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Biomedical subjects

T K Sampath

Publications and source records attributed to T K Sampath.

At least 55 records · Page 3Linked to original sources
PubMed1996

Complete regeneration of bone in the baboon by recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7).

We examined the efficacy of a single application of recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7) for its ability to regenerate large calvarial defects in adult male baboons (Papio ursinus). Recombinant hOP-1, in conjunction with baboon or bovine guanidinium-extracted insoluble collagenous bone matrix (0.1, 0.5 and 2.5 mg per g of collagenous matrix as carrier), was implanted in 46 calvarial defects surgically prepared in 14 baboons, whilst 18 defects were implanted with the carrier matrix without hOP-1. Specimens were harvested on d 15, 30, 90 and 365 and subjected to histomorphometry on serial undecalcified sections cut at 7 microm to study the temporal sequence of tissue morphogenesis after the single application of hOP-1. Histological analysis indicated that the induction of new bone formation proceeded from the periphery to the central core of hOP-1 treated specimens after rapid angiogenesis and mesenchymal cell migration in apposition to the collagenous matrix. Whilst chondrogenesis was limited, newly formed bone has already filled with fully differentiated bone marrow elements as early as d 15, even with the 0.1 mg dose of hOP-1. On d 30 and 90, doses of 0.1 and 0.5 mg of hOP-1 showed greater amounts of bone than controls, and on d 90, they induced complete regeneration of the defects. Doses of 2.5 mg hOP-1 per g of matrix induced extensive osteogenesis initially with heterotopic ossification and displacement of the temporalis muscle above the defects. One year after implantation of hOP-1 there was restoration of the internal and external cortices of the calvaria. These results show that hOP-1 induces complete regeneration of calvarial bone in the adult primate, and suggest that the optimal activity of hOP-1 to achieve regeneration is between 100 and 500 microg of hOP-1 per g of matrix. These results in the primate may form the scientific basis for future clinical applications of hOP-1.

Animals↗
PubMed1995

Effects of recombinant human osteogenic protein-1 on the differentiation of osteoclast-like cells and bone resorption.

Recombinant human OP-1 stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) significantly and in a dose-dependent manner in rat bone marrow cell culture. Newly formed MNCs either induced by hOP-1 alone or with 1,25(OH)2D3 were also positive for vitronectin receptor and carbonic anhydrase II. Moreover, OP-1 markedly increased the capacity of 1,25(OH)2D3 to induce osteoclast-like cell formation and bone resorption in bone marrow cultures. 25 pg/ml of calcitonin significantly inhibited both OP-1- and vitamin D3-induced TRAP-positive MNC-formation in marrow cultures, indicating that in both cases the MNC formation was calcitonin sensitive. OP-1 at 5-100 ng/ml did not have any significant effect on bone resorption as studied by pit formation assay. These studies that OP-1 in concert with 1,25(OH)2D3 could have an important role in bone remodeling by exhibiting its effects not only on osteoblast growth and differentiation but also on the recruitment of osteoclasts.

Actins↗
PubMed1995

Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects.

Proteins in the TGF-beta superfamily transduce their effects through binding to type I and type II serine/threonine kinase receptors. Osteogenic protein-1 (OP-1, also known as bone morphogenetic protein-7 or BMP-7), a member of the TGF-beta superfamily which belongs to the BMP subfamily, was found to bind activin receptor type I (ActR-I), and BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB) in the presence of activin receptors type II (ActR-II) and type IIB (ActR-IIB). The binding affinity of OP-1 to ActR-II was two- to threefold lower than that of activin A. A transcriptional activation signal was transduced after binding of OP-1 to the complex of ActR-I and ActR-II, or that of BMPR-IB and ActR-II. These results indicate that ActR-II can act as a functional type II receptor for OP-1, as well as for activins. Some of the known biological effects of activin were observed for OP-1, including growth inhibition and erythroid differentiation induction. Compared to activin, OP-1 was shown to be a poor inducer of mesoderm in Xenopus embryos. Moreover, follistatin, an inhibitor of activins, was found to inhibit the effects of OP-1, if added at a 10-fold excess. However, certain effects of activin, like induction of follicle stimulating hormone secretion in rat pituitary cells were not observed for OP-1. OP-1 has overlapping binding specificities with activins, and shares certain but not all of the functional effects of activins. Thus, OP-1 may have broader effects in vivo than hitherto recognized.

Activin Receptors↗
PubMed1995

Regulation of insulin-like growth factor system components by osteogenic protein-1 in human bone cells.

Bone morphogenetic proteins (BMPs) have the unique ability to convert mesenchymal cells into matrix-producing osteoblasts. To understand the mechanism(s) by which a BMP produces a multitude of effects on bone cells, we examined the effects of recombinant human osteogenic protein (OP)-1 (referred to as BMP-7) on the insulin-like growth factor (IGF) regulatory system, an important growth factor system in bone. After 48 h of treatment, OP-1 increased the level of IGF-II (3- and 2-fold, respectively, at 100 ng/ml) in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cells with osteoblastic characteristics, whereas IGF-I levels were low to undetectable in the CM of either cell type. OP-1 treatment had no significant effect on the messenger RNA (mRNA) level for type 1 and type 2 IGF receptors. In TE85 and SaOS-2 cells, 100 ng/ml OP-1 increased the level of IGF binding protein (BP)-3 more than 10-fold, decreased the IGFBP-4 level by 50%, and increased the level of the 29-32.5 kDa IGFBP-5 3-fold in the CM as determined by analysis with Western ligand blot, Western immunoblot, and RIA. The effect of OP-1 on IGFBP production was time and dose dependent. The OP-1 induced changes in the levels of IGFBPs were associated with decreased IGFBP-3 and -5 protease activity (29% and 71%, respectively) and proportional changes in IGFBP mRNA levels. OP-1 increased the level of IGFBP-3 mRNA (2- and 10-fold, respectively, after 4 and 24 h of treatment at 100 ng/ml) and of IGFBP-5 mRNA (more than 5-fold after 24 h of treatment) but decreased the level of IGFBP-4 mRNA (> 50% after 24 h at 100 ng/ml). OP-1 treatment had no effect on IGFBP-4 protease activity. These results collectively demonstrate that OP-1 can act locally by modulating the IGF regulatory system, suggesting that the mitogenic/differentiative effect of OP-1 on human bone cells may in part be mediated via IGF-II by increasing its secretion, and by regulating the balance between the stimulatory (e.g. IGFBP-5) and inhibitory (e.g. IGFBP-4) classes of IGFBPs both at the level of production (mRNA) and at the level of degradation but not by up-regulating the IGF receptor.

Bone Morphogenetic Protein 7↗
PubMed1995

Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures.

We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.

Alkaline Phosphatase↗
PubMed1994

Crystallization and preliminary crystallographic data of recombinant human osteogenic protein-1 (hOP-1).

We have obtained trigonal crystals of recombinant human osteogenic protein-1 (hOP-1), a member of the transforming growth factor-beta (TGF-beta) superfamily. hOP-1 (also referred to as BMP-7) is a bone morphogenetic protein and is active as a dimer of M(r) 32 to 36 kDa. The crystals have the symmetry of space group P3(1)21 or the enantiomorph P3(2)21 with unit cell dimensions of a = b = 99.46 A, c = 42.09 A. The crystals diffract to 2.2 A resolution and there is one hOP-1 monomer per asymmetric unit. In this paper we describe the first crystallization of a bone morphogenetic protein and present the results of preliminary X-ray diffraction data from the native protein and two heavy-atom derivatives.

Bone Morphogenetic Protein 7↗
PubMed1994

Evidence that human bone cells in culture contain binding sites for osteogenic protein-1.

Recent studies have shown that osteogenic protein (OP)-1 or bone morphogenetic protein (BMP)-7 increases proliferation and differentiation of human bone cells (HBCs) in culture and modulates production of IGF system components. In order to study the mechanism by which OP-1 causes these effects, we sought to test the hypothesis that the effects of OP-1 are mediated at least in part by specific receptors (for OP-1) in HBCs. Binding studies with serum-free cultures of normal HBCs and human osteosarcoma cells showed a maximum binding of 15-25% for [125I]OP-1; the binding was time- and temperature-dependent in different experiments. Scatchard analysis of [125]OP-1 binding to TE85 human osteosarcoma cells showed at least two binding sites, about 30,000 and 60,000 per cell with apparent Kd of 2.5 x 10(-10)M and 1 x 10(-9)M, respectively. [125]OP-1 binding to TE85 cells was displaced by unlabeled OP-1 (16-1000 ng/ml), with a 50% displacement at 250 ng/ml. BMP-2 effectively displaced [125]OP-1 binding to HBCs while TGF-beta 1 did not. Affinity cross-linking studies showed that [125]OP-1 interacted specifically with three binding sites with apparent M(r) of 34, 65 and > 205kDa. The findings of this study demonstrate that the effects of OP-1 on HBCs may be mediated in part via BMP-specific receptors.

Binding Sites↗
PubMed1994

Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4.

Bone morphogenetic proteins (BMPs) are multifunctional proteins, structurally related to transforming growth factor-beta (TGF-beta) and activin. TGF-beta and activin exert their effects by forming heteromeric complexes of type I and type II serine/threonine kinase receptors. We have previously identified a series of type I serine/threonine kinase receptors, termed activin receptor-like kinase (ALK)-1 to -6. ALK-5 is a TGF-beta type I receptor, whereas ALK-2 and ALK-4 are activin type I receptors. Here we investigated the binding of proteins in the BMP family to ALKs. In transfected COS cells, the binding of osteogenic protein (OP)-1 and BMP-4 to certain ALKs was observed in the absence of type II receptors, and their binding was increased after co-transfection of a BMP type II receptor from Caenorhabditis elegans, DAF-4. OP-1 bound to ALK-2 and ALK-6 efficiently, and to ALK-3 less efficiently, whereas BMP-4 bound to ALK-3 and ALK-6 efficiently. Similarly, OP-1 bound to ALK-2, ALK-3, and/or ALK-6 in various nontransfected cell lines, although the binding profiles were different between different cell types. BMP-4 bound to ALK-3 in MC3T3-E1 osteoblasts and human foreskin fibroblasts. These results suggest that ALK-3 and ALK-6 are type I receptors for OP-1 and BMP-4; in addition, ALK-2 is a type I receptor shared by activin and OP-1, but not by BMP-4.

Activin Receptors↗
PubMed1994

Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes.

Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein subfamily of the transforming growth factor-beta (TGF-beta) superfamily. Since members of the TGF-beta superfamily have a role in tissue development the distribution of OP-1 in developing human embryos (gestational age 5-14 weeks) was examined by immunohistochemical methods. Positive staining for OP-1 was observed in: sclerotome, hypertrophied chondrocytes, osteoblasts, periosteum, epithelial cells of the adrenal "provisional cortex" and the convoluted tubules of developing kidneys. In the developing lungs, pancreas and skin, OP-1 was localized in basement membranes underlying the epithelium. In vitro binding studies of 125I-OP-1 to various extracellular matrix components revealed high affinity of OP-1 for type IV collagen and less for heparin, collagen types I and VI. Present findings suggest that, in addition to bone formation, OP-1 could have other important regulatory roles in human embryogenesis with high binding affinity to a basement membrane component.

Adrenal Glands↗
PubMed1994

Opposite effects of osteogenic protein and transforming growth factor beta on chondrogenesis in cultured long bone rudiments.

Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-beta superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-beta 1 and porcine TGF-beta 1 and beta 2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-beta 1 and beta 2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-beta inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-beta exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-beta have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.

Animals↗
PubMed1994

Osteogenic protein-1, a bone morphogenic protein member of the TGF-beta superfamily, shares chemotactic but not fibrogenic properties with TGF-beta.

We have previously shown that recombinant human osteogenic protein-1 (rhOP-1), a bone morphogenetic protein member of the TGF-beta superfamily, can induce new bone formation when implanted with an appropriate carrier at subcutaneous sites in rats and can restore completely large diaphyseal segmental defects in laboratory animals. The role of OP-1 in the early events of bone induction viz, chemotaxis of phagocytic leukocytes, and fibroblastic mesenchymal cells is currently unknown. In the present study, we examined the effect of rhOP-1 on chemotaxis of phagocytic leukocytes (human neutrophils and monocytes) and fibroblastic mesenchymal cells (infant foreskin fibroblasts). Since OP-1 is structurally related to TGF-beta 1, we assessed the effects of OP-1 on several other fibroblast functions (in addition to chemotaxis) known to be modulated by TGF-beta 1. Our results demonstrated that rhOP-1, like TGF-beta 1, is a potent chemoattractant for human neutrophils, monocytes, and fibroblasts. However, in contrast to TGF-beta 1, OP-1 does not to stimulate fibroblast mitogenesis, matrix synthesis [collagen and hyaluronic acid (hyaluronan)], or production of tissue inhibitor of metalloproteinase (TIMP), i.e., fibroblast functions associated with fibrogenesis. These results clearly demonstrate a dichotomy between these two members of the TGF-beta superfamily with a regard to fibrogenic effects on fibroblasts but a similarity in their chemotactic properties.

Bone Morphogenetic Protein 7↗
PubMed1994

The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects.

A rabbit ulnar non-union model was used to evaluate the effect of recombinant human osteogenic protein-1 on the healing of a large segmental osteoperiosteal defect. A 1.5-centimeter segmental defect was created in the mid-part of the ulnar shaft of adult rabbits. The defect was filled with an implant containing either recombinant human osteogenic protein-1 or naturally occurring bovine osteogenic protein. The recombinant human osteogenic protein-1 implants consisted of a carrier of 125 milligrams of demineralized, guanidine-extracted, insoluble rabbit bone matrix (the collagen carrier), reconstituted with 3.13, 6.25, 12.5, twenty-five, fifty, 100, 200, 300, or 400 micrograms of recombinant human osteogenic protein-1. Animals that received recombinant human osteogenic protein-1 were compared with animals that received an implant of 250 micrograms of a preparation of naturally occurring bovine osteogenic protein mixed with the collagen carrier. Limbs that served as controls received either the collagen carrier alone or no implant at all. The treated and the untreated defects were examined radiographically and histologically at eight or twelve weeks after implantation. Mechanical testing was performed on six animals. All implants of recombinant human osteogenic protein-1, except for those containing 3.13 micrograms of the substance, induced complete radiographic osseous union within eight weeks. The defects that were treated with an implant of bovine osteogenic protein also healed within this time-period. The bone induced by both types of implants had new cortices with advanced remodeling and marrow elements. Histological evaluation of this new bone at eight weeks postoperatively revealed primarily lamellar bone, with the formation of new cortices and normal-appearing marrow elements. The average torsional strength and energy-absorption capacity of the union induced by recombinant human osteogenic protein-1 was comparable with that of intact bone. The control defects that had been implanted with collagen carrier alone and those with no implant showed no bridging of the defect.

Animals↗
PubMed1994

Osteogenic protein-1 (BMP-7) inhibits cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat osteosarcoma (17/2.8) cells.

We recently showed that osteogenic protein-1(OP-1), a bone morphogenetic protein member of TGF-beta superfamily, induces endochondral bone formation in vivo, and stimulates growth and differentiation of osteoblasts in rat calvarial-derived cell cultures. In the present study, we examined the effect of OP-1 on cell growth and expression of markers that are characteristic of osteoblast phenotype using the clonal rat osteosarcoma cells (ROS 17/2.8). A comparison of OP-1 and TGF-beta 1 effects on cell growth showed that, both OP-1 and TGF-beta 1 inhibited DNA synthesis up to 90 percent and 60 percent of the controls at concentrations of 10 ng/ml and 1 ng/ml, respectively, in serum-free medium. In the presence of 5% serum, TGF-beta 1 did not have any significant inhibitory effects while 40 ng OP-1/ml inhibited the DNA synthesis up to 80% of the controls. Examination of collagen synthesis showed that 40 ng OP-1/ml increased the expression of type I collagen mRNA, and thus increased collagen synthesis (4-fold), as examined by collagenase-digestible protein. Evaluation of markers that are characteristic of the osteoblast phenotype demonstrated that OP-1 stimulated cAMP production in response to PTH (10-fold at 200 ng/ml), alkaline phosphatase specific activity (ALPase) (4-fold at 80 ng/ml), and osteocalcin (OC) synthesis (4.5-fold at 40 ng/ml). Northern blot analysis revealed that OP-1 increased mRNA expression for both ALPase and OC in a dose-dependent manner. These data collectively demonstrate that OP-1 suppresses cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat clonal osteoblastic osteosarcoma cells (ROS 17/2.8).

Alkaline Phosphatase↗
PubMed1994

Recombinant human bone morphogenetic protein-7 induces healing in a canine long-bone segmental defect model.

An ulnar segmental defect model was used in adult male dogs to examine the effect of recombinant human bone morphogenetic protein-7 (recombinant human Osteogenic Protein-1 [rhOP-1]; Creative Biomolecules, Hopkinton, Massachusetts) on new bone induction and healing, and to test the mechanical strength of healed 2.5-cm segmental bone defects. The rhOP-1 composites consisted of a carrier of 500 mg of demineralized, guanidine-extracted, insoluble bovine bone matrix (collagen carrier), reconstituted with rhOP-1. Six animals received 1200 micrograms rhOP-1 unilaterally and were killed at 12 weeks for torsional load-to-failure testing using the contralateral side as a control. Two further animals received varying amounts of rhOP-1 bilaterally and were studied histologically. All defect sites receiving rhOP-1 were completely bridged radiographically by eight weeks. A control composite, containing no rhOP-1, failed to induce new bone formation at any time. Histologically, rhOP-1-treated sites examined at 16 weeks had formation of new cortical and cancellous bone, with normal appearing marrow elements in the reconstituted medullary canal. The torsional strength of the rhOP-1-implanted ulnae averaged 72% of control (range, 30-99%). The angular deformation to failure averaged 92% of control (range, 39-122%). The energy absorption to failure averaged 67% of control (range, 27-111%). This study demonstrates the efficacy of rhOP-1 in healing segmental osteoperiosteal defects in a canine model.

Animals↗
PubMed1993

Bovine articular chondrocytes do not undergo hypertrophy when cultured in the presence of serum and osteogenic protein-1.

Osteogenic protein-1 (hOP-1, BMP-7) is a member of the transforming growth factor-beta superfamily. We have recently shown that hOP-1 induces and promotes maturation and hypertrophy of chick sternal chondrocytes, cultured in monolayer or suspension in the presence or absence of serum. In the present study we demonstrate that bovine articular chondrocytes, grown for up to 5 weeks in the presence of 0.5% or 10% serum in combination with 30 ng/ml hOP-1, do not undergo hypertrophy, as determined by cell size, the absence of type X collagen expression and synthesis, and of alkaline phosphatase activity. Treatment with hOP-1 (30 ng/ml) resulted in increased matrix synthesis as measured by [35S]sulfate incorporation and by collagen type II synthesis and expression, without influencing cell proliferation. These data suggest that primary mammalian articular chondrocytes are not able to undergo hypertrophy in conditions previously shown to be permissive for hypertrophy of both chick sternal and chick articular chondrocytes.

Alkaline Phosphatase↗
PubMed1993

Osteogenic protein-1 stimulates proliferation and differentiation of human bone cells in vitro.

The effect of recombinant human osteogenic protein-1 (OP-1) on proliferation and alkaline phosphatase (ALP) activity was studied using human bone cells in culture. TE85, osteosarcoma cells with osteoblastic characteristics and normal bone cells derived from mandible (HBM) were used. OP-1 stimulated 3H-thymidine incorporation in a dose dependent manner in TE85 (5-fold over vehicle control at 3-10 ng/ml) and HBM cells (1.8-fold at 100 ng/ml). In TE85 cells, OP-1 also increased cell number (2.4-fold over vehicle control at 3 ng/ml). OP-1 stimulated ALP activity in TE85 cells (4-fold over vehicle control at 30 ng/ml), but moderately inhibited ALP activity in HBM cells (to 67% of vehicle control at 100 ng/ml). Because 1,25(OH)2D3 has been shown to increase ALP activity in many cell types, we also studied if 1,25(OH)2D3 modulates the effects of OP-1 on ALP activity. In the presence of 10(-8) M 1,25(OH)2D3, a biphasic response occurred in TE85 and HBM cells with stimulation of ALP activity at low dose of OP-1 and inhibition at high dose. Thus, the effect of OP-1 on ALP activity appeared to be modulated by 1,25(OH)2D3. Our results suggest that OP-1 could be an important regulator of osteoblast proliferation and differentiation.

Alkaline Phosphatase↗
PubMed1993

Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals.

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.

Amino Acid Sequence↗