Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I.

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Procollagen I COOH-terminal fragment induces VEGF-A and CXCR4 expression in breast carcinoma cells.

The COOH-terminal fragment of procollagen type I (C3) is produced in tissues with high synthesis of collagen I, such as in breast cancer stroma and in bone. We previously demonstrated that C3 is chemoattractant for breast carcinoma and endothelial cells, and that in tumor cells it induces expression and activation of metalloproteinases (MMP) -2 and -9. Here we demonstrate that C3 induces expression of vascular-endothelial growth factor (VEGF) and of CXCR4, the receptor of the CXCL12/SDF-1 chemokine, in MDA MB 231 breast cancer cells. We show that the changes in gene expression and motility induced by C3 occur in a timely succession and are mediated by multiple and different signaling pathways. C3 induces early phosphorylation of p38/MAPK. Induction of VEGF expression requires continual activity of p38/MAPK and of Protein Kinase C (PKC). Pro-MMP-2 and -9 are induced through a signaling pathway involving G0alpha.i protein, and cell migration requires the activity of a combination of these signaling pathways. Our results suggest that C3 acts as a stromal-derived, cancer-promoting agent active in inducing the migratory phenotype and the survival of cancer cells and determining timely changes in their gene expression that establish conditions promoting tumor angiogenesis and invasion.

FMS*Calciumfluor specifically increases mRNA levels and induces signaling via MAPK 42,44 and not FAK in differentiating rat osteoblasts.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre-osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP-2 and -14), procollagenase C (BMP-1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*-treated osteoblasts than in control cultures. MMP-2 and -14 mRNA are not down-modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP-1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen-activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre-osteoblasts, with a cell differentiation stage-dependent and pertussis toxin (PTX)-sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP-14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)-mediated signaling.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.

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.

Trimer carboxyl propeptide of collagen I produced by mature osteoblasts is chemotactic for endothelial cells.

During the second phase of osteogenesis in vitro, rat osteoblasts secrete inducer(s) of chemotaxis and chemoinvasion of endothelial and tumor cells. We report here the characterization and purification from mature osteoblast conditioned medium of the agent chemotactic for endothelial cells. The chemoactive conditioned medium specifically induces directional migration of endothelial cells, not affecting the expression and activation of gelatinases, cell proliferation, and scattering. Directional migration induced in endothelial cells by conditioned medium from osteoblasts is inhibited by pertussis toxin, by blocking antibodies to integrins alpha(1), beta(1), and beta(3), and by antibodies to metalloproteinase 2 and 9. The biologically active purified protein has two sequences, coincident with the amino-terminal amino acids, respectively, of the alpha(1) and of the alpha(2) carboxyl propeptides of type I collagen, as physiologically produced by procollagen C proteinase. Antibodies to type I collagen and to the carboxyl terminus of alpha(1) or alpha(2) chains inhibit chemotaxis. The chemoattractant is the propeptide trimer carboxyl-terminal to type I collagen, and its activity is lost upon reduction. These data illustrate a previously unknown function for the carboxyl-terminal trimer, possibly relevant in promoting endothelial cell migration and vascularization of tissues producing collagen type I.

FMS*Calciumfluor increases alkaline phosphatase expression during osteogenesis in vitro of tibia-derived rat osteoblasts by activation of G alpha 0/G alpha i proteins.

We studied the involvement of G-proteins in transducing the inductive signal generated by treatment of tibial-derived neonatal rat osteoblasts (ROB) cultured in vitro with FMS*Calciumfluor, a homeopathic preparation utilized in the therapy of osteoporosis. We previously reported that FMS*Calciumfluor acts as inducer and potentiator of osteogenic differentiation in vitro, among other effects, by increasing the expression of Alkaline phosphatase (AP). We utilized Pertussis Toxin (PTX), an inhibitor of G alpha 0/G alpha i proteins, Mastoparan 7, an activator of G alpha 0/G alpha i proteins and Cholera Toxin (CTX), a stimulator of G alpha s protein to show involvement of specific G proteins in the inductive effect on AP of FMS*Calciumfluor. We here show that the increase in AP expression induced by FMS*Calciumfluor is dependent on the activation of G alpha 0/G alpha i proteins, while it is unaffected by the activation stage of the G alpha s protein. Moreover, we show that the expression of endogenous AP during osteogenesis in vitro is regulated independently from G proteins, and unaffected by their activation stage and therefore that treatment with FMS*Calciumfluor activates a new pathway of cellular response.

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.

Osteoblasts modulate secretion of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in human prostate cancer cells promoting migration and matrigel invasion.

Prostate carcinoma (PRCA) cells metastasize to the skeleton with high frequency. Bone stores growth regulatory factors, which are released in active form during bone remodeling. We propose that bone cell-derived growth factors may induce the development of PRCA bone metastasis by recruiting tumor cells and increasing their proliferation in the bone microenvironment. Serum-free conditioned medium harvested from osteoblast cultures (OB CM) stimulated the in vitro chemotaxis of PRCA cells and invasion of a reconstituted basement membrane (Matrigel), suggesting enhanced invasive activity. Preosteoblastic cell CMs were less effective than CMs obtained from mature OB. CMs harvested from differentiated osteoblast cultures capable of matrix mineralization were more active compared to CMs from proliferating osteoblasts. OB CMs stimulated secretion of urokinase (uPA) and matrix metalloproteinase-9 (MMP-9). Inhibition of these matrix-degrading proteases by neutralizing antibodies and/or by inhibitors of their catalytic activity reduced Matrigel invasion. Secretion of uPA and activation of MMP-9 were most prominent by differentiated OB CMs with respect to poorly differentiated cells in vitro. These results are in agreement with several in vivo studies and indicate that factors produced during osteogenesis by bone cells stimulate PRCA cell chemotaxis and matrix proteases expression, thus representing potential targets for alternative therapies deterring the progression of PRCA metastasis to bone.

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.

Integrin synthesis and utilization in cultured human osteoblasts.

This study describes the adhesion of human osteoblasts, cultured in vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77-100%, in 2 h and at 55 nM substrata concentration, and it was accompanied by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the beta 1 integrin and antibodies to the alpha 5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against alpha 2, alpha 3, alpha 5, alpha v, beta 1 and beta 3 integrins we detected synthesis of alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and an alpha v beta 1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus alpha 4, alpha 1 and beta 5 subunits. In cells adhering in the presence of serum we showed organization of beta 3 and alpha v integrins in focal contacts. In cells adhering to fibronectin alpha 5 and beta 1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.

Progesterone receptors are expressed in human osteoblast-like cell lines and in primary human osteoblast cultures.

Expression of progesterone receptors (PR) was studied in human osteoblast-like cell lines and primary human osteoblast cultures at the molecular level. Using the sensitive reverse transcriptase polymerase chain reaction (RT-PCR) and oligonucleotide primers which flank the progesterone-binding domain of human PR, progesterone receptor (PR) mRNA was detected in three osteoblast-like cell lines--HOS-TE85, MG-63, and SAOS-2. When compared with beta-actin gene expression, levels of PRmRNA transcripts varied between cell lines (PRmRNA in HOS-TE85 > MG-63 >> SAOS-2). In addition, RT-PCR confirmed the presence of PRmRNA transcripts in primary human osteoblast cells cultured from collagenase-treated bone. Immunostaining was used to visualize PR protein in cells. All osteoblast-like cell lines showed specific staining for PR. Immunoreactivity was distributed equally in the nucleus and cytoplasm. The level of staining was significantly lower than that detected in PR-positive MCF-7 breast cancer cells though well above background levels obtained for PR-negative HeLa cells. The finding that PR is expressed at both the level of mRNA and protein in several osteoblast-like cell lines as well as in human primary osteoblast cultures indicates that bone-forming osteoblast cells are direct targets for progesterone action.

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.

Modulation of integrins expression during human osteoblasts "in vitro" differentiation.

We here report the modulation of the adhesion of cultured human osteoblasts on Laminin during the acquisition of differentiated phenotype. We also show that interference with the differentiation program caused by treatment with Retinoic acid of the cultures, causes changes of the capability to adhere to Laminin and type I Collagen. The younger or dedifferentiated cells have lower capability to bind to Laminin or Collagen. The maturation associated changes are specific for the adhesion to the above substrata and do not involve the adhesion to FN or plastic. The alpha subunit(s) of the integrin receptor(s) for these proteins is likely to be responsible for the modulation adhesion to Laminin and Collagen.

Differential expression of alkaline phosphatase in clones of human osteoblast-like cells.

We established cultures of cells growing out from adult bone chips and maintained them through 12 passages in culture. The cultures showed osteoblastic phenotype accompanied by synthesis of collagen type I, osteonectin, alkaline phosphatase, and osteocalcin. We report the characterization of 21 clones obtained from three different individual primary cultures. We studied the expression of osteonectin, alkaline phosphatase, collagen, and osteocalcin in the clones. Metabolic labeling showed production of type I collagen and of osteonectin in all clones studied. In two-thirds of the clones and in mass cultures alkaline phosphatase was not detected at passage 2, but it was detected in increasing amounts at later passages in culture. The clones attained different but detectable levels of expression of this marker by passage 8. The different levels in the expression of alkaline phosphatase in positive clones may be because they were derived from cells at different stages of osteoblastic maturation or due to small changes in microenvironment. The alkaline phosphatase-positive clones were tested for osteocalcin, and they showed measurable expression only at passage 10. A third of the clones obtained were negative for alkaline phosphatase during 12 passages in culture. The obtainment of clones unable to produce alkaline phosphatase may be due to loss of differentiating potential under the in vitro culture conditions. The growth rate and potential of all clones studied were similar through 12 passages in culture, regardless of their potential for expression of alkaline phosphatase.

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.

Hypertrophic chondrocytes undergo further differentiation in culture.

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.