During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation.... more During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteo...
Benign encapsulation of living cells within stable, permselective materials holds exciting prospe... more Benign encapsulation of living cells within stable, permselective materials holds exciting prospects for implantable autonomous therapeutics, but is challenged by the demand by such applications for materials bearing an optimal combination of microstructure, stability, and functionality. The ideal capsule would simultaneously protect the cell from immunological attack, efficiently transport nutrients, and rapidly release therapeutic cell metabolites. Recently, we have developed a benign and controllable method for synthesizing stable silica nanoparticles (Lys-Sil) from 10 to 50 nm, and their in situ and ex situ ordering into colloidal crystal arrays and thin films1. Encapsulating living cells within nanoparticle films of controllable thickness (i.e., tens of nanometers) will translate to higher flux of chemical species2. In addition, the porosity of such ordered nanoparticle films, imparted by the interstitial spacing between particles, can be tuned through particle size control and...
Journal of applied physiology (Bethesda, Md. : 1985), 1999
The decrease in cancellous bone formation after estrogen treatment is generally thought to be cou... more The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1-32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bon...
Experiments were carried out to determine the ability of female rats with poorly mineralized skel... more Experiments were carried out to determine the ability of female rats with poorly mineralized skeletons to increase bone mineralization in response to increased dietary Ca consumption. We specifically addressed this question with regard to two different periods of the life cycle: the period of sexual maturation (6-9 weeks of age), and in animals that had attained adult rates of skeletal mineralization (100 days of age). We found that at both stages, increased dietary Ca consumption resulted in increased trabecular bone volume and total bone Ca. In the younger animals, it was found that dietary history influenced the disposition of bone mineral. Animals that were initially Ca-deprived exhibited increased trabecular bone and decreased cortical thickness compared to animals continuously fed 0.5% Ca. Ovariectomy of mature animals reduced but did not eliminate the response to increased Ca intake.
Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular p... more Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. Porcine islets were co-cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7-day co-culture by perifusion glucose-stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (∼10(7)) were implanted under the renal capsule of C57BL/6 mice. No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1-day co-culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or followi...
Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It ... more Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.
Evaluate the ability of osteogenic protein-1 to induce formation of de novo bone in the presence ... more Evaluate the ability of osteogenic protein-1 to induce formation of de novo bone in the presence of bacterial infection and metal in an intramuscular osteoinduction model in the rat. Prospective experimental design with assessment time points of up to 4 weeks. Intramuscular pocket surgically created along each side of the spine. One-hundred-twenty adult male Sprague-Dawley rats. Each intramuscular pocket received 0, 10, or 25 microg of osteogenic protein-1 combined with a lyophilized collagen carrier, and 0 or 5 x 10 colony-forming units of Staphylococcus aureus. Pockets in 48 animals received a metal implant. Animals were killed at 1, 2, 3, or 4 weeks. High-resolution radiographs of resulting nodules of bone/soft tissue were digitized, and areas of newly formed bone were quantified using an image analysis workstation. The nodules were decalcified for histology, and calcium content of the decalcifying solution was quantified by flame atomic absorption spectrophotometry. There were minimal levels of calcium and area of new bone formation in nodules from pockets containing collagen carrier without osteogenic protein-1, for both infection and noninfection conditions. Calcium content and area of newly formed bone were significantly greater: 1) in infected pockets with osteogenic protein-1, compared to infected pockets without osteogenic protein-1; and 2) in noninfected pockets with osteogenic protein-1, compared to infected pockets with osteogenic protein-1. The presence of metal did not have a significant effect. Osteogenic protein-1 maintained its osteoinductive capability in a contaminated intramuscular pocket in the rat, albeit at a lower level than without infection. This finding supports further study using a more clinically realistic model.
The goal of this study was to use a segmental defect model in the rat femur to determine if osteo... more The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein-1 (OP-1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague-Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP-1 (11 or 50 microg), lyophilized bovine type I collagen (carrier for the OP-1), and 10(5) colony-forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP-1, compared with contaminated defects without OP-1. The 50 microg dose of OP-1 induced significantly more bone formation than the 11 microg dose, both with and without bacteria. This investigation has demonstrated that OP-1 maintains its osteoinductive capability in a contaminated segmental defect. OP-1 may potentially be used in the clinical management of contaminated fractures.
Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinkin... more Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinking on bone metabolism are largely uninvestigated. Here, we studied the long-term dose-response (0, 3, 6, 13, and 35% caloric intake) effects of alcohol on cancellous bone in the proximal tibia of 8-month-old female rats. After 4 months of treatment, all alcohol-consuming groups of rats had decreased bone turnover. The inhibitory effects of alcohol on bone formation were dose dependent. A reduction in osteoclast number occurred at the lowest level of consumption but there were no further reductions with higher levels of consumption. An imbalance between bone formation and bone resorption at higher levels of consumption of alcohol resulted in trabecular thinning. Our observations in rats raise the concern that moderate consumption of alcoholic beverages in humans may reduce bone turnover and potentially have detrimental effects on the skeleton.
During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation.... more During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteo...
Benign encapsulation of living cells within stable, permselective materials holds exciting prospe... more Benign encapsulation of living cells within stable, permselective materials holds exciting prospects for implantable autonomous therapeutics, but is challenged by the demand by such applications for materials bearing an optimal combination of microstructure, stability, and functionality. The ideal capsule would simultaneously protect the cell from immunological attack, efficiently transport nutrients, and rapidly release therapeutic cell metabolites. Recently, we have developed a benign and controllable method for synthesizing stable silica nanoparticles (Lys-Sil) from 10 to 50 nm, and their in situ and ex situ ordering into colloidal crystal arrays and thin films1. Encapsulating living cells within nanoparticle films of controllable thickness (i.e., tens of nanometers) will translate to higher flux of chemical species2. In addition, the porosity of such ordered nanoparticle films, imparted by the interstitial spacing between particles, can be tuned through particle size control and...
Journal of applied physiology (Bethesda, Md. : 1985), 1999
The decrease in cancellous bone formation after estrogen treatment is generally thought to be cou... more The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1-32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bon...
Experiments were carried out to determine the ability of female rats with poorly mineralized skel... more Experiments were carried out to determine the ability of female rats with poorly mineralized skeletons to increase bone mineralization in response to increased dietary Ca consumption. We specifically addressed this question with regard to two different periods of the life cycle: the period of sexual maturation (6-9 weeks of age), and in animals that had attained adult rates of skeletal mineralization (100 days of age). We found that at both stages, increased dietary Ca consumption resulted in increased trabecular bone volume and total bone Ca. In the younger animals, it was found that dietary history influenced the disposition of bone mineral. Animals that were initially Ca-deprived exhibited increased trabecular bone and decreased cortical thickness compared to animals continuously fed 0.5% Ca. Ovariectomy of mature animals reduced but did not eliminate the response to increased Ca intake.
Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular p... more Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. Porcine islets were co-cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7-day co-culture by perifusion glucose-stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (∼10(7)) were implanted under the renal capsule of C57BL/6 mice. No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1-day co-culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or followi...
Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It ... more Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.
Evaluate the ability of osteogenic protein-1 to induce formation of de novo bone in the presence ... more Evaluate the ability of osteogenic protein-1 to induce formation of de novo bone in the presence of bacterial infection and metal in an intramuscular osteoinduction model in the rat. Prospective experimental design with assessment time points of up to 4 weeks. Intramuscular pocket surgically created along each side of the spine. One-hundred-twenty adult male Sprague-Dawley rats. Each intramuscular pocket received 0, 10, or 25 microg of osteogenic protein-1 combined with a lyophilized collagen carrier, and 0 or 5 x 10 colony-forming units of Staphylococcus aureus. Pockets in 48 animals received a metal implant. Animals were killed at 1, 2, 3, or 4 weeks. High-resolution radiographs of resulting nodules of bone/soft tissue were digitized, and areas of newly formed bone were quantified using an image analysis workstation. The nodules were decalcified for histology, and calcium content of the decalcifying solution was quantified by flame atomic absorption spectrophotometry. There were minimal levels of calcium and area of new bone formation in nodules from pockets containing collagen carrier without osteogenic protein-1, for both infection and noninfection conditions. Calcium content and area of newly formed bone were significantly greater: 1) in infected pockets with osteogenic protein-1, compared to infected pockets without osteogenic protein-1; and 2) in noninfected pockets with osteogenic protein-1, compared to infected pockets with osteogenic protein-1. The presence of metal did not have a significant effect. Osteogenic protein-1 maintained its osteoinductive capability in a contaminated intramuscular pocket in the rat, albeit at a lower level than without infection. This finding supports further study using a more clinically realistic model.
The goal of this study was to use a segmental defect model in the rat femur to determine if osteo... more The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein-1 (OP-1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague-Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP-1 (11 or 50 microg), lyophilized bovine type I collagen (carrier for the OP-1), and 10(5) colony-forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP-1, compared with contaminated defects without OP-1. The 50 microg dose of OP-1 induced significantly more bone formation than the 11 microg dose, both with and without bacteria. This investigation has demonstrated that OP-1 maintains its osteoinductive capability in a contaminated segmental defect. OP-1 may potentially be used in the clinical management of contaminated fractures.
Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinkin... more Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinking on bone metabolism are largely uninvestigated. Here, we studied the long-term dose-response (0, 3, 6, 13, and 35% caloric intake) effects of alcohol on cancellous bone in the proximal tibia of 8-month-old female rats. After 4 months of treatment, all alcohol-consuming groups of rats had decreased bone turnover. The inhibitory effects of alcohol on bone formation were dose dependent. A reduction in osteoclast number occurred at the lowest level of consumption but there were no further reductions with higher levels of consumption. An imbalance between bone formation and bone resorption at higher levels of consumption of alcohol resulted in trabecular thinning. Our observations in rats raise the concern that moderate consumption of alcoholic beverages in humans may reduce bone turnover and potentially have detrimental effects on the skeleton.
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