Tumor induced by Moloney sarcoma virus causes periosteal osteogenesis engaging osteopontin, fibronectin, stromelysin-1 and tenascin.

Excessive bone formation occurring in such conditions as paravertebral ligamentous ossification, hallux osteophytes or some neoplastic tumors, presents a significant problem, both epidemiological and clinical. Since pathogenesis of this disorder is still unclear, we studied its mechanism in experimental model utilizing inducible orthotopic osteogenesis. Periosteal bone apposition stimulated by Moloney sarcoma is characterized by unusually high volume of new bone tissue appearing subperiosteally in the bone adjacent to the tumor. Genes engaged in this growth have not been characterized so far. Here we show the results of mRNA Representation Difference Analysis in Moloney sarcoma, which reveal high expression of four genes coding extracellular matrix proteins: osteopontin, fibronectin, stromelysin-1 and tenascin. These findings suggest that the uncommon dynamics of the Moloney sarcoma-induced osteogenesis depends on high expression of these extracellular matrix proteins.

Whole body X-irradiation and repopulation with syngeneic bone marrow have no significant effect on basophils and mast cell compartment of murine lymph nodes.

In mice the atrophy of lymph nodes following a whole body X-irradiation with a dose of 800 R is not followed by a depletion of their basophils and mast cells content. Repopulation of irradiated mice with syngeneic bone marrow cells has no significant effect on the basophils and mast cells number in the popliteal lymph nodes.

Heterotopically induced bone does not develop functional periosteal membrane.

Heterotopic bone induced in mice by implantation of demineralized rat bone matrix does not respond to Moloney sarcoma. The biochemical and morphological parameters of osteogenesis (alkaline and acid phosphatase activity, 45Ca-uptake and total calcium content) are similar in the sites of Moloney sarcoma as in the contralateral, unexposed to the sarcoma, side. In contrast, periosteum of implants of syngeneic costal bone in response to Moloney sarcoma proliferates and produces new bone in the same manner, as orthotopic bones at the sites of Moloney sarcoma do. It is concluded that ectopically induced bones do not develop a true periosteum.

Association of giant multinucleated cells with deterioration of osteoinductive potency of bone matrix.

A thirty four intramuscular implants of rat decalcified bone matrix into mice and rats were histologically inspected in order to find out if there is an inverse correlation between the induction of multinucleated giant cell formation and the bone induction activity. It was found that the presence of multinucleated giant cells which avidly resorbed the implanted matrix was almost always combined with the lack of local bone induction. In contrast, the specimens exhibiting intense bone induction were devoided of multinucleated giant cells. Our observation confirm the opinion expressed by Walters and Schneider that multinucleated giant cells induced by mineralized bone chips are not osteoclasts but a type of inflammatory cells, analogous if not identical to the "foreign body cells". It is postulated that formation of multinucleated giant cells is a morphological expression of hyperactivity of mononuclear phagocytes. Hyperactive multinucleated giant cells avidly resorb bone matrix and degrade bone inducing substance(s) like skeletal growth factor and/or bone morphogenetic peptides resulting in the deterioration or lack of bone induction.

Mast cells in regional lymph nodes responding to syngeneic and xenogeneic tumour MSVC and HeLa cell lines in BALB/c mice.

In response to primary and secondary stimulation by xenogeneic HeLa carcinoma cells or by syngeneic sarcoma MSVC cells the regional lymph nodes of BALB/c mice are enlarged, however, their absolute mast cells content does not increase and in relative terms a decrease of mast cells concentration is observed. These results are in accordance with our earlier observations that during antigeneic and T-cell mitogen stimulation the lymphatic mast cells population does not increase but is rather reduced. This may indicate the engagement of these cells in delayed-type hypersensitivity.

Evaluation of locally induced osteoarthritis by the complete and incomplete Freund's adjuvant in mice. The application of DEXA measurements.

The inflammatory reactions elicited in mice by subcutaneous injections of IFA and CFA had opposite effects when tested on local metacarpal shank bones and the distal epiphysis of shank bones. Although the intensity of the immune reactions was similar, IFA induced bone loss, while CFA induced bone formation, which was mostly periosteal in nature. BMC and BMD measurements were assessed by means of high resolution DEXA, using a hologic 4500A bone scanner with software dedicated for the analysis of small animal bones. DEXA scans were evaluated and related to histological and bone ash content analyses. The morphological and quantitative ash weight analyses of bones exposed to the adjuvants were consistent with DEXA bone density scan measurements.

Biochemical parameters of osteogenesis in epiphysis of mice irradiated and repopulated with syngeneic bone marrow.

Whole-body irradiation of BALB/cann mice with 600 R of gamma rays produces a profound atrophy of the spleen and diminishes alkaline phosphatase activity in the homogenates of epiphysis. The acid phosphatase activity remains unchanged until day 10, then slightly declines. Incorporation of 45Ca into epiphysis is practically not impaired following irradiation. Repopulation of irradiated animals with 1-1.5 million nucleated syngeneic bone marrow cells restores spleen weight within 6 days, but until day 11 the activity of alkaline phosphatase of epiphysis remains lower. Full recovery of enzyme activities was not observed until day 14, but on day 28 these activities returned to the normal level. Histological inspection did not reveal a full recovery of bone marrow in the epiphysis of irradiated animals until day 14, possibly because of trapping of the vast majority of stem cells by the spleen and liver. On day 28, however, epiphyses were completely filled with the bone marrow.

Osteoblastic and chondroblastic response to a variety of locally administered immunomodulators in mice.

Administration into mouse shank muscles of various immunomodulators which directly or indirectly activate lymphocytes (BCG, Con A, Carrageenan IV, Dextran, PHA-M, PWM, lipopolysaccharides from Corynebacterium) have a dual effect on local bones, stimulating both periosteal bone formation and bone resorption, the former being dominant. These effects vary in frequency and magnitude, the most potent stimulation of periosteal bone formation being observed after administration of Con A and BCG in complete Freund adjuvant. BCG was also a strong inducer of bone resorption in vivo. Concanavalin A and, to a lesser degree, other immunomodulators applied, when administered subcutaneously into the pinna, also have induced perichondrial chondrogenesis. These novel effects of immunodulators could be applied in the field of skeletal tissues regeneration.

Non-adherent bone marrow cells are a rich source of cells forming bone in vivo.

Syngeneic, allogeneic and xenogeneic (rat) freshly isolated bone marow cells + stromal cell cultures maintained in vitro for 10-30 days, as well as non-adherent cells removed from these cultures on 3rd-4th day were injected into the kidney parenchyma of mice, immunosuppressed with hydrocortisone. In syngeneic grafts the immunosuppression was omitted. In all transplant systems bone tissue was formed inside the kidney with 20% to 32% variation. Bone produced by allogeneic and xenogeneic cells is subject to rejection when immunosuppression ceases, as the bone formed is of donor origin. The "floating" cells, regardless of the transplant system, normally discarded during media replacement, turned out to be efficient bone producers. This notion is of practical implication when bone marrow cells are used for bone healing.

Mast cells and basophils in murine lymph nodes stimulated by B-cell mitogen.

The total number of mast cells and basophilic granulocytes in lymph nodes activated in vivo by a B-cell mitogen (formaldehyde-fixed S. paratyphi B) remains unchanged in spite of a significant enlargement and cellularity of the lymph nodes. This is in contrast to the previous finding showing that T-cell mitogens reduce the number of granular cells in the lymph nodes.

In vivo exposure to sodium fluoride does not modify the yield of viral tumour-induced periosteal bone nor of heterotopic bone induced by human tumour KB cells in mice.

In mice local or systemic administration of fluoride (5-25 mg NaF/kg body weight) during the proliferative phase of bone formation (up to 20 days) has no effect on the yield of bone formed either by local stimulation of periosteal membrane by Moloney sarcoma virus-induced tumour or on bone induced heterotopically by human KB cells. The lack of stimulatory activity of fluoride on rapidly induced osteogenesis in mice is in agreement with recent reports which show that fluoride is not a potent mitogen for human osteoblasts grown in vitro.

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells.

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.

Fluvastatin increases heterotopically induced ossicles in mice.

1. The aim of the present study was to evaluate the influence of fluvastatin (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor) on heterotopic ossification (HO) induced by HeLa cells. 2. C57Bl/6 mice were injected with 3 x 10(6) HeLa cells into right thigh muscles. Mice in the experimental group received fluvastatin 1.2 mg/kg per day for 17 consecutive days, while mice in the control group received placebo. Intact mice served as an additional control. Seventeen days post-HeLa cell grafting, blood samples were collected to measure total serum cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol and alkaline phosphatase (AP). 3. In all animals injected with HeLa cells, the mass of mineral deposited in the induced ossicle was established after hydrolysis of soft tissues surrounding the induced ossicles. In fluvastatin-treated mice, the mass of mineral deposited in heterotopically induced ossicles was significantly increased, when compared to mice receiving placebo. This was followed by a significant decrease of TG concentration; whereas the levels of serum AP were not significantly affected. 4. These results indicate that administration of statins may affect heterotopic ossification. This may also have clinical implication, because patients predisposed to HO and receiving statins during hypocholesterolemic treatment, may be at even greater risk of HO.

Bone histogenesis mediated by nonosteogenic cells.

Intramuscular implantation of several established, nonfibroblastic epithelial cell lines of various origin causes endochondral ossification at the site of cell graft. Heterotopically-induced bone is also a site of myelopoiesis. Myelohistograms of ectopic bone marrow do not differ substantially from that of marrow present in orthotopic (skeletal) bones. Heterotopically-induced bones are not covered by a periosteal membrane with a functioning cambium layer. The nature of the bone-inducing principle of epithelial cells is not known as yet, although it was established that cells able to induce heterotopic bone formation are characterized by a high alkaline-phosphatase activity and have an epithelial status. Cells responding to the osteoinducing principle are distributed among skeletal muscles and are absent in the peritoneal cavity or in the kidney parenchyma. Periosteal membranes of skeletal bones are activated by a variety of agents (Moloney sarcoma virus-induced tumors, numerous nonvirus-induced neoplasms, T-lymphocyte mitogens, and activated lymphocytes) to proliferate and lay down new bone. The same agents can also activate in vivo perichondral membranes. It is theorized that cells involved in antitumor reaction produce a chondroblast/osteoblast activating factor(s), in addition to the osteoclast-activating one.

Orthotopic and ectopic chondrogenesis and osteogenesis mediated by neoplastic cells.

In mice, tumors of various origins have been found to stimulate cambium layer cells of periosteum/ perichondrium of adjacent orthotopic bone or cartilage to proliferate and/or differentiate into osteoblasts or chondroblasts. Tumors may induce new bone and/or cartilage formation. In progressively growing tumors the osteogenic/chondrogenic activity is gradually surpassed by the resorptive processes mediated either by osteoclasts, directly by tumor cells, or by tumor stroma. In regressing tumors, however, the deposits of new bone remained unresorbed, resulting in a permanent gain of bone mass. In human subjects, similar changes were observed in bone adjacent to carcinoma development. Stimulation of periosteal bone formation was observed at earlier stages of the disease, while bone resorption mainly by tumor cells and their stroma was observed in later stages of tumor development. The unresponsiveness of the heterotopically-induced bone to the Moloney sarcoma virus, in contrast to the response of orthotopic bone clearly indicates that ectopic bones do not develop a true periosteum.