Biomedical subjects

C T Brighton

Publications and source records attributed to C T Brighton.

At least 19 recordsLinked to original sources

PubMed1995

Microvessel endothelial cells and pericytes increase proliferation and repress osteoblast phenotypic markers in rat calvarial bone cell cultures.

To investigate the influence of microvessel cells on osteoblasts, we exposed osteoblast-enriched cultures of rat calvarial cells to cultured endothelial cells and pericytes using feeder-layer co-cultures, co-culture dish inserts, and conditioned media experiments. When co-cultured with growth-arrested feeder-layers of endothelial cells or pericytes for 10 days, bone cell cultures showed an increase in cell number and reduction in alkaline phosphatase activity. The response of bone cells to endothelial cells was nearly twice their response to pericytes. A similar response was demonstrated by exposure to microvessel cells in co-culture dish inserts and by exposure to media conditioned by microvessel cells. In long-term cultures of bone cells, the levels of osteocalcin and the number of mineralized nodules both were reduced by exposure to media conditioned by the microvessel cells. Transient exposure to conditioned media from the microvessel cell cultures for 3 days, during the period from initial plating to cell confluence, produced nearly the same effect on the cultures of bone cells as did continuous exposure to these conditioned media. The influence of isolated microvessel cells on osteoblast-enriched calvarial cells was found to be primarily mitogenic, mediated by soluble factors, independent of cell contact, and a cause of prolonged reduction in the expression of early and late markers of the osteoblast phenotype.

Animals↗

PubMed1995

Tibial nonunion treated with direct current, capacitive coupling, or bone graft.

Two hundred seventy-one tibial nonunions of average duration of 23.5 months (range, 9-69 months) were treated with direct current (167 patients), capacitive coupled electrical stimulation (56 patients), or bone graft surgery (48 patients). Logistic regression analysis was used to compare heal rates among the 3 treatment methods, to identify risk factors adversely affecting the heal rate, and to predict the probability of successful healing of a nonunion of any given risk profile treated with each of the 3 forms of therapy. Seven risk factors were identified: duration of nonunion, prior bone graft surgery, prior electrical treatment, open fracture, osteomyelitis, comminuted or oblique fracture, and atrophic nonunion. When no risk factors were present, there were no significant differences among the 3 treatment methods. As progressively more risk factors were present, the predicted heal rates decreased significantly regardless of the treatment method. Some differences among the treatment groups did appear in the heal rates: bone graft surgery yielded a worse heal rate when there was a previous bone graft failure, and capacitive coupling had a worse heal rate in the presence of an atrophic nonunion.

Adult↗

PubMed1995

Real-time calcium response of cultured bone cells to fluid flow.

Using a parallel-plate flow chamber and fura-2 fluorescence microscopy, intracellular calcium was measured cell by cell in preconfluent primary culture rat calvarial bone cells to 18, 35, and 70 dynes/cm2 of fluid-induced shear stress. A heterogeneous response with respect to peak amplitude and latency was observed for the culture, with an overriding dose-dependent relationship between the mean peak amplitude of response and shear-stress magnitude. A dose dependence was observed between the number of responsive cells (responding > 50% over basal levels) and shear-stress magnitude. Not all cells could be restimulated by repeated exposure to flow. The observed cell response appears to be independent of whether cells are clustered together or isolated. Substratum stretch, hydrostatic pressure, and fluid shear stress have been shown in the literature to increase inositol phosphate (IP3) in bone cells, with IP3 causing the release of calcium from intracellular stores such as the endoplasmic reticulum. Therefore, a 6-fold inhibitory effect observed when calcium release from stores was blocked with 8-(n,N-diethylamino)octyl 1-3,4,5-atrimethoxybenzoate hydrochloride implicates an IP3 biochemical pathway mediating the fluid flow response in bone cells.

Animals↗

PubMed1994

Identification of integrin receptors on cultured human bone cells.

The interactions of bone cells with their surrounding extracellular microenvironment may be mediated by integrins, a family of heterodimeric glycoproteins consisting of alpha and beta subunits that noncovalently interact to form cell-substratum adhesion receptors. We previously described the integrins on calvarial bone cells in rats with use of polyclonal antibodies against some integrin subunits. In the present study, we expanded this initial characterization by employing a more complete panel of monoclonal antibodies to identify integrins on human bone cells. Minced fragments of trabecular bone obtained during total knee arthroplasty were grown in culture until bone cells became confluent. The cells then were dissociated, plated again, grown to confluence, and assayed for alkaline phosphatase activity, response of cyclic adenosine monophosphate to stimulation with parathyroid hormone, and osteocalcin content. The percentage of the cells that adhered to various substrates was measured; 60-70% adhered to type-I collagen, fibronectin, vitronectin, and poly-D-lysine; 40-50% adhered to type-IV collagen, laminin, and gelatin; and only 10% adhered to fibrinogen. Flow cytometric analysis with anti-integrin monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates of the human bone cells revealed high levels of alpha 1 beta 1, alpha 3 beta 1, alpha 5, beta 1 and alpha v beta 5 integrins and much lower levels of alpha 2 beta 1, alpha 4 beta 1, alpha v beta 1, and alpha v beta 3 integrins. This description of the integrin repertoire of cultured human bone cells represents the first step toward an understanding of the role played by integrins in the growth, maintenance, and repair of bone.

Aged↗

PubMed1994

Low-power electromagnetic stimulation of osteotomized rabbit fibulae. A randomized, blinded study.

The purpose of this study was to determine whether low-power-consuming symmetrical-waveform electromagnetic stimuli could increase the stiffness of fracture sites in a rabbit fibular-osteotomy model. Both active and placebo devices were used in a blinded study protocol. Dose-response studies of pulse amplitude and pulse width were performed by continuous application (twenty-four hours a day) of repetitive (fifteen-hertz), bursted (five-millisecond-long) symmetrical, rectangular electromagnetic stimulus waveforms. The power consumed by these stimuli is approximately one-fifth that consumed by the pulsing electromagnetic field devices that are in current clinical use. Significant increase of callus bending stiffness was produced by pulse widths of five to seven microseconds and pulse amplitudes of fifty to 100 millivolts.

Animals↗

PubMed1994

Growth plate matrix vesicle biogenesis. The role of intracellular calcium.

Isolated bovine growth plate chondrocytes suspended in a synthetic cartilage buffer containing 10(-7) to 10(-3) M concentrations of calcium were treated with 1 microM ionomycin to induce changes in intracellular ionized calcium as measured by Fura-2 fluorescence. An increase in intracellular calcium of 10(-6) to 10(-4) M calcium resulted in the secretion of vesicles that contain alkaline phosphatase. An increase in intracellular calcium above 10(-4) M resulted in nonspecific cell fragmentation because of cell damage. Electron microscopy of the secreted vesicles demonstrated that their structure resembled matrix vesicles. Isolated cells treated with ionomycin (synthetic cartilage buffer with 10(-6) M calcium) demonstrated intact cell membranes, normal intracellular architecture, and numerous plasma membrane processes undergoing vesiculation. The plasma membrane of these isolated cells and the isolated vesicles demonstrated a positive stain for alkaline phosphatase. Fresh growth plate tissue demonstrated plasma membrane staining for alkaline phosphatase at the lower proliferative and upper hypertrophic cell zone, and an increasing number of alkaline phosphatase stained matrix vesicles in the matrix of the lower hypertrophic cell zone. The data indicate that an increase in intracellular calcium in the lower proliferative and hypertrophic cell zones of the growth plate induces the secretion of plasma membrane derived matrix vesicles.

Alkaline Phosphatase↗

PubMed1994

Analysis of physeal growth in dogs, using biplanar radiography.

Biplanar radiography was used to study normal growth of the left and right radius in 5 Beagles and growth of the left radius alone in 15 additional Beagles. We explored the applicability of this radiographic method in veterinary medicine by measuring the contribution to total radius length from each growth plate. Spherical tantalum markers (0.5 mm) were embedded in the proximal epiphysis, diaphysis, and distal epiphysis of each dog's radius at 10 weeks of age. Simultaneous biplanar radiographic views were obtained every 4 weeks until skeletal maturity was documented. A three-dimensional coordinate system was constructed allowing for measurement of growth (in millimeters). Resolution of the measuring system was 0.074 mm. Mean +/- SEM length of the skeletally mature Beagle's radius, as measured from proximal epiphyseal bead to distal epiphyseal bead, was 95.33 +/- 1.07 mm. The percentage of contribution to the total radius length from the proximal and distal growth plates was 36.76 and 64.73%, respectively, with 95% confidence interval of 2.29%. The percentage of contribution to radius length from the distal radial growth plate increased for each consecutive time segment, with the distal radial physis contributing 61.75% from 10 to 14 weeks of age and increasing to 70.22% from 22 to 26 weeks of age. Significant growth was not observed after 26 weeks of age. The period of most rapid growth was between 10 and 14 weeks of age. Biplanar radiography was accurate and precise in quantifying the relative contribution of the proximal and distal growth plate to radius length in Beagles.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Comparison of asymmetrical and symmetrical pulse waveforms in electromagnetic stimulation.

Pulsing electromagnetic field (PEMF) stimulation is a noninvasive therapeutic modality that has been successfully used to stimulate healing of surgically resistant human bone fracture nonunions. Asymmetry of the stimulus pulse waveform was thought to be necessary for therapeutic effectiveness, but asymmetrical pulses require significant electrical energy that constrains clinical delivery systems to suboptimal designs. Development of low-energy consuming stimuli will enable clinical device improvements and may provide additional information about the interaction of electromagnetic fields with tissues. The objectives of this study were (a) to determine if asymmetry of the stimulus pulse waveform is needed for efficacy and (b) to determine if symmetrical pulse waveform stimuli also can produce a beneficial therapeutic response. The rabbit fibular osteotomy model was used to answer these questions and to identify which components of the clinically used asymmetrical PEMF produce the therapeutic response. The results suggest that asymmetry is not necessary and that a narrow pulse width, symmetrical square wave signal can also stimulate stiffness increases in this model. The data also suggest that the high-amplitude, narrow-pulse portion of the asymmetrical PEMF is the principal component of the signal pulse that is responsible for the clinical therapeutic effect.

Animals↗

PubMed1992

The inositol phosphate pathway as a mediator in the proliferative response of rat calvarial bone cells to cyclical biaxial mechanical strain.

Isolated newborn rat calvarial bone cells grown in monolayer on polyurethane membranes in specially constructed culture chambers and subjected to a cyclical biaxial mechanical strain of 0.17% at a frequency of 1 Hz for 30 min demonstrated a 16% increase in DNA synthesis during the subsequent 24 h. The metabolites of the inositol phosphate pathway, shown to be an important second messenger in many cell types, were shown to be elevated using high-performance liquid chromatography to separate and quantitate the various inositol polyphosphates. Inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1,3,4,5-tetrakisphosphate reached peak accumulations after 20 s of mechanical strain. Inositol 1,3,4-trisphosphate reached a peak accumulation after 2 min, and inositol 1,2,3,4,5,6 phosphate reached a peak accumulation after 60 min of mechanical strain. Neomycin, an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to start the inositol phosphate cascade, completely inhibited accumulation of the above inositol phosphates during mechanical straining of the bone cells. Neomycin also completely abolished the increase in DNA synthesis that was seen after a mechanical strain of 0.17%. It is concluded from this study that the inositol phosphate pathway is activated by mechanical strain in bone cells and that this pathway is an important and primary mediator in the transduction of mechanical strain into cellular proliferation in these cells.

Analysis of Variance↗

PubMed1992

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.

Animals↗

PubMed1992

In vitro bone-cell response to a capacitively coupled electrical field. The role of field strength, pulse pattern, and duty cycle.

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cell's proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.

Alkaline Phosphatase↗

PubMed1992

The pericyte as a possible osteoblast progenitor cell.

Bovine capillary and microvessel pericytes were grown in monolayer in standard tissue culture medium supplemented with 10% newborn calf serum at various oxygen tensions for up to ten weeks. The pericytes synthesized alkaline phosphatase and formed colonies that mineralized. Energy dispersive X-ray spectrometry revealed the presence of calcium and phosphate, showed positive staining for collagen and glycosaminoglycan, and, most importantly, demonstrated the synthesis of osteocalcin. Cell proliferation, hydroxyproline production, and alkaline phosphatase synthesis were greatest in 3% oxygen, whereas osteocalcin production was least in 3% oxygen. These findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent. It is suggested from these findings that the capillary or microvessel pericyte may be an osteoblast precursor cell.

Alkaline Phosphatase↗

PubMed1991

The effect of oxygen tension on proteoglycan synthesis and aggregation in mammalian growth plate chondrocytes.

Growth plate chondrocytes isolated from the proliferative and hypertrophic zones of bovine costochondral junctions were grown in vitro in the presence of various oxygen tensions ranging from 3 to 60%. Using [35S] sulfate as an index of glycosaminoglycan synthesis, incorporation was found to be maximal at 21% O2. In contrast, proteoglycan aggregation under the same conditions was found to be maximal at 3% O2. There were no consistent differences in response between cells from the different morphologic zones even though they are exposed to different oxygen tensions in situ. These results show that proteoglycan synthesis and aggregation in growth plate chondrocytes in vitro are differentially affected by the ambient oxygen environment.

Animals↗

PubMed1991

Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions.

Perinatal rat calvarial bone cells were isolated by sequential collagenase digestion and grown in oxygen tensions ranging from 1 to 60% O2. Cell proliferation as determined by automated cell counting and DNA content was greatest in the lower oxygen tensions (less than or equal to 9% O2), whereas alkaline phosphatase activity and [35S]sulfate and [14C]proline incorporation were greatest in the higher oxygen tensions (greater than or equal to 13% O2). It is concluded that lower oxygen concentrations favor bone cell proliferation, whereas higher oxygen concentrations favor macromolecular synthesis. These findings, when related to the known pO2 of the fracture callus, suggest the following sequence of events: first, at the time of fracture an ingrowth of osteoprogenitor cells, capillary buds, and primitive mesenchymal cells occurs in the fracture site, a region of low pO2; second, a great increase in cellular proliferation accompanied by an initiation of macromolecular synthesis follows; finally, as the pO2 levels begin to increase, cellular proliferation decelerates, accompanied by an increase in macromolecular synthesis.

Alkaline Phosphatase↗

PubMed1991

The proliferative and synthetic response of isolated calvarial bone cells of rats to cyclic biaxial mechanical strain.

Isolated bone cells from the calvaria of newborn rats were grown in monolayer on polyurethane membranes in specially constructed culture chambers. These were subjected to cyclic biaxial mechanical strains of 0.02 per cent (200 microstrain), 0.04 per cent (400 microstrain), and 0.1 per cent (1000 microstrain) at a frequency of one hertz for periods ranging from fifteen minutes to seventy-two hours. DNA content, an index of proliferation, was significantly increased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four and forty-eight hours. DNA content was not increased at the other amplitudes of strain that were evaluated, nor was it increased after prolonged mechanical stimulation for forty-eight hours or longer. Synthesis of collagen, non-collagenous protein, and proteoglycan, as well as activity of alkaline phosphatase, all indicators of macromolecular synthesis, were significantly decreased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four, forty-eight, and seventy-two hours. Macromolecular synthesis was not affected by the other amplitudes of strain that were evaluated in this study. At a strain of 0.04 per cent, prostaglandin E2 content was significantly increased after five, fifteen, and thirty minutes of mechanical stimulation, whereas net cAMP content did not change significantly. This suggests that the described cellular events (increased proliferation and decreased macromolecular synthesis) that occur secondary to mechanical strain are mediated, at least in part, by prostaglandin E2.

Alkaline Phosphatase↗

PubMed1991

Psychosocial, functional, and quality of life assessment of patients with posttraumatic fracture nonunion, chronic refractory osteomyelitis, and lower extremity amputation.

One hundred nine patients with long-bone fracture nonunion, chronic refractory osteomyelitis, or posttraumatic amputation were evaluated for the impact of chronic disability on quality of life, as measured by the Arthritis Impact Measurement Scale (AIMS) and the Psychosocial Adjustment to Illness Scale (PAIS). A self-administered PAIS for spouses assessed psychosocial adjustment of spouses or significant others. A final questionnaire ranked the reasons for either continuing medical therapy or accepting amputation. The PAIS scores differed significantly between osteomyelitis patients and both nonunion and amputation patients (p less than .05). The presence or absence of pain produced significant differences in AIMS and PAIS scores of nonunion and osteomyelitis patients (p less than .05). Subscale analysis of AIMS scores revealed significant differences among the three groups in health perception and scale of orthopedic problem: osteomyelitis patients were more severely affected than nonunion or amputation patients. The PAIS detected no statistically significant difference in psychosocial adjustment of spouses of patients in the three population groups. The most common reason for continuing medical and surgical management of nonunion and osteomyelitis was expectation for cure. The amputee group chose ablation to avoid further treatment. Differences in psychosocial and functional ability were related to disease diagnosis, pain, status of fracture healing, and timing of amputation.

Adaptation, Psychological↗

PubMed1991

Early histological and ultrastructural changes in medullary fracture callus.

Light and electron microscopic studies of early changes in the medullary callus of a fracture of the rib in rabbits revealed the loss of normal architecture of the marrow and the disappearance of blood vessels in the region of high cellular density adjacent to the fibrin clot; the enlargement and transformation of capillary and venous endothelial cells in the region of low cellular density adjacent to the normal, uninjured marrow; the appearance of polymorphic mesenchymal cells throughout the medullary callus; and the appearance of osteoblasts and new-bone formation by twenty-four hours after the fracture. The meaning of these morphological changes is not clear. However, the spatial relationship between the various cells suggests the possibility that the transformed endothelial cells, reticular cells, and polymorphic mesenchymal cells may be interrelated and may either be osteoblast progenitor cells or may in some way lead to the appearance of osteoblasts in the early callus.

Animals↗

PubMed1990

Mechanism of action of parathyroid hormone-induced proteoglycan synthesis in the growth plate chondrocyte.

In the growth plate chondrocyte, parathyroid hormone (PTH) stimulates phosphoinositol 4,5 bisphosphate (PIP2) degradation, which results in the rapid production of inositol (1,4,5) triphosphate (IP3). IP3 induced the release of calcium from an intracellular store, which caused a rapid increase in the cytosolic ionized calcium concentration. Parathyroid hormone also induced a 30-50% increase in proteoglycan synthesis. Phorbol esters, which pharmacologically activate protein kinase C, resulted in a 70-80% increase in proteoglycan synthesis. Treatment of the chondrocytes with retinoic acid (0.2 microM) inhibited the parathyroid hormone and phorbol ester-induced increase in intracellular ionized calcium and the increase in proteoglycan synthesis. From this data we postulate that the stimulation of proteoglycan synthesis in growth plate chondrocytes by PTH is mediated by the breakdown of membrane phosphoinositides, which results in the production of IP3 and an increase in ionized intracellular calcium. It is suggested that the degradation of membrane phosphoinositides also results in production of diacylglycerol and, thereby, an activation of protein kinase C, which has a large stimulatory effect on proteoglycan synthesis. The increase in cytosolic calcium most likely acts synergetically with diacylglycerol to activate protein kinase C. Retinoic acid blocks the effect of PTH and phorbol ester-induced proteoglycan synthesis and may act through the inhibition of protein kinase C. The overall effect of PTH on the growth plate chondrocyte appears to be a stimulation of proteoglycan synthesis that is mediated by the degradation products of membrane phosphoinositides.

Animals↗