Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Transdifferentiation of hypertrophic chondrocytes into osteoblasts in murine fetal metatarsal bones, induced by co-cultured cerebrum.

The fate of hypertrophic chondrocytes in 17-day-old metatarsal bones of fetal mice was studied in a culture system in which these cells were kept confined to their lacunae. Because the periosteum had been stripped off, osteoclasts could not invade the long bone and resorb the lacunar walls. The majority of the hypertrophic chondrocytes stayed alive and dedifferentiated gradually into cells with the appearance of stromal cells. When the long bones were co-cultured with pieces of cerebrum, the chondrocytes transdifferentiated into osteoblasts. We followed this process from day to day. The cells produced bone matrix that immunostained for collagen type I and osteocalcin. To exclude with certainty the possibility that the intralacunar osteoblasts had derived from remaining periosteal osteoprogenitor cells that invaded the lacunae, the long bones were pre-cultured with cytochalasin D, which inhibits cell proliferation and migration. After removal of the drug this effect persisted until after transdifferentiation had occurred. This proved that the bone matrix producing osteoblasts inside the cartilage lacunae were transdifferentiated chondrocytes. The transdifferentiation stimulating factor from brain tissue is still unknown.

Osteoclast formation in vitro from progenitor cells present in the adult mouse circulation.

The development of multinucleated cells with tartrate-resistant acid phosphatase (TRAP) activity was studied in coverslip cultures of murine blood leukocytes and in cocultures of blood leukocytes with murine fetal bone rudiments. Cells with TRAP activity were not present among the leukocytes before culture and were absent in the bone rudiments at the time of explanation. After 14 days, macrophages with only tartrate-sensitive acid phosphatase activity developed in cultures of leukocytes without long bones. Multinucleated cells were not seen. In cocultures of leukocytes with bone rudiments, however, multinucleated cells with a strong TRAP activity had formed after 10-14 days of coculture. These TRAP-positive cells had invaded the bones and resorbed part of the calcified matrix. Electron microscopy revealed ruffled borders on the resorbing cells. In cocultures, TRAP-positive cells also formed from leukocyte fractions depleted of strongly adherent cells. Also on the cellophane supports of the cocultures, mononuclear cells with a stellate appearance and a strong TRAP activity were seen. We suggest that, in the cocultures, osteoclasts developed from a TRAP-negative, circulating progenitor cell. The presence of osteoclast progenitor cells in the circulation is discussed in light of the descent of osteoclasts from hematopoietic stem cells. That appearance of TRAP activity was always seen in resorbing cells and was not acquired in monocytes present in the leukocyte fraction by mere culture means that in the mouse TRAP is a useful marker for osteoclasts.

Stimulation of bone resorption by inflamed nasal mucosa, dermonecrotic toxin-containing conditioned medium from Pasteurella multocida, and purified dermonecrotic toxin from P. multocida.

The effects of inflamed nasal mucosa from pigs with atrophic rhinitis (AR), cell extract from Bordetella bronchiseptica, conditioned medium from Pasteurella multocida, and purified dermonecrotic toxin (DNT) from P. multocida on mouse fetal long bones in organ culture were studied. Inflamed nasal "AR mucosa" stimulated the release of 45Ca from prelabeled cultures, while histologically the formation of calcified matrix was impaired as well. B. bronchiseptica cell extract only transiently increased 45Ca release, but also impaired the formation of matrix. 45Ca release was also stimulated by DNT-containing conditioned medium from P. multocida and by purified DNT. The effect of DNT was biphasic: low doses (1 to 25 ng/ml) slightly stimulated bone resorption, higher doses were inhibitory. The stimulatory action of DNT on 45Ca release was accompanied by an increase in numbers of preosteoclasts and osteoclasts. The significance of these findings for the pathogenesis of AR is discussed.

Osteoclast generation from human fetal bone marrow in cocultures with murine fetal long bones. A model for in vitro study of human osteoclast formation and function.

Osteoclast formation in vitro from human progenitor cells was studied in cocultures of periosteum-free murine long-bone rudiments and human fetal tissues. No osteoclasts were generated from chorionic villi or from fetal liver, but bone marrow and purified bone-marrow fractions gave rise to multinucleated cells that resorbed calcified cartilage matrix. These polykarya react very strongly for tartrate-resistant acid phosphatase (TrAP) and upon ultrastructural examination show large ruffled borders in areas of resorption. Resorption of murine calcified cartilage matrix by human osteoclasts was less than resorption. by osteoclasts formed from murine fetal bone-marrow cells. Our results show that the murine long-bone rudiment can be used to generate osteoclasts from human sources of progenitor cells and to assess the biological activity of the formed osteoclasts. This coculture system thereby offers possibilities to study human osteoclast pathology in vitro. The use of TrAP as marker for osteoclasts in cell cultures is discussed.

Influence of beta-D-xyloside on growth and histological aspect of long bones in chicken embryos.

It is known that beta-D-xylosides interfere with the proteoglycan synthesis in several tissues. A possible influence of this disturbed synthesis on the matrix formation of bone and cartilage has not been described light microscopically. In the present study we used 10-day-old chicken embryos which were exposed in ovo to a final concentration of 0.5 mM beta-D-xyloside. After 3, 6, 9, 20, 25, 31, 35 and 40 days, lengths of several skeletal elements were determined and the middle metatarsal bones were processed for light microscopical demonstration of acidic groups. The results demonstrate that beta-D-xyloside inhibits growth of long bones and induces synthesis of a cartilage matrix with a very low concentration of chondroitin sulphate. It has no noticeable influence on the amount of acidic groups in the organic bone matrix. Despite the absence of chondroitin sulphate, the cartilage matrix becomes mineralized normally.

Skin inhibits cartilage proliferation and calcification. Tissue culture of fetal mice bones.

In an in vitro study, fetal mice metatarsal bones were cultured with skin or muscle fragments at some distance. We found that skin fragments inhibit growth in length and progress of calcification. Muscle fragments had no significant effects. Apparently, skin cells may produce factors that inhibit the development of metatarsal bones.

Formation sites and distribution of osteoclast progenitor cells during the ontogeny of the mouse.

The presence of osteoclast progenitor cells in embryonic, fetal, young growing, and adult murine tissues and organs was investigated in a coculture system with fetal metatarsal bones stripped of periosteum and not yet invaded by osteoclasts. Osteoclasts were found to originate from the early yolk sac and from every tissue tested in the fetus and young mouse. In the adult mouse they were formed only from tissues with a large mononuclear phagocyte population. No osteoclasts could be generated from the young embryo proper, prior to establishment of the vascular connection with the yolk sac. Progenitors of osteoclasts or their stem cells therefore do not develop from undifferentiated mesenchyme outside the yolk sac, but are distributed from the yolk sac to embryonic tissues and hematopoietic organs through the vascular circulation. The embryonic distribution of osteoclast progenitors coincides with the distribution of immature macrophages. Furthermore, they are present before the formation of monocytes in the fetus. The results also indicate that osteoclast precursor cells are not identical with mature, differentiated macrophages, but are cells with little capacity to phagocytose and therefore are, at the most immature progenitors of macrophages or cells of an early diverging lineage. In view of these results the derivation of osteoclasts is discussed.

Fusion disability of embryonic osteoclast precursor cells and macrophages in the microphthalmic osteopetrotic mouse.

Osteoclast formation in the microphthalmic osteopetrotic (mi) mouse was studied from very early embryonic to newborn stages. Embryonic and fetal milmi osteoclasts, generated during the period before bone marrow is formed in the long bones, were predominantly mononuclear and lacked ruffled borders. These cells did, however, show many osteoclastic morphologic and functional properties, such as an abundance of mitochondria, positive succinic dehydrogenase and acid phosphatase reactions, and close contact with and resorption of the calcified cartilage matrix (though diminished). These osteoclastic mononuclear cells appeared in vivo as well as in organ cultures of fetal metatarsal bones with their intact periostea. They also were observed in cocultures of periosteum-free fetal metatarsal bones, with several extraneous sources of osteoclast precursors: yolk sacs and abdominal regions of 9- and 11-day-old embryos, fetal livers, and precultured mononuclear phagocytes isolated from the fetal liver. In contrast, +/+ osteoclasts were always multinuclear, functioned normally in resorbing the calcified cartilage matrix, and had ruffled borders in vivo as well as when derived from the above-mentioned sources. Fetal liver-derived milmi macrophages also failed to form multinuclear foreign body giant cells as opposed to +/+ macrophages in granulomas on implanted pieces of Melinex. The fusion failure of cells derived from embryonic and fetal extramedullary milmi monocyte/macrophage sources contrasted with the occurrence of multinuclear osteoclasts and foreign body giant cells derived from precursors from the bone marrow in young milmi mice. We conclude that the fusion defect of milmi osteoclast precursor cells is already present in their ancestry in blood cell-forming organs of very young embryos and that these cells differentiate into mononuclear osteoclasts that function inefficiently in prenatal bone. We presume that in fully developed bone marrow, local factors are favorable for abolishing the fusion defect.

The role of mesenchyme in embryonic long bones as early deposition site for osteoclast progenitor cells.

Metatarsal bone rudiments taken from 12- to 17-day-old mouse embryos were cultivated as organ cultures and/or transplanted on to the chorioallantoic membranes of Japanese quail embryos, with or without the adhering surrounding mesenchyme. In cultivated explants the presence of mesenchyme was essential for the development of osteoclasts. This mesenchyme contained small blood vessels. In transplants with adhering mesenchyme, graft (mouse)-derived osteoclasts predominated, whereas in transplants without surrounding mesenchyme the osteoclasts originated from host (quail) cells. Distinction could be made between mouse- and quail-derived osteoclasts because of the specificity of the chromatin pattern of quail cell nuclei. Precartilaginous anlagen of metatarsals precultured before transplantation, displayed mouse-derived osteoclasts, thus indicating that osteoclast progenitor cells home into the long bone anlage very early, in this case at least 6 days before the appearance of osteoclasts in vivo. During embryonic development, osteoclast progenitor cells could very well be (as in the adult situation) hematopoetic cells conveyed to the site of long bone development by the circulating blood as soon as distribution of these cells starts from central blood-cell forming organs to the periphery. Mesenchyme in and around the long bone region seems to play a role as early deposition site of these cells where proliferation, differentiation, and fusion of osteoclast progenitor cells take place and are controlled.

Accumulation and localization of gallium-67 in various types of primary lung carcinoma.

The uptake and location of Ga-67 were investigated in 15 primary pulmonary carcinomas. The accumulation in the tumor was determined by scintigraphy of the patient, grain counts over fields of tumor cells in autoradiographs of tumor-tissue samples, and gamma counts in specimens of the tumor. Good correlation was found between the results obtained with these three methods. The relationship between accumulation of Ga-67 in the tumor and the histologic type of tumor was also studied. Undifferentiated carcinomas, and tumor cells in squamous-cell carcinomas showed significantly more Ga-67 than tumor cells in adenocarcinomas. No correlation was found between the presence of inflammatory infiltrates in or around the tumor and the grade of the scintigraphic images. In the autoradiograms, lymphocytes, plasma cells, granulocytes, and macrophages showed less radioactivity than the tumor cells--or none at all. Collagen fibers appeared to have bound some Ga-67, but necrotic areas showed no uptake.

Autoradiographic model experiments with 67Ga and 99mTc.

In order to evaluate the feasibility to localize correctly 67Ga citrate and 99mTc pertechnetate in tissues, the resolution of these radioactive compounds were measured in a model system using four different autoradiographic techniques, wet as well as dry. Wet autoradiographic techniques gave an almost complete loss of 67Ga. In deparaffinized sections of fixed and paraffin-embedded tissue the remaining 67Ga, which was probably bound to proteins, gave a resolution of 2.6 mu. 99mTc was either completely lost in wet techniques or the procedure could not be performed at all because of the very short half life of 99mTc. The resolution of 67Ga in a dry autoradiographic technique (according to Stumpf) was 6.9 mu and the resolution of 99mTc 22.6 mu. The technique in which frozen sections are thawed on dry film and consequently dryed, gave slightly better resolutions than the dry technique (according to Stumpf) with 67Ga as well as 99mTc. It is concluded that for the histological localization of 67Ga and 99mTc a dry technique is required. However, the use of a wet technique can be considered, provided a loss of the radioisotope is acceptable and the procedure is controlled by a dry technique.