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Biomedical subjects

A H Reddi

Publications and source records attributed to A H Reddi.

At least 55 records · Page 3Linked to original sources
PubMed1995

Bone morphogenetic proteins, bone marrow stromal cells, and mesenchymal stem cells. Maureen Owen revisited.

In postnatal mammals, there are persistent molecular signals and responding cells in bone to initiate osteogenesis and repair in response to trauma. The responding osteogenic precursor cells are of 2 categories: determined and inducible. The latter can be induced by demineralized bone matrix to form bone. Demineralized bone matrix consists of extracellular matrix and tightly associated bone morphogenetic proteins. The genes for bone morphogenetic proteins have been cloned, the recombinant proteins have been expressed, and currently their mechanism of action is being explored. Bone morphogenetic proteins are pleiotropic initiators of inducible osteogenic precursor cells. Bone morphogenetic proteins govern the 3 key steps in the osteogenic cascade: chemotaxis, mitosis, and differentiation. The receptors for bone morphogenetic proteins have been cloned and expressed and consist of 2 classes, Types I and II, that are membrane bound serine/threonine protein kinases. Bone morphogenetic proteins bind to extracellular matrix and their collaborative action on osteogenic cells culminates in the terminal differentiation of the osteoblast-osteocyte continuum. Bone morphogenetic proteins are currently on the threshold for clinical applications.

Animals↗
PubMed1994

Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development.

Partially purified extracts from newborn calf articular cartilage were found to induce cartilage and bone when subcutaneously implanted in rats. This activity showed characteristics of bone morphogenetic proteins (BMPs). Degenerate oligonucleotide primer sets derived from the highly conserved carboxyl-terminal region of the BMP family were designed and used in reverse transcription-polymerase chain reactions with poly(A)+ RNA from articular cartilage as template to determine which BMPs are produced by chondrocytes. Two novel members of the transforming growth factor-beta (TGF-beta) superfamily were identified and designated cartilage-derived morphogenetic protein-1 (CDMP-1) and -2 (CDMP-2). Their carboxyl-terminal TGF-beta domains are 82% identical, thus defining a novel subfamily most closely related to BMP-5, BMP-6, and osteogenic protein-1. Northern analyses showed that both genes are predominantly expressed in cartilaginous tissues. In situ hybridization and immunostaining of sections from human embryos showed that CDMP-1 was predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bones, whereas CDMP-2 expression was restricted to the hypertrophic chondrocytes of ossifying long bone centers. Neither gene was detectable in the axial skeleton during human embryonic development. The cartilage-specific localization pattern of these novel TGF-beta superfamily members, which contrasts with the more ubiquitous presence of other BMP family members, suggests a potential role for these proteins in chondrocyte differentiation and growth of long bones.

Amino Acid Sequence↗
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

Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage.

The three ingredients for the successful tissue engineering of bone and cartilage are regulatory signals, cells, and extracellular matrix. Recent advances in cellular and molecular biology of the growth and differentiation factors have set the stage for a symbiosis of biotechnology and biomaterials. Recent advances permit one to enunciate the rules of architecture for tissue engineering of bone and cartilage. The purification and cloning of bone morphogenetic proteins (BMPs) and growth factors such as platelet derived growth factors (PDGF), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGF-I) will allow the design of an optimal combination of signals to initiate and promote development of skeletal stem cells into cartilage and bone. Successful and optimal bone and cartilage formation is a synergy of inductive and conductive strategies governed by biomechanics, optimal load bearing, and motion. BMPs function as inductive signals. Biomaterials (both natural and synthetic) mimic the extracellular matrix and play a role in conduction of bone and cartilage. Examples of biomaterials include hydroxyapatite, polyanhydrides, polyphosphoesters, polylactic acid, and polyglycolic acid. The prospects for novel biomaterials are immense, and they likely will be a fertile growth industry. Cooperative ventures between academia and industry and technology transfer from the federal government augur well for an exciting future for clinical applications.

Animals↗
PubMed1994

Regulatory role of transforming growth factor-beta, bone morphogenetic protein-2, and protein-4 on gene expression of extracellular matrix proteins and differentiation of dental pulp cells.

The expression of developmental stage-specific genes during pulp cell differentiation into preodontoblasts was examined in bovine adult pulp cell culture. When proliferation was down-regulated after 14 days of primary culture, expression of fibronectin and type I and type III collagen mRNAs was increased. Expression of alkaline phosphatase was gradually increased, and mRNA for osteocalcin, a marker of preodontoblast, appeared just before the onset of mineralization. Contrarily, in expanded culture, the expression of mRNA for the extracellular matrix proteins was gradually increased from the beginning of culture up to Day 28. Similarly, mRNA levels of alkaline phosphatase and osteocalcin were also increased gradually. Expression of TGF-beta 1 mRNA disappeared on Day 21 in the primary culture when expression of alkaline phosphatase mRNA was increased. BMP-4 mRNA was expressed on Day 14 when the expression of the extracellular matrix proteins was increased. BMP-2 mRNA was expressed on Day 28 when osteocalcin appeared. Recombinant TGF-beta 1 inhibited alkaline phosphatase activity, while BMP-2 and BMP-4 stimulated it. BMP-4 increased expression of alpha 1(I) collagen mRNA, and BMP-2 increased osteocalcin synthesis. These results demonstrate the regulatory role of these TGF-beta superfamily members on the gene expression of extracellular matrix proteins and the differentiation of pulp cells into preodontoblasts.

Alkaline Phosphatase↗
PubMed1994

Recombinant bone morphogenetic protein-4, transforming growth factor-beta 1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro.

Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-beta (TGF-beta) supergene family and is characterized by its ability to induce singly de novo cartilage and bone in vivo. The influence of recombinant bone morphogenetic protein-4 and some related members, TGF-beta 1, activin A, and inhibin A, on articular chondrocyte metabolism in the presence and absence of extracellular matrix has been examined. BMP-4 and TGF-beta 1 stimulated [35S]-sulfate incorporation in a dose-dependent manner in short-term monolayer, micromass, and explant cultures. Activin A showed a slight but significant stimulation of proteoglycan synthesis while inhibin A decreased metabolic activity. The effects observed were most pronounced in the explant culture system. Although the relative influence of the growth factors was less apparent in chondrocytes isolated from adult cartilage, the qualitative responses were similar with cells obtained from young animals. The maintenance and enhancement of the cartilage phenotype was further investigated by Northern blot analysis. BMP-4 and TGF-beta 1 increased the levels of expression of type II collagen and proteoglycan aggrecan in short-term cultures, while activin A and inhibin A did not affect these parameters significantly when compared to serum-free control cultures. Binding experiments with 125I-BMP-4, revealed the presence of specific, high-affinity binding sites with an apparent dissociation constant of 110 pM and about 6000 receptors per cell. Chemical cross-linking showed the presence of three components (apparent size 200, 90, and 70 kDa), demonstrating the presence of functional receptors for BMP-4 on primary articular chondrocytes.

Activins↗
PubMed1994

Bone and cartilage differentiation.

Recent progress in the study of regulation of bone and cartilage differentiation has come from the isolation, cloning, and expression of genes encoding bone morphogenetic proteins (BMPs). BMPs initiate cartilage and bone formation in a sequential cascade. Their pleiotropic effects on chemotaxis, mitosis, and differentiation are based on concentration-dependent thresholds. The existence of multiple members of the BMP family raises issues concerning functional redundancy. Current work in progress in different laboratories has revealed that BMP-2 or BMP-4 gene knockout by homologous recombination results, surprisingly, in embryonic lethality. Cartilage and bone differentiation during endochondral development involves a continuum of steps: initiation, promotion, maintenance, modeling, and termination. The signaling factors for initiation and maintenance are being defined at the molecular level, and future studies will focus on the gene regulation of initial signaling molecules such as BMPs. Critical progress in the determination of the role of BMPs in bone development has been accomplished by systematic study of skeletal mutations such as short ear and brachypodism in mice. The accelerating pace of advance in this area augurs well for the resolution of the molecular basis of morphogenesis of bone and cartilage.

Animals↗
PubMed1994

Osteoclast recruiting activity in bone matrix.

An activity that recruits osteoclasts has been identified and partially characterized from bone matrix. Bone-derived osteoclast recruiting activity (BORA) was co-purified with osteogenin, a bone inductive protein. Osteogenin was extracted from bovine bone with 6 M urea and purified by chromatography on hydroxyapatite, heparin-Sepharose and Sephacryl S-200 gel filtration. The biologically active osteoclast formation-stimulating material was further purified by C18 reverse phase HPLC. BORA is obviously distinct from osteogenin and transforming growth factor beta (TGF-beta), since further purified osteogenin and pure TGF-beta did not stimulate the formation of osteoclast-like cells. BORA (0.1-10 micrograms/ml) stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in a dose-dependent manner. These multinucleated cells resorbed bone when cultured on bovine bone slices. The effect of BORA is primarily directed to differentiate osteoclast precursors, since it did not stimulate osteoclast function in in vitro resorption assay where disaggregated rat osteoclasts were cultured on bovine bone slices. However, after 24 h preincubation with 50 nM PTH in the mouse calvaria assay, BORA at 10 micrograms/ml significantly stimulated bone resorption.

Animals↗
PubMed1994

Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium.

Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defined cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis.

Animals↗
PubMed1994

Periodontal regeneration: potential role of bone morphogenetic proteins.

Initiation of osteogenesis and cementogenesis is a problem central to periodontal regeneration. A major advance in the understanding of bone formation has been the identification of an entirely new family of protein initiators, the bone morphogenetic proteins, that regulate cartilage and bone differentiation in vivo. The purification, genetic cloning and expression of recombinant human bone morphogenetic proteins (BMPs) have laid the foundation for the cellular and molecular dissection of bone development and regeneration. The striking evolutionary conservation of the BMP genes indicates that they are critical in the normal development and function of animals. In addition to postfetal osteogenesis, the BMPs may play multiple roles in embryonic development and organogenesis, including skeletogenesis and the development of craniofacial and dental tissues. The availability of recombinant human BMPs provides several challenges and opportunities to gain insights into the mechanisms regulating the regeneration of bone and cementum for optimal outcome in the periodontal patient.

Animals↗
PubMed1994

The effect of osteogenin (a bone morphogenetic protein) on the formation of bone in orthotopic segmental defects in rats.

We studied the effects of partially purified, natural osteogenin, a bone morphogenetic protein, on the formation of bone in rats. An osteoperiosteal segmental defect, eight millimeters wide, in the middle of the femoral diaphysis was created bilaterally in thirty-six adult male Fischer rats and stabilized with a polyacetyl plate and threaded Kirschner wires. One defect was filled with a cylinder of 60 per cent hydroxyapatite and 40 per cent tricalcium phosphate ceramic (pore diameter, 250 to 400 micrometers) containing 100 micrograms of partially purified bovine osteogenin, and the contralateral defect was filled with a hydroxyapatite-tricalcium ceramic cylinder without osteogenin. Eighteen animals (six animals each at one, two, and four months after the operation) were studied histologically and histomorphometrically. The implants from eighteen additional animals (six animals each at one, two, and four months after the operation) were subjected to biomechanical testing. Histomorphometry revealed that the total area of bone, the area of bone outside of the implant, and the amount of bone within the pores of the implant were all significantly (p < or = 0.05) greater in the femora that had an implant with osteogenin than in those that had an implant without osteogenin at most time-periods. The presence of osteogenin had no significant effect on the biomechanical parameters measured in this study.

Animals↗
PubMed1994

Bone grafts and bone induction substitutes.

The use of bone grafts is the basis of all craniofacial surgery. Bone grafts are used to stabilize the open segments, expand the patient's structural boundaries, and fill defects created by trauma or congenital malformations. Bone grafts are harvested from local or distant sites as indicated. Biomaterial implants are used as bone substitutes when it is not possible to use bone grafts.

Adult↗
PubMed1993

Changes in the expression of insulin-like growth factor II/mannose-6-phosphate receptor during endochondral bone development.

Endochondral bone development can be induced by subcutaneous implantation of demineralized bone matrix (DBM) in rats. We used this in vivo model to study the relationship between endochondral bone formation and expression of IGF-II/M-6-P receptor, a multifunctional protein which binds not only IGF-II, but also lysosomal enzyme bearing mannose-6-phosphate motif. We found that IGF-II/M-6-P receptor was present in implants from day 1 to day 21; the highest levels were expressed on day 11 during bone differentiation. IGF-II/M-6-P receptor mRNA content was highest on day 9. We conclude from these data that IGF-II/M-6-P receptor expression is developmentally regulated during endochondral bone formation. This regulation occurs in part at the level of IGF-II/M-6-P receptor mRNA. The relatively high level of IGF-II/M-6-P receptor during ossification suggests that this receptor might play a role in bone formation and remodeling.

Animals↗
PubMed1993

The functional equivalence of demineralized bone and tooth matrices in ectopic bone induction.

The objective of this study was to determine whether demineralized rat incisor matrices were a more potent inducer of ectopic endochondral bone formation than demineralized diaphyseal bone matrices derived from the same donors. Twenty-five-milligram disks of demineralized bone or tooth matrix obtained from adolescent Long-Evans rats were implanted in a standardized ectopic site. Biochemical and histometric measurements of bone formation revealed that the two matrices were functionally equivalent inducers of endochondral bone formation. The induced pellicle of bone reached a maturation point 18 days after implantation. Dentin matrix implants generated a significantly greater amount of mineralized tissue than did bone matrix implants. This difference could be explained on the basis of remineralization of the dentin particles to a greater degree than the bone matrix particles. Initial observations suggesting a more robust osteoinductive activity in demineralized incisor matrix can be attributed to the decreasing activity of bone matrix from older donors when compared to younger donors. The extent of osteoinduction by the two substrata was equivalent when the matrices were matched for age.

Acid Phosphatase↗
PubMed1993

Initiation and promotion of bone differentiation by bone morphogenetic proteins.

The presence of growth and differentiation factors in bone has been demonstrated by subcutaneous implantation of demineralized bone matrix that initiates new cartilage and bone morphogenesis. The genes for bone morphogenetic proteins (BMPs) have been cloned and expressed. Recombinant BMPs induce endochondral bone formation in vivo. The multistep sequential developmental cascade consists of chemotaxis, mitosis, and differentiation of cartilage and bone. The pleiotropic response has been well characterized. BMPs stimulate osteogenic and chondrogenic phenotypes. Natural bovine osteogenin (BMP-3) and recombinant BMP-4 are equipotent in chemotaxis, limb bud chondrogenesis, cartilage maintenance, and in vivo bone induction. There are multiple isoforms of BMPs, raising the biologic relevance of the redundancy. The mode of action and second messengers are not clear. BMPs appear to have cognate receptors as demonstrated by iodinated BMP-2B (BMP-4). Other novel members of the BMP family include osteogenic protein 1 (BMP-7) and osteogenic protein 2 (BMP-8). Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily and include three distinct subfamilies: BMP-2, BMP-3, and BMP-7. Native BMP-3 and recombinant BMP-4 bind type IV collagen of the basement membrane. This novel connection may be the long elusive mechanistic explanation for the requirement of angiogenesis and vascular invasion for bone morphogenesis. BMPs may have a role in fracture repair, periodontal regeneration, and alveolar ridge augmentation.

Animals↗