RESUMO
OBJECTIVE: To ascertain, using specific inhibitors, the potential role of calcium-related signal transduction pathways in the mechanism of cartilage matrix protein gene induction and metalloproteinase gene suppression by capacitively coupled electric fields. METHODS: Articular chondrocytes were isolated from adult bovine patellae and cultured in high density for 7 days. To study matrix protein expression, cells cultured in the absence or presence of specific calcium pathway inhibitors were exposed to a capacitively coupled electrical field (60 kHz, 20 mV/cm): for aggrecan 1h at 50% duty cycle and for type II collagen 6h at 8.3% duty cycle. To study metalloproteinase expression in the presence of interleukin 1 beta (IL-1beta), cells were cultured as above but exposed for only 30 min to a 100% duty cycle signal. At harvest, total mRNA was isolated and aggrecan, type II collagen, matrix metalloproteinase (MMP-1, -3 and -13) and aggrecanase [a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTS-4 and -5)] mRNA expression were measured by quantitative real-time polymerase chain reaction (qPCR). RESULTS: (1) In the absence of inhibitors, appropriate electrical stimulation induces a 3-4-fold up-regulation of both aggrecan and type II collagen mRNA and a 3.7-9.6-fold down-regulation of IL-1beta-induced metalloproteinases; (2) the presence of inhibitors alone does not affect any target mRNA levels; (3) inhibitors of intracellular calcium regulation and inositol 1,4,5-triphosphate (IP(3)) formation [8-(diethylamino)octyl-3,4,5,-trimethoxybenzoate hydrochloride (TMB-8) and neomycin, respectively] have no effect on regulation of target mRNA levels by electrical stimulation; and (4) inhibitors of voltage-gated calcium channels (verapamil), calmodulin activation (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride, W-7), calcineurin activity (cyclosporin A), phospholipase C activity (bromophenacyl bromide, BPB) and prostaglandin E(2) (PGE(2)) synthesis (indomethacin) completely inhibit the effects of electrical stimulation. CONCLUSIONS: The results are consistent with the effects of electrical stimulation involving a pathway of extracellular Ca(2+) influx via voltage-gated calcium channels rather than from intracellular Ca(2+) repositories; and with downstream roles for calmodulin, calcineurin and nuclear factor of activated T-cells (NF-AT) rather than for phospholipase C and IP(3).
Assuntos
Sinalização do Cálcio , Condrócitos/metabolismo , Estimulação Elétrica , Matriz Extracelular/metabolismo , Transdução de Sinais , Agrecanas/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Bovinos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Metaloproteinases da Matriz/metabolismo , RNA Mensageiro/análiseRESUMO
BACKGROUND: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells. METHODS: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined. RESULTS: All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca(2+) through voltage-gated calcium channels leading to an increase in cytosolic Ca(2+) (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca(2+) leading to an increase in cytosolic Ca(2+) (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7). CONCLUSIONS: The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca(2+) ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca(2+) from intracellular stores. The final pathway, however, is the same for all three signals-that is, there is an increase in cytosolic Ca(2+) and an increase in activated cytoskeletal calmodulin.
Assuntos
Osteoblastos/fisiologia , Transdução de Sinais , Animais , Divisão Celular , Células Cultivadas , Estimulação Elétrica , Camundongos , Osteoblastos/citologia , Transdução de Sinais/efeitos dos fármacosRESUMO
We investigated the effect of newborn bovine serum on the intracellular calcium [Ca(2+)](i) response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca(2+)](i) responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10-15min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca(2+)](i) response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca(2+)](i) flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca(2+)](i) response in bone cells subjected to fluid flow-induced shear stress.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Crânio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Concentração Osmolar , Ratos , Crânio/citologia , Crânio/efeitos dos fármacos , Estresse MecânicoRESUMO
Multipotential mesenchymal stem cells capable of chondro-osseous induction contribute to the endochondral callus of healing fractured bone. Microvascular pericytes serving the role of multipotential mesenchymal stem cells are considered osteoprogenitors because they express type I collagen, alkaline phosphatase enzyme activity, osteocalcin immunoreactivity, and bone sialoprotein mRNA. Previous electron microscopic studies indicate that this cell type has a contribution to the fracture callus. Limited data suggest that pericytes may also assume a chondrogenic phenotype. We undertook in vitro studies to understand how the chondro-osseous phenotype of the pericyte might be regulated. Using Northern analysis and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that cultured pericytes produce aggrecan and type II collagen mRNA indicating their chondrogenic potential. Aggrecan message is elevated by BMP-2 as analyzed by both Northern hybridization and RT-PCR. This finding suggests a regulatory role for this morphogen on this phenotype in pericytes. RT-PCR amplified versican product was also associated with pericyte cultures but was not affected by BMP-2. Our data strongly support a chondrogenic role for the pericyte and that the phenotype is regulated at least in part by BMP.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas da Matriz Extracelular , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Proteoglicanas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta , Agrecanas , Animais , Sequência de Bases , Proteína Morfogenética Óssea 2 , Bovinos , Células Cultivadas , Condrogênese/fisiologia , Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/genética , Primers do DNA/genética , Consolidação da Fratura/fisiologia , Expressão Gênica/efeitos dos fármacos , Lectinas Tipo C , Microcirculação/citologia , Fenótipo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VersicanasRESUMO
STUDY DESIGN: A randomized double-blind prospective comparison with a placebo control. This report of the results is the first in an ongoing study. OBJECTIVES: To evaluate the effect of noninvasive capacitively coupled electrical stimulation on the success rate of lumbar spine fusion surgery, and to compare active with placebo stimulators as adjuncts to contemporary fusion techniques. SUMMARY OF BACKGROUND DATA: Previous studies have established the effectiveness of direct current and electromagnetic field stimulation as adjuncts for some forms of spinal fusion. None of the previous placebo-controlled studies on external bone stimulation included posterolateral fusion techniques, and most were conducted with prior generations of internal fixation hardware. METHODS: The investigation was conducted by 28 U.S. surgeons. Patients with a primary diagnosis of degenerative disc disease with or without other degenerative changes were selected. The study protocol defined success as a clinical outcome rated as excellent or good and a fusion documented as solid by both the investigator and the blinded independent radiologist. Disagreements on radiographic success were resolved by a second blinded independent reviewer. RESULTS: For the 179 patients who completed treatment and evaluation, the overall protocol success rate (both clinical and radiographic results rated as successes) was 84.7% for the active patients and 64.9% for the placebo patients. This difference is highly significant according to the Yates corrected chi-square test (P = 0.0043). Best improvements in patient outcomes (20% or greater success rate) occurred when active stimulation was used in conjunction with posterolateral fusion (P = 0.006) and when internal fixation also was incorporated (P = 0.013). DISCUSSION: This study was consistent in that active stimulation improved results for each stratification, although some strata had insufficient numbers of patients for the results to have statistical significance. Improved success rates when capacitively coupled stimulation is added to internal fixation are hypothesized to result from overcoming the biochemical effects of stress shielding. CONCLUSIONS: Capacitively coupled stimulation is an effective adjunct to primary spine fusion, especially for patients with posterolateral fusion and those with internal fixation.
Assuntos
Terapia por Estimulação Elétrica , Disco Intervertebral , Vértebras Lombares/cirurgia , Doenças da Coluna Vertebral/terapia , Fusão Vertebral , Adulto , Parafusos Ósseos , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/cirurgia , Vértebras Lombares/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Estudos Prospectivos , Curva ROC , Radiografia , Fusão Vertebral/métodos , Resultado do TratamentoRESUMO
Bovine brain microvessel pericytes, bone cells, and fibroblasts were grown in tissue culture in 3%, 21%, or 60% oxygen for 7 weeks. Alkaline phosphatase activity was highest in bone cells and pericytes grown in 3% oxygen, with the activity higher in the former than the latter. Alkaline phosphatase activity was very low in fibroblasts at every oxygen concentration. Osteocalcin concentration was higher in bone cells than in pericytes, was not detected in fibroblasts, and in bone cells and pericytes the concentration was highest in 21% oxygen. Other bovine brain microvessel pericytes were grown in 3% or 21% oxygen for 3 to 24 days in the presence or absence of bone morphogenetic protein 2 and in the presence or absence of parathyroid hormone. At Day 3 of culture, alkaline phosphatase activity was highest in 21% oxygen in the presence of bone morphogenetic protein 2. By Day 17 of culture, alkaline phosphatase activity was highest in 3% oxygen whether bone morphogenetic protein was present or not. Cyclic adenosine monophosphate production in pericytes in response to parathyroid hormone stimulation was very modest when compared with that of bone cells, and this response was not found to be significantly altered by bone morphogenetic protein 2, duration of culture, or the oxygen concentration during incubation. These findings show that the microvessel pericyte is capable of exhibiting several oxygen dependent, phenotypic characteristics ascribed to osteoblasts.
Assuntos
Microcirculação/metabolismo , Osteócitos/metabolismo , Fator de Crescimento Transformador beta , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Microcirculação/citologia , Microcirculação/efeitos dos fármacos , Osteocalcina/metabolismo , Osteócitos/efeitos dos fármacos , Osteócitos/enzimologia , Oxigênio/fisiologia , Hormônio Paratireóideo/farmacologia , Radioimunoensaio , Fatores de TempoRESUMO
Rat calvarial bone cells or mouse MC3T3-E1 bone cells subjected to a capacitively coupled electric field of 20 mV/cm consistently showed significant increases in cellular proliferation as determined by deoxyribonucleic acid content. Verapamil, a membrane calcium channel blocker; W-7, a calmodulin antagonist; indocin, a prostaglandin synthesis inhibitor; or bromophenacyl bromide, a phospholipase A2 inhibitor, each at a concentration that did not interfere with cell proliferation in control cultures, inhibited proliferation in those cultures subjected to the electric field. In contrast, neomycin, an inhibitor of the inositol phosphate cascade, did not inhibit this electrically induced cellular proliferation. Prostaglandin E2 production also was increased significantly with electrical stimulation, and this increase was inhibited by verapamil or indocin but not by neomycin. Thus, the data suggest that the signal transduction mediating the proliferative response of cultured bone cells to a capacitively coupled field involved transmembrane calcium translocation via voltage gated calcium channels, activation of phospholipase A2, and a subsequent increase in prostaglandin E2. Increases in cytosolic calcium and activated calmodulin are implied. The inositol phosphate pathway, unlike its dominant role in signal transduction in mechanically stimulated bone cells, does not appear to play a role in signal transduction in the proliferative response of bone cells to electrical stimulation.
Assuntos
Osso e Ossos/citologia , Transdução de Sinais/fisiologia , Animais , Divisão Celular , Linhagem Celular , Células Cultivadas , Condutividade Elétrica , Fosfatos de Inositol , Camundongos , Ratos , Estresse MecânicoRESUMO
Delayed union represents an ongoing failure of initial fracture management. It still occurs partly because the precise reason why a patient's fracture does not heal frequently is unknown. This article aims to outline the limited material available on the pathophysiology of delayed healing. The systemic status of the patient, local limb status before injury, the nature of the traumatic injury, local host response to the injury, potential negative impact of orthopaedic fracture care, and pharmacologic variables are considered.
Assuntos
Consolidação da Fratura/fisiologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Osso e Ossos/cirurgia , Colágeno/fisiologia , Feminino , Consolidação da Fratura/efeitos dos fármacos , Fraturas Ósseas/classificação , Fraturas Ósseas/imunologia , Fraturas Ósseas/fisiopatologia , Fraturas Ósseas/cirurgia , Nível de Saúde , Humanos , MasculinoRESUMO
Mechanical stimulation can prompt healing of bone fractures. However, it is largely unknown how osteogenesis is promoted by mechanical stimulation. In this study, we found that mechanical strain-induced proliferation of osteoblastic cells (MC3T3-E1) accompanied increased levels of platelet-derived growth factor-A (PDGF-A) mRNA, determined by quantitative reverse transcription/polymerase chain reaction. In addition, neomycin and W-7, which blocked mechanical strain-induced proliferation of the osteoblast cells, also blocked mechanical stimulation-induced elevation of PDGF-A mRNA. Finally, an antibody against PDGF can inhibit physical stimulation-induced proliferation of MC3T3-E1 cells, suggesting that the increased MC3T3-E1 cells produced by mechanical stimulation at least partially depends on the increased activity of PDGF.
Assuntos
Divisão Celular , Osteoblastos/citologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Estresse Mecânico , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular , Fosfatos de Inositol/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/metabolismoRESUMO
It is well-known that electrical stimulation can prompt healing of bone fractures. However, the mechanism involved is less clear. In this study, we found that capacitively coupled electric field-induced proliferation of osteoblastic cells (MC3T3-E1) accompanied increased levels of transforming growth factor-beta 1 (TGF-beta1) mRNA determined by quantitative reverse transcription/polymerase chain reaction. Previous reports have shown that verapamil and W-7, both of which block voltage gated calcium channels and inhibit the activation of cytosolic calmodulin, respectively, blocked capacitively coupled electric field-induced proliferation of the osteoblast cells. Interestingly, we found that verapamil and W-7 can also block capacitively coupled electric field-induced elevation of TGF-beta1 mRNA. This result suggested that electrical stimulation induces the level of TGF-beta1 mRNA in osteoblastic cells by a mechanism involving calcium/calmodulin pathway. The potential roles of TGF-beta in the electrical signal-induced osteogenesis was discussed.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Osteoblastos/metabolismo , RNA Mensageiro/genética , Fator de Crescimento Transformador beta/genética , Células 3T3 , Animais , Divisão Celular , Estimulação Elétrica , Camundongos , Osteoblastos/citologia , Osteoblastos/fisiologia , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: To document early histological and ultrastructural changes in periosteal fracture callus blood vessels. DESIGN: Rabbit control and fractured ribs, after healing for three, six, and twelve hours and daily for seven days, were evaluated by light and electron microscopy. RESULTS: Control periosteal microvessels were formed mainly by endothelial cells and occasionally by pericytes. Only these cells displayed basal lamina within the periosteum. Three to twelve hours postfracture, periosteal microvessels were little changed. By two days postfracture, dramatic increases in size and population of microvessel cells resulted in a smaller lumen and thicker wall. Microvessel cells, while retaining their basal lamina, had transformed to mesenchymal cells. Transformed pericytes, as evidenced by their basal lamina, had extravasated. Three to four days postfracture, additional transformed pericytes had extravasated. Within the distal periosteal callus, a close spatial relationship among transformed microvessels, extravascular mesenchymal cells (some with basal lamina), and osteoblasts was present. Four to five days postfracture, within the proximal periosteal callus, a close spatial relationship among transformed microvessels (rapidly disappearing because of continued extravasation), extravascular mesenchymal cells (some with basal lamina), and chondroblasts (some with basal lamina) was present. CONCLUSIONS: New evidence showed that after fracture, periosteal microvessel endothelial cells and pericytes increased in population and transformed to mesenchymal cells. These changes, their subsequent extravasation as mesenchymal cells, and their development into chondroblasts were verified by basal lamina evidence. New evidence also suggested that continued extravasation of transformed microvessel cells rendered the fracture callus cartilage avascular.
Assuntos
Calo Ósseo/irrigação sanguínea , Consolidação da Fratura , Periósteo/irrigação sanguínea , Animais , Calo Ósseo/ultraestrutura , Cartilagem/irrigação sanguínea , Cartilagem/citologia , Endotélio Vascular/anatomia & histologia , Endotélio Vascular/ultraestrutura , Consolidação da Fratura/fisiologia , Masculino , Microcirculação/citologia , Microcirculação/ultraestrutura , Microscopia Eletrônica , Periósteo/ultraestrutura , Coelhos , Fatores de TempoRESUMO
It is well known that mechanical stimulation can prompt healing of bone fractures. However, the mechanism involved is less clear. In this study, we found that a 0.17% cyclic, biaxial mechanical strain delivered at 1 Hz increased proliferation of MC3T3-E1 cells, a clonal osteoblastic cell line. Mechanical strain also increased the level of TGF-beta 1 mRNA determined by quantitative reverse transcription/ polymerase chain reaction. Previous reports have shown that neomycin and W-7, which are inhibitors in the inositol phosphate/calmodulin pathway, blocked mechanical strain-induced proliferation of the osteoblast cells. Interestingly, we found that neomycin and W-7 can also block mechanical stimulation-induced elevation of TGF-beta 1 mRNA. Finally, using an antibody which blocked the action of TGF-beta 1, we found that the increased MC3T3-E1 cell proliferation induced by mechanical strain did not depend on the action of TGF-beta 1.
Assuntos
Divisão Celular/genética , Osteoblastos/citologia , RNA Mensageiro/genética , Fator de Crescimento Transformador beta/genética , Células 3T3 , Animais , Calmodulina/metabolismo , Fosfatos de Inositol/metabolismo , Camundongos , RNA Mensageiro/metabolismoRESUMO
Cultured cells subjected to fluid flow are exposed to mechanical forces and electrokinetic forces. The convective current establishes an electrokinetic force created by the flow-dependent transport of mobile ions in the media over the charged cell surfaces. This current can be expressed as a current density, the current normalized by the cross-sectional area in which it exists. In this study, we hypothesized that the convective current density has no role in the bone cell real-time intracellular calcium response to fluid flow. Our hypothesis was tested by incorporating electrokinetic measurements and classical electrokinetic double-layer theory to estimate the value of convective current density in a parallel-plate flow chamber and then to apply an external current during the presence of fluid flow that would alter convective current density. There was no difference between the mean peak calcium response of cells exposed to flow with an altered (canceled or doubled) convective current density versus flow with an unmodified convective current density, as was measured with fura-2 fluorescence microscopy. These results suggest that mechanical forces, such as fluid-induced shear stress, rather than concomitant electrokinetic forces are the primary stimuli in eliciting the observed calcium response of bone cells to fluid flow.
Assuntos
Osso e Ossos/metabolismo , Cálcio/metabolismo , Análise de Variância , Animais , Osso e Ossos/citologia , Células Cultivadas , Condutividade Elétrica , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Reologia , Estresse MecânicoRESUMO
In this study, we sought to determine if there is a requirement for calcium entry from the extracellular space as well as calcium from intracellular stores to produce real-time intracellular calcium responses in cultured bone cells subjected to fluid flow. Understanding calcium cell signaling may help to elucidate the biophysical transduction mechanism(s) mediating the conversion of fluid flow to a cellular signal. An experimental design which utilized a scheme of pharmacological blockers was employed to distinguish between the biochemical pathways involved in this cell signaling. A parallel-plate flow chamber served as the cell stimulating apparatus and a fluorescence microscopy system using the calcium-sensitive dye fura-2 measured the intracellular calcium changes. In the present study, evidence for a role by the inositol-phospholipid biochemical pathway, specifically inositol trisphosphate (IP3) was obtained using neomycin which completely inhibited the calcium response to flow. Additionally, a concomitant role of extracellular calcium was demonstrated through experiments performed in calcium-free medium which also eliminated the flow response. Experiments conducted with gadolinium, a stretch-activated channel blocker, partially inhibited (approximately 30%) the flow response while verapamil, a type-L voltage sensitive channel blocker, had no effect on the flow response. These results suggest a requirement of extracellular calcium (or calcium influx) as well as IP3-induced calcium release from intracellular stores for generating the intracellular calcium response to flow in bone cells.
Assuntos
Osso e Ossos/fisiologia , Cálcio/metabolismo , Análise de Variância , Animais , Osso e Ossos/efeitos dos fármacos , Bradicinina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Gadolínio/farmacologia , Neomicina/farmacologia , Ratos , Ratos Sprague-Dawley , Reologia , Estresse Mecânico , Verapamil/farmacologiaRESUMO
Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation. When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E2 had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [3H]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E2 synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [3H]-thymidine. Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain. The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca2+, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium. With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca2+ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126 +/- 15.2 compared with 16 +/- 8.0 nanomolar, mean and standard deviation). The addition to the medium of neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration completely blocked the increase in the concentration of cytosolic Ca2+ in these cells; this concentration of neomycin had been shown to have no effect on proliferation in control bone cells. There was also a dose-dependent relationship between the duration of the stimulus and the cellular proliferation. Remarkably, one cycle of pressure at ten pounds per square inch (sixty-nine kilopascals) and a frequency of approximately one hertz produced a 57 per cent increase in the incorporation of [3H]-thymidine at twenty-four hours (p < 0.001). From these findings, we hypothesized that the inositol phosphate cascade-cytosolic Ca(2+)-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells, at least under the conditions reported here.
Assuntos
Osso e Ossos/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Cálcio/análise , Cálcio/metabolismo , Calmodulina/análise , Calmodulina/antagonistas & inibidores , Calmodulina/biossíntese , Divisão Celular/efeitos dos fármacos , Técnicas de Cultura , Inibidores de Ciclo-Oxigenase/administração & dosagem , Inibidores de Ciclo-Oxigenase/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , DNA/análise , DNA/biossíntese , Dinoprostona/análise , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Corantes Fluorescentes , Fura-2/análogos & derivados , Pressão Hidrostática , Indometacina/administração & dosagem , Indometacina/farmacologia , Fosfatos de Inositol/antagonistas & inibidores , Neomicina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Crânio , Estresse Mecânico , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Timidina/metabolismo , Fatores de Tempo , TrítioRESUMO
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.
Assuntos
Transplante Ósseo , Terapia por Estimulação Elétrica/métodos , Fraturas não Consolidadas/terapia , Fraturas da Tíbia/terapia , Adulto , Feminino , Consolidação da Fratura , Humanos , Modelos Logísticos , Masculino , Fatores de Risco , Fatores de Tempo , Transplante Autólogo , Falha de TratamentoRESUMO
The biosynthetic and migratory response of bone cells to changes in both surface composition and morphology of polystyrene (PS) substrates was examined. A system was devised wherein micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either 0.5- or 5.0- microns-deep surface grooves. Smooth replicas (0% S) served as the control surfaces. The chemical and morphologic characteristics of the nine unique model biomaterial surfaces (MBSs) produced using this system were documented and were found to be distinct. For the biosynthetic studies, bone cells isolated from neonatal rat calvaria were plated onto the MBSs and labeled at postconfluence with [14C]proline for 24 h. Total DNA per surface, total newly synthesized collagenous (CP), and noncollagenous protein (NCP) (cell associated and secreted) were determined. Cell-associated CP was found to increase significantly for the bone cells cultured on the substrates with 0.5-micron grooves and 2% S (P < .05). Cell-associated NCP was found to be elevated for all 2% S substrates and for the 0.5-micron grooves substrates with 1% S. For the migration studies, bone cells were plated first onto 5-mm nitrocellulose disks that were attached to standard Petri dishes using a plasma clot. At confluence, the disks were removed aseptically and placed on the replicas. The cellular area occupied as a result of the outward migration of the bone cells was measured after 4 days of culture using an image analysis system. An average velocity for the leading edge of bone cell populations on each of the nine MBSs was calculated: Cells on surfaces with either 1% S or 5.0-microns grooves displayed significantly higher velocities than did the control cultures. A significant interaction effect between chemistry and morphology was observed. The biosynthetic and migratory responses of in vitro cultures of bone cells were not predictable from the observations of the cellular responses to the individual features, but appeared to depend on cellular responses to more than one substrate factor.
Assuntos
Materiais Biocompatíveis , Colágeno/biossíntese , DNA/metabolismo , Biossíntese de Proteínas , Crânio/citologia , Crânio/fisiologia , Animais , Animais Recém-Nascidos , Autorradiografia , Radioisótopos de Carbono , Movimento Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/fisiologia , Matriz Extracelular/ultraestrutura , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteoclastos/ultraestrutura , Prolina/metabolismo , Ratos , Ratos Sprague-Dawley , Crânio/metabolismo , Propriedades de SuperfícieRESUMO
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.
Assuntos
Endotélio Vascular/fisiologia , Osteoblastos/fisiologia , Crânio/citologia , Animais , Comunicação Celular , Divisão Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Técnicas Citológicas , Endotélio Vascular/citologia , Marcadores Genéticos , Microcirculação , Fenótipo , Ratos , Ratos Sprague-DawleyRESUMO
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.