Biomedical subjects

A H Reddi

Publications and source records attributed to A H Reddi.

At least 109 records · Page 6Linked to original sources

PubMed1990

Radiation-sterilized insoluble collagenous bone matrix is a functional carrier of osteogenin for bone induction.

The influence of gamma radiation on the role of the collagenous substratum as a carrier for proteins which cause bone induction was examined. Osteoinductive demineralized bone matrix was extracted by 4 M guanidinium hydrochloride. The insoluble collagenous bone matrix (ICBM) obtained was not osteoinductive; however, when reconstituted with partially purified osteogenin, bone induction was restored. In order to apply the principle of bone induction to clinical use, methods of sterilization must be optimized to maintain the osteoinductive activity of bone allografts. The inactive substratum was irradiated and reconstituted with an active, partially purified bone extract and bioassayed. Irradiation of the ICBM by a Cobalt 60 source at a dose of 1 and 3 Mrads had no deleterious effect on the functional role of the substratum.

Alkaline Phosphatase↗

PubMed1990

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development.

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.

Animals↗

PubMed1990

Temporal changes in the response of chick limb bud mesodermal cells to transforming growth factor beta-type 1.

Transforming growth factor beta (TGF beta) has been found in adult and developing bone in vivo and has varied effects on chondrocytes and osteoblasts in culture. We investigated the effects of TGF beta-type 1 on embryonic chick endochondral bone precursors in culture. Stage 24 chick limb bud mesoderm cells were cultured at high density. Under these conditions a dense mat containing nodules of cartilage surrounded by alkaline phosphatase-positive cells formed. Exposure of mesodermal cells to TGF beta-type 1 over the course of 4-7 days or during Days 3 and 4 caused a reduction in alkaline phosphatase activity and [35S]sulfate incorporation into proteoglycans. When the TGF beta-type 1 was applied during Days 1-2, it caused an increase in both parameters: these increases were observed in the absence of a corresponding change in [3H]thymidine incorporation. The specificity of the effects of TGF beta-type 1 was confirmed by neutralizing antibodies. These studies show that TGF beta-type 1 modulates the phenotype of embryonic endochondral bone precursors. The influence may depend on the window of exposure to the growth factor and, therefore, on the state of differentiation of the cells.

Alkaline Phosphatase↗

PubMed1990

The osteogenic potential of two composite graft systems using osteogenin.

The purpose of this study was to determine the quantity of new bone formation in critical sized calvaria defects in rats treated with two composite graft systems. The systems consisted of either a combination of the bone inductive protein (osteogenin) plus type I collagen (Os + C) or the combination of osteogenin with coralline hydroxyapatite (Os + HA). Additional treatments consisted of coralline hydroxyapatite (HA) or untreated control defects. After 28 days the calvaria were recovered and processed for quantitative radiography (radiomorphometry) and histomorphometry. Histomorphometric results were based on quantitation of regenerated trabecular bone. Results indicated that the Os + C combination produced substantially more bone than the Os + HA, HA, or control groups (P less than 0.05). Radiomorphometric assessment was based on the detection of radiopacity in the calvarial wounds. Due to the radiopaque property of HA, it was not possible to accurately quantitate the radiopacity of the regenerating bone from HA and host bone. Therefore, conclusions about the efficacy of the treatments must be derived from histomorphometric data. Results from histometric measurements of healing indicate that the Os + C combination has the greatest potential for regenerating calvarial bone defects. The potential for osteogenin in regenerating alveolar bone lost due to periodontal disease is suggested by these studies.

Animals↗

PubMed1990

Growth and differentiation factors: role in bone induction and potential application in craniofacial surgery.

Repair of craniofacial bone often requires autogenous or allogeneic bone. Natural materials deliver bone-inducing substances to wound beds and provide scaffolding for osteoconduction, but not without risk of morbidity. We are developing a synthetic material consisting of proteins which stimulate bone repair encased within a moldable, biodegradable delivery system. The stages of bone induction include chemoattraction of stem cells, proliferation and differentiation of these cells to competent chondrocytes and osteoblasts, angiogenesis, mineralization, and remodeling. Bovine osteogenin induces bone formation in soft tissue in rats, and accelerates bone repair in cranial defects. The protein has been purified and partially sequenced. Growth factors may augment the inductive effects of osteogenin by attracting preosteoblasts, accelerating their proliferation, and stimulating angiogenesis. A composite material of osteogenin and growth factors released from a biodegradable delivery system in a time-dependent fashion is proposed to decrease the necessity for autogenous and allogeneic bone implants.

Bone Morphogenetic Protein 3↗

PubMed1990

Repair of calvarial nonunions by osteogenin, a bone-inductive protein.

Efforts were taken to determine the dose of bovine osteogenin (OG) that would induce more bone than that induced by 20 mg of rat particulate demineralized bone matrix (DBM), the amount allowed by the confines of an 8-mm rat craniotomy defect. Dose-response studies were performed for demineralized bone matrix alone and osteogenin, partially purified from bovine demineralized bone matrix, plus rat insoluble collagenous bone matrix (M). Demineralized bone matrix alone (2.5, 5.0, 10, 20, or 40 mg) or osteogenin (0.0625, 0.125, 0.250, 0.50, or 1.0 mg) plus 25 mg insoluble collagenous bone matrix was implanted into the pectoralis muscle for 3, 5, and 7 weeks. Both materials induced time- and dose-dependent formation of bone. The three highest dosages of osteogenin (plus insoluble collagenous bone matrix) induced more bone than 20 mg demineralized bone matrix and seemed to accelerate bone repair. However, when implanted into the 8-mm rat craniotomy defect for 4 weeks, 20 mg demineralized bone matrix and 0.5 mg osteogenin (plus insoluble collagenous bone matrix) induced comparable amounts of bone. These results suggest different mechanisms for bone formation in heterotopic and orthotopic sites.

Animals↗

PubMed1990

Bone induction by osteogenin and bone morphogenetic proteins.

Bone induction is a multistep process that includes chemotaxis and attachment of mesenchymal stem cells, proliferation of progenitor cells and differentiation into cartilage and bone. Endochondral bone formation is the predominant sequence. Bone induction can be operationally divided into the following phases: initiation, promotion and maintenance. The initiation of bone induction is primarily regulated by osteogenin and bone morphogenetic proteins. Recent work has led to the isolation, purification and cloning of osteogenin and other bone morphogenetic proteins. Other growth factors such as platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta isoforms, insulin like growth factors (IGF 1 and 2), and fibroblast growth factor (FGF) may promote and perhaps maintain the newly induced bone.

Animals↗

PubMed1989

Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation.

Osteogenin was purified from bovine bone matrix and its activity monitored by an in vivo bone induction assay. The purification method utilized extraction of the bone-inducing activity with 6 M urea, followed by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Active fractions were further purified by preparative sodium dodecyl sulfate gel electrophoresis without reduction. Osteogenin activity was localized in a zone between 30 and 40 kDa. The amino acid sequences of a number of tryptic peptides of the gel-eluted material were determined. Reduction and alkylation of purified osteogenin in 7 M guanidine hydrochloride resulted in the total loss of biological activity. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a broad band with an apparent molecular mass of 22 kDa.

Amino Acid Sequence↗

PubMed1989

Affinity of osteogenin, an extracellular bone matrix associated protein initiating bone differentiation, for concanavalin A.

Subcutaneous implantation of demineralized bone matrix results in bone differentiation. The bone inductive protein, osteogenin, was isolated recently by heparin affinity chromatography. The affinity of osteogenin for various lectins was examined to attain further purification and characterization. Osteogenin extracted from bovine bone matrix binds to concanavalin A (Con A) but not to wheat germ agglutinin or soybean lectin. The present data indicate that the bone inductive protein, osteogenin, is a glycoprotein. The use of a Con A Sepharose affinity column followed by preparative gel electrophoresis resulted in a greater than 250,000 fold purification of osteogenin.

Animals↗

PubMed1989

In vivo analysis of the half-life of the osteoinductive potential of demineralized bone matrix using diffusion chambers.

Subcutaneous implantation of demineralized bone matrix (DBM) from rat initiates a sequence of developmental events that results in endochondral bone formation. This investigation examined the modification of the osteoinductive potential of DBM during the initial stages of this developmental cascade. Diffusion chambers (DC), constructed with filters of known pore size, permitting or excluding cells from entering the chambers, and containing DBM were subcutaneously implanted into Long-Evans male rats for specific time periods (1-7 days). DC were recovered and the osteoinductive potential of the matrix from these chambers was then tested by subcutaneous implantation and assaying the resulting day 11 plaque tissue enzymatically for alkaline phosphatase activity, and histologically for evidence of chondrogenesis and osteogenesis. The possible modification of DBM by local systemic factors (enzymatic degradation) or contact by polymorphonuclear leukocytes (PMNs) was also investigated. We have concluded from this study that the osteoinductive potential of DBM has a half-life of 5-7 days following implantation and although the enzymes collagenase, elastase, and trypsin abolished this activity, pepsin significantly enhanced it. Culture of PMNs with matrix prior to its implantation appeared to have little effect. Furthermore, during the initial stages of matrix-induced endochondral bone formation, DBM serves as both the instructive inducer and permissive substratum required in this process.

Alkaline Phosphatase↗

PubMed1989

Influence of testosterone and dihydrotestosterone on bone-matrix induced endochondral bone formation.

A bone matrix-induced endochondral bone development model has been used to study the effects of androgens on different stages of bone development in castrated young adult rats. Androgen treatment, especially with dihydrotestosterone (DHT) for 7 days, inhibited 35SO4 incorporation by the developing cartilage in the induced plaques. Castrated control animals maintained for 11 days after implantation of bone matrix showed significantly lower calcium levels in the induced implant than was observed earlier in the unoperated controls. DHT treatment for 11 days caused dramatic increases in levels of calcium in the implants. Testosterone had little effect. When androgen treatment was continued for 21 days, while levels of alkaline phosphatase in the implants were unaffected, levels of calcium in the implants were significantly higher than on day 11 for both castrated control and androgen-treated animals. Peak alkaline phosphatase activity (day 10) is known to precede peak calcium mineralizing activity (day 12) in this model and it is also known that calcium levels remain high thereafter. Evaluation of calcium and alkaline phosphatase levels in the proximal tibial metaphyses of castrated control and androgen-treated groups of animals showed no changes after 11 days treatment. Prolonged treatment (21 days) elevated the levels of alkaline phosphatase whereas no change was observed in calcium levels in the tibial metaphyses. These findings demonstrate that androgens stimulate mineralization and that DHT is more active when used for short periods of time and in early stages of bone development in matrix-induced implants.

Alkaline Phosphatase↗

PubMed1989

Calvarial bone regeneration using osteogenin.

This experimental study reports on the application of a partially purified noncollagenous protein extracted from cortical bone to restore craniotomy defects in Papio species (baboon). The partially purified protein (osteogenin) produced significantly more bone formation in calvarial wounds than was found in the untreated controls. There were no adverse tissue reactions from the implanted xenogeneic osteogenin.

Animals↗

PubMed1989

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

Osteogenin was recently purified and the amino acid sequences of tryptic peptides were determined. Osteogenin in conjunction with insoluble collagenous 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.

Alkaline Phosphatase↗

PubMed1989

Initiation of bone development by osteogenin and promotion by growth factors.

The cellular and molecular basis of bone development and its regulation by differentiation and growth factors is an exciting area of current research. This article briefly reviews the historical progress in the isolation of osteogenin, a novel bone differentiation factor, and its modulation by well known growth factors. Endochondral bone development is a multistep sequential cascade and the process must be operationally dissected. It has been accomplished with the demineralized bone matrix-induced bone formation model. The reproducible development of cartilage and bone in an extraskeletal site permits the study of the initiation of the first cycle of endochondral bone formation and mineralization. Recent progress in the isolation of osteogenin, a specific bone differentiation factor, by heparin affinity chromatography permits the further investigation of the commitment and clonal expansion of the putative osteoprogenitor stem cells. Once initiated, bone formation is promoted by growth factors such as platelet derived growth factor, fibroblast growth factor, insulin like growth factor, transforming growth factor beta and a plethora of non specific cytokines. Finally bone development is further modulated by systemic hormones and nutrition and a host of physical signals including electrical, gravitational and mechanical forces.

Animals↗

PubMed1989

Biochemical parameters of osteogenesis in epiphysis of mice irradiated and repopulated with syngeneic bone marrow.

Whole-body irradiation of BALB/cann mice with 600 R of gamma rays produces a profound atrophy of the spleen and diminishes alkaline phosphatase activity in the homogenates of epiphysis. The acid phosphatase activity remains unchanged until day 10, then slightly declines. Incorporation of 45Ca into epiphysis is practically not impaired following irradiation. Repopulation of irradiated animals with 1-1.5 million nucleated syngeneic bone marrow cells restores spleen weight within 6 days, but until day 11 the activity of alkaline phosphatase of epiphysis remains lower. Full recovery of enzyme activities was not observed until day 14, but on day 28 these activities returned to the normal level. Histological inspection did not reveal a full recovery of bone marrow in the epiphysis of irradiated animals until day 14, possibly because of trapping of the vast majority of stem cells by the spleen and liver. On day 28, however, epiphyses were completely filled with the bone marrow.

Acid Phosphatase↗

PubMed1988

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography.

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.

Animals↗

PubMed1988

Influence of irradiation on the osteoinductive potential of demineralized bone matrix.

Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1-15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring 45Ca incorporation to bone mineral, and 40Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3-5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.

Acid Phosphatase↗

PubMed1988

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation.

Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.

Animals↗