The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

Osteogenesis in vitro in rat tibia-derived osteoblasts is promoted by the homeopathic preparation, FMS*Calciumfluor.

We studied the effects of in vitro treatment of differentiating osteogenic cells with FMS*Calciumfluor, to determine whether it caused changes in proliferative or differentiation potential of osteoblasts. FMS*Calciumfluor was developed for the therapy of post-menopausal and age-related osteoporosis on the basis of the principles of resonance homeopathy and VTR Vega test. Its daily prescribed therapeutical usage is about 30,000-fold less in fluoride concentration than that recommended for NaF associated with calcium monophosphate. Rat tibial osteoblast (ROB) primary cultures represent populations of early osteoblasts and their derivative cultures of more than 60 cumulative population doubling (CPD) represent more mature osteogenic cells. Both these populations were shown to undergo in vitro differentiation, as monitored by the sequential expression of markers that define the stages of the osteogenic progression. Here we report that continual treatment of ROB during osteogenesis with FMS*Calciumfluor modulated the expression of critical osteogenic markers: alkaline phosphatase (AP), an indicator of osteoblast maturation, and(45)Ca incorporation into the matrix and nodule formation, events of the last phase of osteogenesis and a measure of osteoid mineralization. Treatment did not affect proliferation, or expression and activation of metalloproteinases (MMP). AP activity and levels of AP mRNA were increased by treatment with FMS*Calciumfluor; the incorporation of radiolabelled Ca into the matrix was also increased and the formation of nodules occurred in a shorter time and with higher frequency than in untreated control cultures. The effects of FMS*Calciumfluor were concentration dependent and specific for its modalities of preparation, and were observed at a concentration about three orders of magnitude lower than similar effects reported in the literature by treatment of osteoblast cultures in vitro with NaF.

Rat tibial osteoblasts III: propagation in vitro is accompanied by enhancement of osteoblast phenotype.

Postproliferative confluent cultures of primary rat tibial osteoblasts (ROB), cultured in medium supplemented with ascorbic acid and beta-glycerophosphate (AS-bGP, differentiation medium) express, in sequence, specific bone markers which identify a succession of maturation stages, and eventually form mineralized noduli. We report an investigation on the effect of extensive proliferation in vitro in unsupplemented medium on the osteogenic potential of mass cultures of ROB. The growth rates of the populations, derived from two independent primary cultures, was constant throughout 110 cumulative population doublings (CPD) in culture. Propagated cells maintained features similar to osteoblasts in primary cultures with respect to serum and anchorage dependence for growth and to the chemokinetic effect on endothelial cells exerted by their conditioned media (CM). Propagated populations, set at confluence in differentiation medium, were tested for the expression of early [alkaline phosphatase (AP)] and late [osteocalcin (OC); bone sialoprotein (BSP); 45Ca incorporation and mineralization] osteogenic markers. We observed an increase, parallel to the increase in CPD, in both the level of maximal expression of AP (enzyme/microgram cellular DNA) and in the frequency of nodules, reaching five- to sixfold (at 78 CPD) and eightfold (at 60 CPD), respectively, the levels of primary cultures. AP expression (enzyme and mRNA) persisted during mineralization and 45Ca incorporation. The time required by propagated cultures for the formation of nodules decreased with increase of CPD, and was reduced to less than one third at 87 CDP. Nodules became mineralized over a similar lapse of time as in primary cultures and were positive by histochemistry for BSP and OC. We also obtained osteogenic clones from two independent cultures after 72 CPD. 90% of these showed an osteoblast phenotype, expressing AP and forming nodules positive for OC and BSP, which mineralized. Timing of formation and frequency of nodules/plated cells in clones was similar to that found in propagated cultures of equivalent CPD. In summary, propagated ROB populations and derived clones showed enhanced osteoblast phenotype, possibly due to an increase in osteogenic cells and enrichment of proliferating mature osteoblasts, consequent to extended propagation in culture.

Osteoblastic cells from rat long bone. II: Adhesion to substrata and integrin expression in primary and propagated cultures.

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immunohistochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, alpha 5 beta 1, alpha v beta 3, alpha 3 beta 1, alpha 6 beta 1 and alpha 1 beta 1 integrin heterodimers; furthermore beta 5, alpha 2 and alpha 4 chains were detected by immunoblots and indirect immunofluorescence. alpha v, alpha 1, alpha 6 subunits in most cells, and beta 3 and beta 1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III(COL III) by ROB at different doubling (1.5-11) was dependent on substratum concentration, and after 2.5 h at 55 nM 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell-matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.

Effect of osteoblast supernatants on cancer cell migration and invasion.

Malignant tumor cells of different origin seem to have preferential sites for metastasis. Breast cancer, prostate cancer and certain melanomas have bone as one of their preferential targets for metastasis. Bone is continuously being remodelled, a process largely controlled by local growth factors. A possible explanation for malignant cell recruitment to bone is that osteoblast products, directly secreted or released from the matrix by osteoclast resorbing activity, are able to stimulate cancer cell migration. To test this hypothesis we have utilized an in vitro system of differentiating osteoblasts which in culture progress all the way to the formation of mineralized nodules. Conditioned media obtained from these osteoblast cultures at different stages were able to induce chemotactic migration and invasion of both melanoma and breast cancer cells. The migratory and invasive phenotype was accompanied by enhanced gelatinolytic activity of osteoblast stimulated cancer cells. Our data suggest that osteoblasts secrete potent factors able to direct tumor cell migration towards remodelling bone.

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture.

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA hy 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and beta-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. 45Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured tibial rat osteoblasts: in vitro production and topography of osteonectin, biglycan and decorin.

Rat osteoblasts in culture undergo differentiative changes culminating in the formation of mineralized foci. We here report on the pattern of temporal expression and compartmentalization of osteonectin and of the two small proteoglycans, byglican and decorin. They were constitutively synthesized during in vitro differentiation of rat osteoblasts. The 3 proteins were detected in the conditioned medium and associated with the cell-matrix compartment. Within this compartment they showed prevalent cytoplasmic location and differential distribution on unmineralized noduli was detected for osteonectin and byglican, while decorin was detected throughout the nodules. Along with known functions in the matrix, a possible role in the cytoplasm may have to be sought for these bone cells components.