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Biomedical subjects

M E Bolander

Publications and source records attributed to M E Bolander.

62 records · Page 4Linked to original sources
PubMed1989

Estrogen receptor mRNA expression in callus during fracture healing in the rat.

Estrogen has profound effects on bone metabolism, yet its precise mechanism of action remains unknown. Several recent investigations have located the estrogen receptor (ER) on osteoblast-like cells, suggesting a potential direct action of estrogen via its own receptor on bone cells. The protective role of estrogen on bone in osteoporosis is well known; however, neither the existence nor the mechanism of an estrogenic role in fracture healing has been well studied. In this investigation we used a modification of polymerase chain reaction (PCR) amplification to detect and quantify ER messenger RNA (mRNA) levels in rat fracture callus previously undetectable by northern analysis. Verification of correctly amplified cDNA fragments was provided by restriction digestion. PCR failed to detect ER mRNA in total RNA extracts from tendon and fibrocartilage. The ER message was present in fracture callus at levels up to 70% of that found in rat uterus, and demonstrated a transient 13-fold increase in transcription by day 14 with return to baseline by day 31 of fracture healing. These data demonstrate that the rat estrogen receptor gene is expressed in fracture callus and suggest that estrogen may play an important role in the normal fracture healing process. In addition, this work adds to the growing body of evidence which supports a possible direct action of estrogen via its receptor on osteogenic cells.

Animals↗
PubMed1989

Monoclonal antibodies against osteonectin show conservation of epitopes across species.

Several monoclonal antibodies were produced to bovine osteonectin, a major noncollagenous protein in the extracellular matrix of bone, and four were characterized. These antibodies showed different reactivities in Western immunoblots, immunoprecipitation, and indirect immunofluorescence, indicating that they recognize different epitopes on the protein. The data indicate that an epitope recognized by one of the antibodies is masked in interactions of osteonectin within cells and in the extracellular matrix. The high degree of cross-species immunoreactivity observed against bone osteonectin with these monoclonal antibodies indicates that these common epitopes have been conserved during evolution.

Animals↗
PubMed1989

Isolation and analysis of ribonucleic acids from skeletal tissues.

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.

Animals↗
PubMed1989

Defects of early fracture-healing in experimental diabetes.

Diabetes has been implicated as a cause of impaired fracture-healing. To test this hypothesis, we tested the tensile strength of femora from normal rats and from untreated and insulin-treated diabetic rats two weeks after the production of a closed fracture. One week before the fracture, diabetes was induced by administration of streptozotocin (sixty-five milligrams per kilogram of body weight). The concentration of serum glucose increased from 6.1 +/- 0.3 millimoles per liter (110 +/- 5 milligrams per deciliter) in the control animals to 31.1 +/- 0.8 millimoles per liter (560 +/- 15 milligrams per deciliter) in the untreated diabetic animals. After two weeks of healing, fracture callus from the untreated diabetic animals had a 29 per cent decrease in tensile strength and a 50 per cent decrease in stiffness compared with the controls. Treatment of the diabetic animals with insulin resulted in a mean concentration of serum glucose of 14.4 +/- 0.6 millimoles per liter (260 +/- 10 milligrams per deciliter) and restored the tensile strength and stiffness of the callus to a value that was not statistically different from that of the controls. Between the fourth and eleventh days of healing, there was a 50 to 55 per cent decrease in the collagen content of the callus of the untreated diabetic animals compared with the controls. In addition, on the fourth day of healing, DNA content, an indicator of cellularity of the callus, was decreased 40 per cent in the untreated diabetic group. Between the fourth and eleventh days of healing, the collagen-to-DNA ratio, which was determined as an indicator of net collagen synthesis per cell, was decreased 15 to 50 per cent in callus from the untreated diabetic animals.

Animals↗
PubMed1988

Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).

Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.

Amino Acid Sequence↗
PubMed1986

Osteonectin mRNA: distribution in normal and transformed cells.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.

Animals↗
PubMed1986

The use of demineralized bone matrix in the repair of segmental defects. Augmentation with extracted matrix proteins and a comparison with autologous grafts.

A soluble protein component of bone, bone morphogenetic protein, and decalcified bone matrix have been shown to induce the formation of bone in extraosseous tissue. Clinical and animal studies investigating the use of these materials as bone grafts have shown radiographic and histological evidence of formation of bone, but the clinical usefulness of these grafts remains unknown. This study compared the healing processes when plasma-coated demineralized bone matrix and autologous cancellous bone were used to graft segmental defects of bone. A standard procedure was used to make a two-centimeter defect bilaterally in the ulna of forty-eight skeletally mature New Zealand White rabbits. In each rabbit, one ulnar defect was grafted with autologous citrated plasma-coated demineralized bone matrix while the other defect served as a control and was grafted with either autologous cancellous bone from the iliac crest, demineralized bone matrix, or demineralized bone matrix augmented with bone proteins that had been extracted with guanidinium hydrochloride. The ulnar defect was stabilized by the intact radius, and no supplemental device was necessary for fixation. To examine spontaneous healing in this model, one group of rabbits had a control defect that was not grafted. The grafts were periodically evaluated by radiographs, and twelve weeks after surgery the grafts were harvested and tested to failure in a standard torsion-test machine. The mechanical parameters were calculated, and histological examination of major fragments of the grafts was performed. The results of the radiographic and histological evaluation showed that all of the grafted ulnae healed, with fusion of the graft to the cut ends of the defect and reformation of approximately normal anatomy. No ungrafted ulnar defects healed. The results from the mechanical tests were evaluated by comparing the defect that was grafted with plasma-coated demineralized bone matrix with the control graft in each animal. These data showed that: twelve weeks after grafting, the normal ulnae were significantly stronger than the ulnae that had been grafted with plasma-coated demineralized bone matrix; the ulnae that had been grafted with plasma-coated demineralized bone matrix and those that had been grafted with autologous bone were equivalent in strength; and twelve weeks after grafting, grafts of demineralized bone matrix that were augmented with extracted bone proteins were significantly stronger than those that had not been so augmented.

Amino Acids↗