.

Effect of fibroblast growth factor on cultured fetal rat calvaria

Normal rat or human serum causes a greater incorporation of 3H-proline into bone collagenase digestible protein (CDP) and noncollagen protein (NCP) than does serum from hypophysectomized animals or hypopituitary humans. In the present study, we have tested fibroblast growth factor (FGF), a peptide isolated from bovine pituitary glands that has been shown to stimulate RNA and DNA synthesis in various mesodermal cells, for its effects on cultured fetal rat calvaria. The major effect of FGF appeared to be a stimulation of periosteal fibroblastic cell proliferation. Incorporation of 3H-thymidine into DNA was increased at concentrations of 10–1000 ng/ml; the effect appeared after 12 hr, was sustained for 96 hr, and could not be ascribed to an effect on 3H-thymidine uptake. Total DNA content was increased and histologic sections showed an increase in the number of mitoses in periosteal fibroblasts after colcemid arrest. These effects were accompanied by an increase in the uptake and incorporation of 3H-uridine, a decrease in the incorporation of labeled proline into CDP, and a small and variable increase in the incorporation of proline into NCP. Cortisol opposed the effects of FGF on 3H-thymidine and 3H-uridine incorporation. Insulin did not alter the effect of FGF on 3H-thymidine incorporation, but FGF decreased the stimulatory effect of insulin on the labeling of CDP. The effect of FGF on thymidine incorporation and collagen synthesis was not altered by indomethacin. The major effect of FGF in calvaria is to increase DNA synthesis and stimulate the proliferation of periosteal fibroblasts. It does not appear to be the pituitary-dependent factor in serum that stimulates 3H-proline incorporation into CDP and NCP in calvaria.

Endocytosis of sugars in embryonic skeletal tissues in organ culture. II. Effect of sucrose on cellular fine structure.

ABSTRACT A study of the fine structure of embryonic chick limb-bone rudiments in organ culture has shown that exposure to 0·08 M sucrose causes intense cytoplasmic vacuolation of the peripheral cells, while the structure of the cytoplasmic organelles is apparently unaltered. The lamellae and vesicles of the Golgi apparatus are more abundant than in the controls and the vesicles are no longer confined to the Golgi zone. The first vacuoles appear in the articular chondrocytes after exposure to sucrose and are autophagic vacuoles or clear vacuoles, probably containing sucrose. After 24 h or more the vacuoles are more heterogeneous and contain a great variety of membranous, granular and fibrous material. These vacuoles appear to be formed by fusion between endocytotic coated vesicles, autophagic vacuoles, multivesicular bodies, clear vacuoles and vesicles derived from the Golgi zone. After 2 days some vacuoles contain fibrils identical in appearance to those in the surrounding cartilage matrix. The development of vacuoles in the chondrocytes in the inner regions of the epiphyses is much slower and autophagic vacuoles are only seen after 6 days’ exposure to sucrose. Similar changes are observed in the fine structure of the perichondrial and periosteal fibroblasts, and after 2 days in the presence of sucrose many fibroblasts have cytoplasmic vacuoles containing banded fibres similar to the extracellular collagen fibres. In shafts of 11-day embryonic chicks exposed to sucrose for 8 days the cytoplasmic vacuoles in the osteoblasts contain banded fibres and dense crystallites resembling those of the bone matrix. When rudiments are transferred to normal medium after 2 days’ exposure to sucrose the vacuoles are gradually lost; some of the vacuoles appear to move to the periphery of the cell and then fuse with the plasma membrane, thus releasing their contents. Throughout this process the fine structure of the cytoplasmic organelles is unaltered. It is suggested that exposure to sucrose stimulates an increased production of lysosomal enzymes which are transported in small vesicles from the enlarged Golgi region to the clear vacuoles and to the autophagic vacuoles. It seems possible that during this process enzymes are released into the surrounding matrix and modify the ground substance sufficiently to enable some of the connective tissue cells to become actively phagocytic and to ingest collagen fibres and bone crystallites.

Comportamento dell'acido desossiribonucleico e dell'istone nel nucleo delle cellule ossee

1. A quantitative investigation of both DNA and histone has been performed in nuclei of the cells directly or indirectly involved in the ossification process, i.e. preosteoblasts, osteoblasts, osteocytes, as well as periosteal fibroblasts. 2. The histophotometric technique was applied on sections of cranial vaults of newborn rats. The sections were stained according to the Feulgen method for the DNA and the “fast green” method for the histone. 3. Preosteoblasts, osteoblasts and periosteal fibroblasts are characterized by a diploid content of DNA. The preosteoblasts only reveal some tetraploid nuclei according to their mitotic activity. These findings suggest that in spite of their different functional activity the three types of cells have the same content of DNA. 4. On the contrary the DNA content of the osteocyte nucleus only amounts to 87% as regards to that of the osteoblast nucleus. The exact meaning of such a finding is at present not easily interpretable. One might speculate that it is depending upon the peculiar biological reduced activity of the osteocyte. 5. Preosteoblasts and periosteal fibroblasts show the same nuclear affinity for the “fast green”. Consequently the DNA/histone ratio in the two cell types remains unchanged. On the contrary the nuclear affinity for the “fast green” increases in the osteoblast and decreases in the osteocyte. Probably the finding concerning the osteoblast is not to be considered as a true increase in histone content but as the result of the release by the trichloroacetic acid of some radicals previously linked to the RNA, the amount of which in the nucleus of this cell is considered quantitatively high. On the other hand the finding concerning the osteocyte might be considered the result either of a true decrease in the histone content or of a decreased release of radicals depending upon a seemingly lower content of RNA.

Studies on the Growth of Tissues in Vitro

ABSTRACT In a previous paper of this series (Trowell & Willmer, 1939) an account was given of the effects on the growth of chick periosteal fibroblasts of the addition of saline extracts of various organs from the growing and fully grown bird. Attention was called to the fact that those organs and tissues which yielded growth-promoting extracts were just those which have been shown by Warburg and his school (Warburg, 1930) to possess considerable powers of glycolysis. The parallelism is illustrated in Fig. 1. In the upper series are plotted the mean mitotic indices for the fibroblast cultures during the period from the tenth to the twentieth hour after the addition of the tissue extracts, at which time the effects produced by such extracts have been shown to be most comparable. In the lower series, the anaerobic glycolytic activities, as measured by lactic acid production, of the corresponding tissues are plotted. Comparison of these two sets of figures at once suggests that the organ extracts may exert their effects by influencing the carbohydrate metabolism of the tissues to which they are applied. The only apparent exception is the thyroid, and the effects of extracts of that organ on the growth of cultures were very variable and probably other factors were involved.

Studies on the Growth of Tissues in Vitro

ABSTRACT Tissue extracts have been made from several organs of growing and adult fowls, and the growth-promoting properties of these extracts have been tested on periosteal fibroblasts growing in vitro. The growth-promoting properties are most pronounced in brain extract and diminish in the sequence : thyroid, thymus, testis, ovary, bone marrow, liver, kidney and muscle extracts. Extracts made from the spleens of adult cocks were found to be far more efficient growth-promoting agents than extracts made from the spleens of young birds. The growth-promoting power of a tissue extract cannot always be correlated with the age of the tissue from which the extract was made, with the rate of growth of that tissue, nor with its nuclear content.