In vitro establishment of a murine Moloney sarcoma cell line. I. Its morphology and transplantation.
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Biomedical subjects
A H Reddi
Publications and source records attributed to A H Reddi.
Importance of skeletal muscle environment for ectopic bone induction in mice.
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Changes in pyrophosphatase activity during the de novo mineralization associated with cartilage and bone formation.
Pyrophosphatase was extracted from implants undergoing de novo mineralization in an in vivo model of matrix-induced endochondral bone formation. Before the onset of the mineralization of the plaques and after the mineralization process had been completed only one form of pyrophosphatase activity was observed. During the active deposition of calcium phosphate, however, a new, higher molecular weight form of pyrophosphatase activity was produced suggesting that this enzyme activity is associated with biological mineralization. This observation gives support to the earlier suggestion that inhibitors of calcium phosphate precipitation, such as pyrophosphate, must be removed from the site of mineralization before calcification can occur. This high-molecular-weight activity also appears to be associated with alkaline and/or acid phosphatase activity as determined by molecular exclusion chromatography.
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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.
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Implant-stimulated interface reactions during collagenous bone matrix-induced bone formation.
The sequential cellular reactions in the interface of collagenous bone matrix implants are described. The multistep cascade in response to bone matrix implantation include: binding of fibrin and fibronectin to the implanted matrix, chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasions, bone formation, remodeling, and bone marrow differentiation. The mechanisms of action is not known. However, several properties governing the implant-cell interface are described. It is possible that bone matrix is a suitable biomaterial with potential applications in periodontal and orthopedic practice.
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Decalcified tooth matrix powder induces new bone formation and hematopoietic microenvironment in the mouse.
Implants of bone and tooth matrix powder were placed subcutaneously (s.c.) on intraperitoneal (i.p.) Mitex or Polyvic membranes. Implants were removed for histology after 1-24 weeks. Macrophages, fibroblasts, and vascular sinusoids infiltrated around bone and tooth matrix particles after one week. In the s.c. tooth matrix implants, a few sites of cartilage formation and ossification developed at two weeks, and by three weeks granulocytopoiesis and megakaryocytopoiesis developed adjacent to new bone; erythropoiesis was not observed. In s.c. bone matrix implants and in the i.p. artificial membranes coated with bone or tooth matrix powder, ossification or hematopoiesis was not observed. Small numbers of CFU-s, CFU-nm, BFU-e, and CFU-e appeared 10-20 days after s.c. implantation of tooth matrix; none were detected in s.c. bone matrix implants.
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Induction of endochondral bone by demineralized bone matrix from diabetic rats.
In this investigation we examined the osteoinductive potential of demineralized bone matrix derived from chronically diabetic (streptozotocin-induced) rats. Long-Evans rats (28-31 days) were made diabetic with a single injection of streptozotocin (65 mg/kg) and provided food and water ad lib for 2 months. Diaphyseal shafts of femurs and tibias removed from the diabetic rats and their sibling controls were dehydrated, pulverized, sieved to 74-420 micron particles, and demineralized. Matrix was then bioassayed for its ability to induce endochondral bone on day 11 following subcutaneous implantation over the thorax of Long-Evans rats. The resulting plaques of tissue were subjected to histological analysis, determination of alkaline phosphatase activity, and calcium content. Bone matrix derived from diabetic animals proved to be a significantly better inducer of endochondral bone than did control matrix.
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Bone matrix-induced local bone induction.
The sequential cellular changes in the implants in response to collagenous bone matrix-induced local bone formation include: binding of fibronectin to matrix, chemotaxis and attachment of progenitor cells, proliferation and differentiation of progenitor cells into chondrocytes, and finally osteogenesis and marrow differentiation. The cellular origin of osteogenic proteins is not clear. The present study compares the osteogenic potential of demineralized rat and porcine bone matrix by dissociative extraction and reconstitution. Judging from the Sephacryl S-200 gel filtration profiles of the dissociative extracts of rat and porcine matrix, the latter appears to be smaller. Under identical experimental conditions, the rat chondrosarcoma and osteosarcoma were examined for chondrogenic and osteogenic properties and found to be devoid of inductive potential. It is noteworthy that gel filtration fractions of rat chondrosarcoma on Sepharose CL-6B are inhibitory to bone inductive potential of demineralized rat bone matrix.
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Age-dependent decline in extracellular matrix-induced local bone differentiation.
Implantation of demineralized bone matrix in subcutaneous sites in the rat results in new bone induction. This model system was examined with bone matrix prepared from young (4-month-old) and old (24-month-old) rats. The implants were removed on Days 9, 14 and 21 and examined for alkaline phosphatase activity and 45Ca incorporation. There is a significant decline in the osteo-inductive potential of the bone matrix derived from 24-month-old rats, as quantitated by the enzyme activity and 45Ca incorporation.
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Does lymphopoiesis regulate tissue mast cells?
An almost twenty-fold increase in mast cell concentration was observed in the spleens of BALB/c mice after sublethal X-irradiation. Repopulation with bone marrow cells abolished this effect.
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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.
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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↗
Changes in the extracellular matrix components fibronectin and laminin during immune rejection of skeletal muscle.
Extracellular matrix is known to play an important role during development and maintenance of various tissues. In the present study, changes in two extracellular matrix glycoproteins, fibronectin and laminin, were investigated in skeletal muscle undergoing immune rejection. Purified antibodies against fibronectin and laminin were used to analyze the matrix by indirect immunofluorescence at various intervals after transplantation of extensor digitorum longus muscle in rats. Fibronectin and laminin were localized in the pericellular basement membrane zone of the normal myofibers; however, the cytoplasm was devoid of both glycoproteins. Transplanted muscle grafts underwent a process of degeneration and then an initial regeneration during the first 7 days. This regeneration effort ceased with the onset of muscle rejection in 14-day transplants. At this time, fibronectin was seen in the cytoplasmic region as well as the extracellular matrix of myofibers and myotubes. At later time intervals, an increased intensity of staining for fibronectin was seen throughout the rejected muscle. In muscle grafts undergoing regeneration but not rejection (i.e., nonantigenic grafts), such an increase in the presence of fibronectin was not seen ( Gulati et al., 1982). The distribution of laminin did not change during the rejection process and was localized in the basement membrane zone of myofibers and myotubes, although the overall configuration of the basement membranes was deformed and collapsed. It appears that the basement membranes are resistant to degradation, and staining for laminin persists in rejected muscle. These results show marked changes in the extracellular matrix of muscle undergoing rejection. The appearance of fibronectin during the initial stages of muscle rejection may have a causal relationship to the process of immune defence mechanism; however, the exact role of fibronectin remains elusive.
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Local mechanisms that regulate bone formation: the subject of a recent NIH-sponsored workshop.
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Animals↗
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.
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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↗
Decreased bone formation and mineralization in virus-induced diabetes mellitus.
Severe hyperglycemic and hypoinsulinemic mice were selected at 30 and 180 days after infection with the D variant of encephalomyocarditis (EMC) virus. In these animals, although the concentration of calcium and phosphate in serum was not changed, the alkaline phosphatase activity and the rate of mineralization in the proximal tibial epimetaphysis were markedly decreased in the diabetic mice for 30 and 180 days compared with those of the age-matched uninfected animals. Viral specific antigens were not found in these tissues at either earlier or later infection with the virus. It is concluded that endochondral bone formation and mineralization are greatly impaired in EMC virus-induced diabetic mice, and the decreased bone formation and mineralization are not due to the virus-induced tissue damage, but to the persistent metabolic alterations.
Alkaline Phosphatase↗
Establishment of the hematopoietic microenvironment in the marrow of matrix-induced endochondral bone.
Implantation of demineralized diaphyseal bone matrix (DBM) into subcutaneous sites in allogeneic Long-Evans rats results in new endochondral bone formation accompanied by hematopoietic bone marrow differentiation in the newly formed ossicles. In the present study, we investigated the relationship between the time of appearance of hematopoietic stem cells (CFU-S) and those of certain postulated elements of the hematopoietic microenvironment, i.e., fibroblast colony-forming cells (CFC-F), colony-stimulating factors (CSF), and collagen types. CFU-S were first detected at 16 days' postimplantation in the developing ossicle. Their numbers increased exponentially until day 24, decreased slightly between days 27 and 33, and then slowly increased in number again until day 36. CFC-F were present at day 10, and their numbers increased exponentially until day 15, decreased dramatically until day 24, and then remained constant till day 35. Thus, the transient growth of CFC-F preceded the appearance and growth of CFU-S in the ossicle by two or three days.
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