Search PubMed⌕ Search

Biomedical subjects

M E Bolander

Publications and source records attributed to M E Bolander.

At least 19 recordsLinked to original sources
PubMed1996

Identification of a dual leucine zipper kinase involved in rat fracture repair.

Complete cDNA sequence was obtained for a gene found to be expressed at a high level during fracture healing in the rat. Comparison of the amino acid sequence (deduced from the cDNA sequence) with known sequences showed 99% identity with mouse Dual Leucine Zipper Kinase (mDLK) and 96% identity with human Zipper Protein Kinase (hZPK). Relative quantitation by RT-PCR indicates abundant expression of this gene in cultured chondrocytes, growth plate and brain tissue. Further, regulated expression of this gene has been observed during fracture healing in rat femur fracture model; maximum gene expression in this model appears to coincide with chondrogenesis and bone formation. Since DLK has been shown to function in the mitogen activated signaling (MAP) pathway, these results provide the first evidence for the involvement of this signaling pathway in fracture healing.

Amino Acid Sequence↗
PubMed1996

Ultrasound-mediated transfection of mammalian cells.

Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of beta-galactosidase (beta-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37 degrees C), plasmid concentrations, and initial cell populations. Additional experiments were performed performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 +/- 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 +/- 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 micrograms/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells.

Animals↗
PubMed1996

A differential scanning calorimetry study of retrieved orthopedic implants made of ultrahigh molecular weight polyethylene.

Differential scanning calorimetry (DSC) was used to examine thermal and thermooxidative properties of ultrahigh molecular weight polyethylene (UHMW-PE) of five acetabular components of failed orthopedic implants retrieved at revision of total hip arthoplasty. The results were compared with controls (unimplanted acetabular cups, a 20-year-old slab of UHMW-PE, and raw material). Profiles of exothermic peaks indicated increased levels of oxidation in all retrieved cups. In three retrieved cups, DSC revealed an additional peak of endotherm that was not seen in control samples. The additional endotherm peaks were not artifacts due to oxidation during scanning, heat buildup during cutting of the samples, or the sterilization method after retrieval. The additional peak was associated with the bulk of the polymer that was extracted with hexane. It varied in relative area, depending on its original location of the sample in a cup, implicating local variability in the extent of changes in material property. The distribution of the changes suggests that, during implantation, tissue exposure and friction affected the level of oxidation and degree of crystallinity in the UHMW-PE to a greater degree than did loading alone. Overall results showed that DSC may be a useful tool in evaluating changes in the properties of UHMW-PE orthopedic components in vivo.

Acetabulum↗
PubMed1996

Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model.

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long-Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm2 spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm2 ultrasound signal, the average maximum torque (223.5 +/- 50.5 Nmm compared with 172.6 +/- 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 +/- 3.4 Nmm/degree compared with 9.5 +/- 2.9 Nmm/degree, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm2 ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound-treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm2 ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). alpha 1(II) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone-related proteins, including alpha 1(I) procollagen, bone gamma-carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor-beta 1, did not differ between ultrasound-treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.

Aggrecans↗
PubMed1995

Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum.

Estrogen deficiency is well recognized as a cause of bone loss in rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method. cDNA was synthesized from 2 micrograms total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensity were uterus, liver, bone, and spleen. Importantly, a similar time-course for estrogen-induced down-regulation of steady-state mRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated.

Animals↗
PubMed1995

Hip arthroplasty in hemophilic arthropathy.

As hemophilic arthropathy infrequently affects the hip joint, we performed a multicenter retrospective study to determine the results of hip arthroplasty in hemophilic patients. Thirty-four hip arthroplasties were performed in twenty-seven male patients at four major hemophilia centers from October 1972 through September 1990. Twenty-six patients had classic hemophilia and one had factor-IX deficiency. The mean age of the patients at the time of the operation was thirty-eight years (range, fifteen to seventy-three years). The mean duration of follow-up was eight years, with a minimum of two years for all patients who were still alive at the time of this review. Four patients were seropositive for the human immunodeficiency virus at the time of the operation, and sixteen patients were seropositive at the time of the most recent follow-up examination. Nine patients (33 per cent) died before the time of this review; seven had been seropositive for the human immunodeficiency virus. There were twenty-six total hip arthroplasties performed with cement, six total hip arthroplasties performed without cement, one total hip arthroplasty in which the femoral component was inserted with cement and the acetabular component was inserted without it (so-called hybrid arthroplasty), and one bipolar arthroplasty performed with cement. There were no early infections after these thirty-four primary arthroplasties. There were three late infections around prostheses inserted with cement, and all led to a resection arthroplasty. Six (21 per cent) of the twenty-eight cemented femoral components and six (23 per cent) of the twenty-six cemented acetabular components were revised because of aseptic loosening.(ABSTRACT TRUNCATED AT 250 WORDS)

Activities of Daily Living↗
PubMed1994

Low intensity ultrasound treatment increases strength in a rat femoral fracture model.

Bilateral closed femoral shaft fractures were made in 22 male Long-Evans rats. In 16 animals, ultrasound was applied to one limb for 15 minutes daily 10 times within the first 14 postoperative days. The treated limbs received a 200 microseconds burst of 1.5 or 0.5 MHz sine waves repeated at 1.0 kHz at a spatial average and temporal average intensity of 30 mW/cm2. The contralateral limb of each animal served as a nontreated control. Six remaining animals with fractures and six additional animals without fractures received sham ultrasound treatment to control for the effects of anesthesia and handling. Fracture repair was evaluated on postoperative day 21 by radiography, mechanical testing in torsion, and histology. Five of 16 ultrasound-treated fractures showed obliteration of the fracture gap on radiographs, whereas none of the 28 controls did. The average maximum torque of fractures treated with either signal was 22% greater than that of the contralateral controls (p < 0.05). The stiffness of treated fractures was greater than that of control fractures, but the difference was significant only in animals treated with the 1.5 MHz signal (p < 0.02). Sham treatment did not affect repair in the control group. These results indicate that low-intensity pulsed ultrasound at either 0.5 or 1.5 MHz can accelerate fracture repair at 21 days in this highly controlled model.

Animals↗
PubMed1994

Induction of growth plate cartilage ossification by basic fibroblast growth factor.

In mammals, longitudinal bone growth results from the precise coupling of chondrogenesis and osteogenesis within the epiphyseal growth plate, a process termed endochondral ossification. The mechanisms coupling chondrogenesis and osteogenesis are unknown. Previous studies have shown that both basic fibroblast growth factor (bFGF) and acidic FGF are expressed by growth plate chondrocytes. Here we show that bFGF, infused directly into the rabbit proximal tibial growth plate, accelerates vascular invasion and ossification of growth plate cartilage. Our results suggest the possibility that bFGF (or a related member of the FGF family) couples osteogenesis to chondrogenesis by attracting vascular and bone cell invasion from the adjacent metaphyseal bone.

Animals↗
PubMed1994

Type X collagen in fracture callus and the effects of experimental diabetes.

Studies of fracture repair in diabetes have shown decreased mechanical strength and total collagen in the callus. Type I and Type II collagen are synthesized by bone and cartilage cells, respectively, while Type X collagen is synthesized by hypertrophic chondrocytes in endochondral ossification. A standardized fracture model was chosen to investigate extracellular matrix changes during fracture healing of normal and diabetic rats. Histological examination of the fracture callus in both normal and diabetic animals showed progression from a fibrous tissue to cartilage to bone. An antiserum to Type X collagen was prepared, and immunostaining was observed in the matrix surrounding the hypertrophic chondrocytes. Fracture calluses from streptozotocin induced diabetic rats had similar histology and immunostaining to controls. Radiolabelled proteins were extracted from the calluses of normal and diabetic rats to measure Type X collagen. Type X collagen expression in the fracture callus of normal rats reached a maximum at day fourteen and was decreased by between 54% and 70% in the fracture callus of diabetic rats. The decrease in Type X collagen synthesis may have a role in the defect of fracture healing in diabetes.

Amino Acid Sequence↗
PubMed1993

Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus by using universal primers.

Fast acquisition of unknown nucleotide sequences around a known sequence has important implication in molecular biology, especially in genome mapping. We have developed a method, termed restriction site polymerase chain reaction (RS-PCR), that utilizes specially designed primers that recognize, anneal, and sustain PCR. These primers, termed restriction site oligonucleotides (oligonucleotide primers specific for a given restriction enzyme recognition sequence or RSOs), could be generated corresponding to any restriction enzyme irrespective of the length of the recognition site and used as PCR primers corresponding to the unknown region of a DNA segment. In this method a first round of PCR is carried out in different tubes with a set of RSOs and a primer specific to the known region. A second round of PCR is then performed on the products of the first PCR with the same RSOs and another specific primer internal to the first one. Subsequently, the products of the last round of PCR are transcribed with an appropriate RNA polymerase and sequenced with a reverse transcriptase with an end-labeled specific primer internal to the second specific PCR primer. To demonstrate the applicability of RS-PCR in retrieving unknown sequences around a known sequence, we have used a set of four RSOs and three specific primers representing the known sequence and have successfully obtained hitherto unknown factor IX sequences (12 of 12 times) from three species starting from genomic DNA. The sequences obtained indicate the presence of a conserved stretch of 20 nucleotides in the 3' noncoding region of the factor IX gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Gene identification by arrested primer extension.

This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identified by the length of the extension products on gel electrophoresis. This simple procedure eliminates the much-demanding process of sequencing. We term this process Arrested Primer Extension (APE). As a demonstration of the feasibility of this method, we have used APE to speciate a known set of cultured mycobacteria. There should be many other applications of this method.

Base Sequence↗
PubMed1993

Improved design of riboprobes from pBluescript and related vectors for in situ hybridization.

The pBluescript family of plasmids and phagemids are sophisticated multi-purpose cloning vectors that allow convenient production of single-stranded sense and anti-sense RNA probes corresponding to DNA sequences inserted into a large multiple cloning site array. We have observed that in many applications sense (control) probes generated from genes cloned into pBluescript II KS(-) give high background signals on in situ hybridization to human tissue sections. Our studies indicate that this spurious hybridization is due to sequences contained within both strands of the multiple cloning site between the SmaI and SacI sites that are similar to human 28S rRNA. This information is useful in construct design in order to minimize nonspecific background problems, as demonstrated by in situ hybridization of sense and anti-sense probes corresponding to a portion of human stromelysin-3 to sections of human lung carcinoma.

Base Sequence↗