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T K Sampath

Publications and source records attributed to T K Sampath.

69 records · Page 4Linked to original sources
PubMed1993

Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.

Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

Alkaline Phosphatase↗
PubMed1992

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.

Alkaline Phosphatase↗
PubMed1992

Anti-ischaemic and endothelial protective actions of recombinant human osteogenic protein (hOP-1).

Osteogenic protein one (hOP-1), a member of the transforming growth factor-beta (TGF-beta) supergenic family, was studied for its anti-ischaemic properties in rats subjected to myocardial ischaemia and reperfusion. Ten minutes after ligation (i.e., just prior to reperfusion) of the left coronary artery, 2 or 20 micrograms/rat recombinant human (hOP-1) or its vehicle, was given intravenously. hOP-1 at 20 micrograms significantly reduced reperfusion injury 24 h later compared to rats receiving only vehicle (i.e., 0.9% NaCl). hOP-1 was also found to preserve rat coronary endothelial function (i.e., release of endothelium-derived relaxing factor, EDRF) in perfused hearts following global ischaemia and reperfusion. Moreover, hOP-1 also significantly inhibited adherence of rat neutrophils to rat vascular endothelium in vitro. Thus, hOP-1 exerts significant anti-ischaemic effects. Some of this cardioprotection may be related to the ability of hOP-1 to preserve endothelial function and inhibit neutrophil adherence to the endothelium.

Animals↗
PubMed1992

Use of bovine osteogenic protein to promote rapid osseointegration of endosseous dental implants.

An enriched bovine osteogenic protein preparation in combination with bone collagen matrix (osteogenic protein device) was used to effect new bone growth in extraction sites in the presence or absence of titanium dental implants. Incisor and canine teeth were extracted from each of three cynomolgus monkeys, and implants were inserted directly into the sockets without surgical site preparation. The osteogenic protein device induced new bone formation in close apposition to the titanium implants in all trials within 3 weeks. A lesser amount of new bone formation in both sets of control sites was limited to the bony socket walls and not closely apposed to the implant. These data show that the osteogenic protein device is capable of inducing new bone formation in tooth sockets within 3 weeks in the presence or absence of titanium implants. This is the first known demonstration of the therapeutic induction of bone formation in close apposition to metallic implants.

Animals↗
PubMed1990

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily.

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.

Alkaline Phosphatase↗
PubMed1990

OP-1 cDNA encodes an osteogenic protein in the TGF-beta family.

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.

Amino Acid Sequence↗
PubMed1987

Isolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography.

Implantation of demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. Demineralized bovine bone matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the bone-inductive proteins were purified greater than 12,000-fold. The purification steps include affinity chromatography on heparin-Sepharose, hydroxyapatite chromatography, gel filtration, and C18 reverse-phase HPLC. Since the purified protein in conjunction with insoluble collagenous bone matrix induced new bone differentiation in vivo we have designated this component osteogenin. The osteogenic potential is specific for osteogenin and is not exhibited by previously isolated growth factors.

Animals↗
PubMed1985

Comparison of bone inductive proteins of rat and porcine bone matrix.

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.

Animals↗
PubMed1984

Extracellular matrix proteins involved in bone induction are vitamin D dependent.

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.

Animals↗
PubMed1984

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix.

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.

Alkaline Phosphatase↗
PubMed1984

In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone.

Subcutaneous implantation of demineralized diaphyseal bone matrix into rats induces cartilage and bone formation in vivo. When minced skeletal muscle is cultured on hemicylinders of demineralized bone in vitro, mesenchymal cells are transformed into chondrocytes. In the present investigation, the potential of extracellular matrix components of bone to trigger cartilage differentiation in vitro was examined. Extraction of bone hemicylinders with 6 M guanidine X HCl resulted in the absence of chondrogenesis in vitro and endochondral bone formation in vivo. Biologically inactive hemicylinders of bone were then reconstituted with the guanidine extract and also with partially purified components extracted from bone matrix and bioassayed. Reconstitution completely restored the ability to elicit chondrogenesis in vitro and endochondral bone differentiation in vivo. Reconstitution of the whole guanidine extract on Millipore filters coated with gels of tendon collagen (type I) and subsequent culture with minced skeletal muscle also resulted in cartilage induction in vitro. These observations show that the extracellular matrix of bone is a repository of factors that govern local cartilage and bone differentiation.

Animals↗
PubMed1984

Importance of geometry of the extracellular matrix in endochondral bone differentiation.

Subcutaneous implantation of coarse powders (74-420 micron) of demineralized diaphyseal bone matrix resulted in the local differentiation of endochondral bone. However, implantation of matrix with particle size of 44-74 micron (Fine matrix) did not induce bone. We have recently reported that the dissociative extraction of coarse matrix with 4 M guanidine HCl resulted in a complete loss of the ability of matrix to induce endochondral bone; the total loss of biological activity could be restored by reconstitution of extracted soluble components with inactive residue. To determine the possible biochemical potential of fine matrix to induce bone, the matrix was extracted in 4 M guanidine HCl and the extract was reconstituted with biologically inactive 4 M guanidine HCl-treated coarse bone matrix residue. There was a complete restoration of the biological activity by the extract of fine matrix upon reconstitution with extracted coarse matrix. Polyacrylamide gel electrophoresis of the extract of fine matrix revealed similar protein profiles as seen for the extract of coarse matrix. Gel filtration of the 4 M guanidine HCl extract of fine powder on Sepharose CL-6B and the subsequent reconstitution of various column fractions with inactive coarse residue showed that fractions with proteins of 20,000-50,000 mol wt induced new bone formation. These observations demonstrate that although fine bone matrix contains, osteoinductive proteins, matrix geometry (size) is a critical factor in triggering the biochemical cascade of endochondral bone differentiation. Mixing of coarse matrix with Fine results in partial response and it was confined to areas in contact with coarse particles. The results imply a role for geometry of extracellular bone matrix in anchorage-dependent proliferation and differentiation of cells.

Alkaline Phosphatase↗
PubMed1983

Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix.

Allogeneic implantation of rat extracellular demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. However, xenogenic subcutaneous implantation of human, monkey, and bovine extracellular bone matrix into rat showed that bovine matrix had only a weak capacity to induce bone, whereas human and monkey matrix had none at all. This suggested that extracellular matrix-induced bone differentiation is apparently species-specific. We recently reported that the extraction of matrix with 4 M guanidine X HCl resulted in complete removal of the ability to induce endochondral bone differentiation, with the biological activity of the matrix being again restored when the extracted active matrix components (less than 50,000 daltons) were reconstituted with the inactive residue. To define the possible biochemical basis of species specificity, human, monkey, and bovine extracellular bone matrices were extracted with 4 M guanidine X HCl and the extracts were reconstituted with biologically inactive rat residue and bioassayed. The results were similar to those obtained with intact matrices and showed that total extracts of bovine matrix had a weak capacity to induce bone, whereas corresponding extracts of human and monkey matrix did not induce bone. However, partial purification by gel filtration of 4 M guanidine X HCl extracts from each species followed by reconstitution of the different fractions with inactive rat residue resulted in bone induction by all species from fractions containing proteins of less than 50,000 daltons. These observations demonstrate that species specificity of xenogenic extracellular bone matrix is due to immunogenic or inhibitory components (or both) in the guanidine X HCl residue and solubilized extracellular matrix components of greater than 50,000 daltons. These results imply that there is homology in the bone inductive proteins from human, monkey, bovine, and rat extracellular bone matrices.

Animals↗
PubMed1981

Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation.

Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn . HCl), 8 M urea/1 M NaCl, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extracts resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn. HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins less than 50,000 daltons were reconstituted with inactive 4 M Gdn . HCl-treated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.

Alkaline Phosphatase↗