Formation of bone by isolated, cultured osteoblasts in millipore diffusion chambers

Simmons, David J.; Kent, G.N.; Jilka, Robert L.; Scott, Douglas M.; Fallon, Michael D.; Cohn, David V.

doi:10.1007/bf02411253

Cited by 71 publications

(36 citation statements)

References 20 publications

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“…Increased alkaline phosphatase activity, PTH-stimulated cAMP production, and production of type I collagen are features characteristic of the osteoblastic phenotype in bone and osteosarcomaderived cells (32,34). Cells expressing these characteristics are capable of synthesis and assembly of mineralized matrix in vitro (35)(36)(37)(38) or in vivo (39). Osteogenin significantly enhanced the expression of the osteoblastic markers in vitro in primary osteoblast cultures.…”

Section: Discussionmentioning

confidence: 99%

Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.

Vukičević

Luyten

Reddi

1989

Proc. Natl. Acad. Sci. U.S.A.

208

100

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Osteogenin was recently purified and the amino acid sequences of tryptic peptides were determined. Osteogenin in conjunction with insoluble coliagenous bone matrix induces cartilage and bone formation in vivo. To understand the mechanism of action of osteogenin, we examined its influence on periosteal cells, osteoblasts, fibroblasts, chondrocytes, and bone marrow stromal cells in vitro. Osteogenin stimulated alkaline phosphatase activity and collagen synthesis in periosteal cells. The cAMP response to parathyroid hormone in periosteal cells was increased by osteogenin. In primary cultures of calvarial osteoblasts, osteogenin stimulated alkaline phosphatase activity, the cAMP response to parathyroid hormone, and the synthesis of collagenous and noncollagenous proteins; however, cell proliferation was not affected. Osteogenin increased the production of sulfated proteoglycans in fetal rat chondroblasts and in rabbit articular chondrocytes. The present experiments demonstrate the significant influence of osteogenin in the stimulation of osteogenic and chondrogenic phenotypes in vitro.Bone has a considerable potential for regeneration. However, the cellular and molecular mechanisms of bone regeneration are not clear. The presence of growth and differentiation factors in bone has been demonstrated by implantation of demineralized diaphyseal bone matrix in intramuscular and subcutaneous sites and results in local cartilage and bone formation (1, 2). The developmental cascade involves the following sequential steps: chemotaxis and attachment of mesenchymal stem cells, proliferation of progenitor cells, and differentiation of cartilage, bone, and hematopoietic marrow (3-5). Osteogenin, a bone-inductive protein, was isolated by heparin affinity chromatography (6). It was further purified and the amino acid sequences of tryptic peptides were determined and found to be unique (7). The amino acid sequence of bovine osteogenin (7) was similar to the amino acid sequence deduced from the cDNA clones of one of the human bone morphogenetic proteins, bone morphogenetic protein 3 (8). Whereas recombinant human bone morphogenetic proteins induced only cartilage (8), osteogenin purified from bovine bone matrix induces cartilage and bone formation in vivo (7, 9). To understand the mechanism of action of osteogenin, we have examined its influence on periosteal cells, osteoblasts, fibroblasts, chondrocytes, and bone marrow stromal cells in vitro. We report that osteogenin stimulates the expression of the osteogenic phenotype in periosteal cells, osteoblasts, and bone marrow stromal cells and of the chondrogenic phenotype in chondroblasts. MATERIALS AND METHODSPreparation of Purified Osteogenin. Osteogenin was purified as described (7). The method utilized extraction of demineralized bovine bone matrix with 6 M urea and purification by hydroxyapatite, heparin-Sepharose affinity chromatography, and molecular sieve chromatography on Sephacryl S-200. The most active fractions were further purified by preparative SDS/PAGE under nonr...

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Section: Discussionmentioning

confidence: 99%

Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.

Vukičević

Luyten

Reddi

1989

Proc. Natl. Acad. Sci. U.S.A.

208

100

View full text Add to dashboard Cite

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“…Preparation of Neonatal Calvarial Osteoblasts-Murine calvarial cells were prepared using a modification of a protocol described previously (15)(16)(17)(18). Briefly, calvaria from 2-to 3-day neonatal mice were pretreated with 10 mM EDTA in phosphate-buffered saline (PBS) for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…The calvaria were then subject to sequential collagenase digestions, and cells were collected. Fractions 3-5 were used as the starting population that consisted of ϳ95% OB or OB precursors, as determined by a variety of criteria (15,17,18). For experiments with MKs, OBs were seeded at 2 ϫ 10 4 cells/ml in 6-well plates or in 10-cm 2 dishes.…”

Section: Methodsmentioning

confidence: 99%

Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts

Kacena

Eleniste

Cheng

et al. 2012

Journal of Biological Chemistry

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Background: Osteoblast proliferation and differentiation are critical for bone formation. Results: Megakaryocytes increase osteoblast number and BrdU incorporation and induce cytoskeletal remodeling and the differential expression of Pyk2 isoforms in osteoblasts. Conclusion: Megakaryocytes stimulate osteoblast proliferation and alter the ratio of alternatively spliced Pyk2 isoforms. Significance: Pyk2 may be an important target for treatments aimed at increasing skeletal bone formation.

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“…Based on an underlying deficiency in Nf1 þ/À osteoprogenitors, 13,14 as well as questionable clinical responsiveness to BMPs, 24 we hypothesized that mice deficient in Nf1 would show reduced sensitivity to BMP treatment. We undertook experiments to examine this ex vivo using an osteoblast primary cell culture system 28 and in vivo using surgical implantation of recombinant human BMP-2. 29 To test whether bisphosphonate treatment could preserve BMP-2-induced bone in our surgical implantation model, wild-type (Nf1…”

Section: Introductionmentioning

confidence: 99%

Modeling bone morphogenetic protein and bisphosphonate combination therapy in wild‐type and Nf1 haploinsufficient mice

Schindeler

Ramachandran

Godfrey

et al. 2007

Journal Orthopaedic Research

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Recombinant bone morphogenetic proteins (BMPs) show promise in treating the orthopedic complications associated with neurofibromatosis type 1 (NF1), such as congenital pseudarthrosis of the tibia. Minimal scientific information regarding the effects of BMP in the context of NF1 is available. As abnormalities in both bone formation and resorption have been documented in Nf1-deficient mice, we hypothesized that inadequate BMP-induced bone formation could be augmented by cotreatment with the bisphosphonate zoledronic acid (ZA). First, primary osteoblasts isolated from wild type (Nf1) mice were cultured in the presence and absence of BMP-2. While Nf1 þ/À cells exhibited less osteogenic potential than Nf1cells, alkaline phosphatase expression and matrix mineralization for both genotypes were enhanced by BMP-2 treatment. To model this response in vivo, 20 mg BMP-2 was implanted intramuscularly into the quadriceps of mice to induce heterotopic bone. Radiographs revealed significantly less net bone formation in Nf1 þ/À mice compared to Nf1 þ/þ controls. To test the effect of an antiresorptive agent, mice were cotreated twice weekly from postoperative day 3 with 0.02 mg/kg ZA or with saline. ZA treatment led to a synergistic increase in the amount of heterotopic bone in both Nf1 þ/þ and Nf1 þ/À mice compared with saline controls, as measured by DEXA and histomorphometry. Thus, the anabolic deficiency noted in Nf1 þ/À mice is amenable to stimulation by BMP-2, but mineralized tissue formation remains below that of Nf1 þ/þ controls. Bisphosphonate combination therapy is superior to BMP therapy alone in terms of net bone production in vivo in both wild-type and Nf1-deficient mice. ß

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Formation of bone by isolated, cultured osteoblasts in millipore diffusion chambers

Cited by 71 publications

References 20 publications

Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.

Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.

Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts

Modeling bone morphogenetic protein and bisphosphonate combination therapy in wild‐type and Nf1 haploinsufficient mice

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