In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical fields

Isolated articular cartilage chondrocytes from 1- to 3-week-old male Holstein calf knee joints were formed into pellets containing 4 X 10(6) isolated cells and were grown in tissue culture medium (minimum essential medium/NCTC 135) containing either 1 or 10% newborn calf serum (NBCS) in plastic Petri dishes in 5% CO2 and air at 37 degrees C in saturation humidity. On the 4th postisolation day either [35S]sulfate or [3H]thymidine was added to the medium, and the pellets were exposed for 24 h to capacitively coupled electrical fields (10, 100, 250, and 1,000 V peak-to-peak, 60 kHz, sine wave signals). The pellets were then harvested, dialyzed, hydrolyzed, and assayed for DNA, protein, [35S]sulfate incorporation, and [3H]thymidine incorporation. Results indicated that at 250 V peak-to-peak there was a statistically significant increase in [35S]sulfate in 1% NBCS and a statistically significant increase in [3H]thymidine in 10% NBCS. At potentials above or below 250 V no changes were noted. Thus, articular cartilage chondrocytes grown in pellet form can be stimulated to increase glycosaminoglycan synthesis or to increase cell proliferation by an appropriate capacitively coupled electrical field. The importance of the serum concentration in the medium in evaluation of biosynthesis in vitro is noted.     

Increased cAMP production after short-term capacitively coupled stimulation in bovine growth plate chondrocytes

Growth plate chondrocytes from newborn calf costochondral junctions grown in monolayer were subjected to a capacitive AC signal of 500 V peak to peak (P-P) at 60 kHz for 48 h and were analyzed for [3H]thymidine uptake. The stimulated chondrocytes showed a 130% greater uptake over unstimulated controls. Other newborn calf growth plate chondrocytes were stimulated at 500 V P-P at 60 kHz for 2.5, 5.0, 10.0, and 20.0 min and were analyzed for cAMP. Chondrocytes stimulated for 2.5 and 5.0 min showed a 142.8% (p less than 0.05) and 394.5% (p less than 0.01) increase over controls, respectively. The chondrocytes stimulated for 10.0 and 20.0 min showed no significant difference from the controls. It is concluded that short-term exposure of growth plate chondrocytes to an appropriate capacitively coupled field stimulates cAMP production, but longer-term application of the electrical field is ineffective.     

Epiphyseal cartilage cAMP changes produced by electrical and mechanical perturbations.

Epiphyseal cartilage from chick embryo was subjected in vitro for one to 15 minutes to static compressive forces or oscillating electric fields. The same stimuli were applied to suspensions of cells isolated from the epiphyses. At the end of the perturbation cAMP was measured in the tissue or cell extracts by radioimmunoassay. It was found that a physiological (60 g/cm2) static pressure reduced the cAMP content in the tissue and the separated cells. An oscillating electric field above 900 V/1.5 cm, 5Hz, enhanced the cAMP accumulation in the intact tissue. This effect was produced only when the long axis of the bones was oriented parallel to the electric field. In isolated cells an electric field above 750 V/1.5 cm, 5Hz, caused a decrease in cAMP content. Charged matrix macromolecules and orientation of the cells within the cell matrix may have a modulating effect on the initiation of this response. It is postulated that the change in cAMP is the early cellular signal in the response to an electrical or mechanical perturbation which leads to bone remodeling.     

Proliferative and synthetic response of bovine growth plate chondrocytes to various capacitively coupled electrical fields

In vitro monolayer cultures of growth plate chondrocytes isolated from newborn calf costochondral junctions were subjected to capacitively coupled electrical fields for 48 h. In part A, the electrical signal was a 60-kHz sine wave applied at different voltages so as to produce electrical fields at the pericellular level of 7, 20, 50, and 126 mV/cm. Incorporations of [3H]thymidine and [35S]sulfate were assayed to determine the effect of the above fields on cells proliferation and matrix synthesis, respectively. Proliferation was increased by 47% in the 20 mV/cm field whereas the same field decreased [35S]sulfate incorporation by 21%. These changes were significant at p less than 0.05 in both instances. In part B, the 20 mV/cm field was applied in a pulsed fashion to produce daily duty cycles of 100, 25, 2, and 0.25%. Incorporation of [3H]thymidine, [35S]sulfate, and [14C]proline per DNA were assayed. Results indicated that the 100, 25, and 0.25% percent duty cycles showed significantly (p less than 0.01-0.05) increased proliferation, whereas the 0.25% signal (5 ms on/495 ms off for 6 h/day) significantly decreased [14C]proline incorporation. We conclude that the biologic response of cells in vitro is signal specific, and that the total amount of electrical energy required to stimulate the growth plate chondrocyte to increased proliferation is very small since the total time the 0.25% duty cycle signal was only 3.6 min of a 24-h period.     

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.     

Effect of direct current on cultured growth cartilage cells in vitro

Cultured growth cartilage cells of young rabbits were stimulated by three levels (10, 1, and 0.1 microA) of constant direct current (DC). The effect of DC was examined by measuring the activities of proteoglycan synthesis (uptake of [35S]sodium sulfate and toluidine blue staining) and DNA synthesis (uptake of [3H]thymidine). DC increased syntheses of both proteoglycans and DNA when 1 microA (calculated current density of 1 microA/cm2) was applied for 3 days. A longer stimulation resulted in a larger difference between stimulated and unstimulated cells. Ten-microampere stimulation devitalized the cells, while 0.1 microA had no effect on cell activities.     

Glucosamine oral bioavailability and plasma pharmacokinetics after increasing doses of crystalline glucosamine sulfate in man

OBJECTIVE: Pharmacokinetic data on glucosamine are scant, limiting the understanding of glucosamine sulfate mechanism of action in support of its treatment effects in osteoarthritis. This study investigated the oral pharmacokinetics and dose-proportionality of glucosamine after administration of the patented crystalline glucosamine sulfate in man. METHODS: Twelve healthy volunteers received three consecutive once-daily oral administrations of glucosamine sulfate soluble powder at the doses of 750, 1,500, and 3,000 mg, in an open, randomised, cross-over fashion. Glucosamine was determined in plasma collected up to 48 h after the last dose by a validated Liquid Chromatography method with Mass Spectrometry detection. Pharmacokinetic parameters were calculated at steady state. RESULTS: Endogenous plasma levels of glucosamine were detected (10.4-204 ng/ml, with low intra-subject variability). Glucosamine was rapidly absorbed after oral administration and its pharmacokinetics were linear in the dose range 750-1,500 mg, but not at 3,000 mg, where the plasma concentration-time profiles were less than expected based on dose-proportionality. Plasma levels increased over 30-folds from baseline and peaked at about 10 microM with the standard 1,500 mg once-daily dosage. Glucosamine distributed to extravascular compartments and its plasma concentrations were still above baseline up to the last collection time. Glucosamine elimination half-life was only tentatively estimated to average 15 h. CONCLUSIONS: Glucosamine is bioavailable after oral administration of crystalline glucosamine sulfate, persists in circulation, and its pharmacokinetics support once-daily dosage. Steady state peak concentrations at the therapeutic dose of 1,500 mg were in line with those found to be effective in selected in vitro mechanistic studies. This is the only glucosamine formulation for which pharmacokinetic, efficacy and safety data are now available.     

Interactions of low density lipoprotein with rat mesangial cells

Hyperlipidemia may contribute to the pathogenesis of glomerular sclerosis. We therefore studied binding and uptake of low density lipoprotein (LDL) by cultured rat mesangial cells. In addition effects of LDL on PGE2 synthesis and cell proliferation were determined. At 4 degrees C mesangial cells bound [125I] LDL in a time- and concentration-dependent manner with half-maximal binding observed at 5 micrograms/ml of LDL protein. Binding was blocked by excess unlabeled LDL and by heparin. Uptake (binding plus internalization) of LDL at 37 degrees C markedly exceeded binding at 4 degrees C, continued to increase even with longer periods of incubation, and showed no saturability, consistent with uptake of LDL by mesangial cells. Further evidence for LDL uptake by mesangial cells was obtained by use of the fluorescent probe 1,1'-dioactadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labeled LDL (Dil-LDL). Incubation of mesangial cells with Dil-LDL at 37 degrees C showed positive fluorescence for all mesangial cells, indicating uptake of the Dil-LDL. LDL had a biphasic effect on mesangial cell proliferation as determined by [3H] thymidine incorporation. LDL at 10 micrograms/ml enhanced [3H] thymidine uptake modestly, but significantly, whereas a progressive and marked inhibition occurred at LDL concentration from 100 to 500 micrograms/ml. While LDL at 10 and 100 micrograms/ml significantly stimulated PGE2 production, inhibition of PGE2 by meclofenamate did not influence the effects of LDL on [3H] thymidine incorporation. We conclude that mesangial cells show specific binding and uptake of LDL and that high concentrations of LDL markedly decrease mesangial cell proliferation. These findings may pertain to the pathogenesis of glomerular lesions in hyperlipidemia of renal disease.     

In vivo growth plate stimulation in various capacitively coupled electrical fields

The right proximal tibial growth plates of adolescent New Zealand white rabbits were stimulated with various capacitively coupled electrical fields. Capacitor plates attached to plastic jigs placed over the proximal tibiae were connected to function generators which supplied sine wave signals of 60 kHz frequency and various voltages (2.5, 5, 10, and 20 V peak-to-peak). At 0 h and at 48 h, each animal was labeled with intravenously injected oxytetracycline. For the next 48 h, each right proximal tibial growth plate was stimulated with one of the above electrical signals. At the end of the 48 h of stimulation, the animals were sacrificed, and the tibiae were excised; histological sections of the proximal growth plate in each tibia were made, and the distance the labels moved away from the bone-cartilage junction down into the metaphysis was measured under fluorescent microscopy. Results indicate that the rabbit growth plate can be consistently stimulated to statistically significant accelerated growth in a capacitively coupled electrical field. A dose-response effect was noted, with 5 V peak-to-peak exhibiting maximum growth acceleration. Thus, the application of the proper capacitively coupled electrical field significantly stimulated the rabbit growth plate at voltage and current levels that are safe for human use.     

Evidence for mitogen-associated protein kinase activation and transduction of mitogenic signals by platelet-derived growth factor in human meningioma cells

OBJECT: Coexpression of platelet-derived growth factor (PDGF)-BB and activated PDGF-beta receptor in meningioma cells indicates that this cytokine may act as an autocrine or paracrine stimulant of meningioma growth. The intracellular events transducing signals from PDGF-beta receptor tyrosine kinases are unknown. In this study the authors evaluated whether or not mitogen-activated protein kinases (MAPKs) are expressed in meningiomas, regulate their growth, and transduce mitogenic signals of PDGF-BB. METHODS: Ten human meningioma tumors as well as cells cultured from two normal leptomeninges and 10 additional human meningiomas were evaluated using Western blot analysis to determine the presence of MAPK and phosphorylated (activated) MAPK. The effects of PD098059, a selective inhibitor of MAPK phosphorylation/activation, on proliferation of meningioma cells stimulated with 10% fetal bovine serum was also evaluated. Last, the authors evaluated whether PDGF-BB stimulation of meningioma cells was associated with activation of MAPK. Western blots of lysates from meningiomas and from cultured leptomeningeal and meningioma cells demonstrated MAPK and phosphorylated MAPK. Treatment with PD098059 produced a 52 to 84% (x = 69.8) loss in [3H]thymidine incorporation, which was associated with a partial or complete loss of phosphorylated MAPK after 3 days of treatment. The PDGF-BB produced a significant increase in [3H]thymidine incorporation and phosphorylation of MAPK at 1 and 3 days. Coadministration of PD098059 completely blocked PDGF-BB's stimulation of [3H]thymidine incorporation and cell proliferation concomitant with reduced MAPK phosphorylation. CONCLUSIONS: The findings indicate that MAPK is constitutively expressed in leptomeningeal and meningioma cells and transduces mitogenic signals of PDGF, contributing to the growth of human meningiomas.     

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.     

Amelioration of oxygen-induced osteoporosis in the in vitro fetal rat tibia with a capacitively coupled electrical field

Near-term fetal rat tibiae were grown in M.E.M. Eagle/NCTC 135/15% newborn calf serum in 5% carbon dioxide and 5, 10, 21, 35, 60, and 90% oxygen for 3, 7, 10, and 14 days. Linear growth of the explants, as measured from macrophotographs of the explants at day zero and each of the days above, was greatest in the lower oxygen concentrations and least in the higher oxygen concentrations. Breaking strengths of the tibial diaphyses were significantly reduced in those explants grown in 60 and 90% oxygen. When the fetal rat tibiae were grown in 60% oxygen for 7 days and were subjected to a capacitively coupled electrical signal (sine wave, 60 kHz, 10 V peak-to-peak output signal; current density and field in the culture dish calculated to be 5.2 microA/cm2 and 0.32 mV/cm, respectively), the breaking strengths and middiaphyseal widths were statistically significantly greater than control tibiae grown in 60% oxygen alone. It is concluded that an appropriate capacitively coupled electrical field can inhibit an oxygen-induced osteoporosis in an in vitro mammalian long bone model.     

Effect of electric currents on DNA synthesis in rat osteosarcoma cells: dependence on conditions that influence cell growth

Effects of sinusoidal 60-Hz electric currents on DNA synthesis in rat osteosarcoma cell (ROS 17/2.8) in culture were investigated using agar electrodes. This exposure system eliminates electrochemical effects at electrode surfaces, minimizes the contribution of magnetic fields and produces no detectable temperature changes (less than +/- 0.1 degrees C) at current densities of up to 1,500 microA r.m.s. (root mean square)/cm2. Thirty-four hours exposure to electric current at intensities of 300-400 microA r.m.s./cm2 enhanced DNA synthesis by 20% relative to controls. The effect depended on serum concentration, cell seeding density, and the age of the cell population at the time of seeding: effects were seen at 0.2% fetal bovine serum, in cells seeded 4 days after previous passage, at 20,000 cells/cm2. These findings indicate that sinusoidal electric currents can midly enhance DNA synthesis in these cells under growth-limiting conditions.     

The treatment of heparin resistance with Antithrombin III in cardiac surgery

PURPOSE: To report three patients who developed heparin resistance during cardiac surgery which was successfully managed with 1000 U Antithrombin III (AT III). CLINICAL FEATURES: We observed heparin resistance prior to cardiopulmonary bypass (CPB) in one patient and during the CPB in two patients. In the first patient who was scheduled for mitral valve replacement, although heparin was administered sequentially up to 500 U x kg(-1) prior the CPB, the ACT value was 354 sec. After 1,000 U ATIII were administered the ACT was 395 sec and CPB was initiated. The ACT remained between 496 and 599 sec throughout CPB and a total of 260 mg protamine sulfate was given. In the other two patients following 300 U x kg(-1) heparin, the ACT was up to 400 sec and CPB was initiated. During CPB, ACT were decreased 360 sec and 295 sec in patients II and III respectively. Although heparin was added 1,500 U, ACT increased to > or = 400 sec could not be achieved. In the second patient ATIII activity was found 10%. After the administration of 1,000 U ATIII, ATIII activity was found to be 67% 40 min later and ACT were increased up to 400 sec. There was no thrombosis within the extracorporeal circuit, additional heparin was not required, less protamine was administered (< or = 3 mg x kg(-1)) and no excessive postoperative bleeding was observed in all patients. CONCLUSION: We recommend that AT III supplementation should be considered to manage heparin resistance prior or during CPB in patients undergoing open heart surgery.     

Pharmacokinetics of Human Cerebral Opioid Extraction: A Comparative Study on Sufentanil, Fentanyl, and Alfentanil in a Patient after Severe Head Injury

Background: The pharmacodynamic differences in time to onset and dissipation of effect of sufentanil, fentanyl, and alfentanil probably result from different rates of blood-brain equilibration. The authors investigated this hypothesis in humans.

Methods: After simultaneous central venous bolus application of sufentanil (10 [mu]g), fentanyl (100 [mu]g), and alfentanil (1,000 [mu]g), arterial and jugular bulb blood samples were drawn simultaneously at 20, 30, 45, 60, 75, 90, 105, 120, 140, 160, 180, 210, 240, 300, 360, and 420 s from 19 patients during the postacute stage of head injury with normal intracranial pressure, cerebral perfusion pressure, and cerebral oxygen metabolism during normocapnia.

Results: Peak brain concentration, indicated by equilibrium between arterial and jugular bulb opioid concentrations, was achieved for alfentanil at 45 s, for sufentanil at 5 min, and for fentanyl at 6 min. The corresponding median time intervals (fifth and ninety-fifth percentiles) to reach 50% of peak brain concentration were 15 (14-18), 25 (18-38) and 35 (25-45) s, respectively. Uptake was highest 20 s after bolus and decreased continuously for fentanyl and sufentanil, whereas alfentanil uptake was biphasic. The ratio of the relative amounts of sufentanil, fentanyl, and alfentanil retained in the brain at peak brain concentration was 1x:x6x:x90.     

Electrostimulation of rat callus cells and human lymphocytes in vitro

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The [3H]thymidine incorporation of the callus cells and 5-[125I]iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of [3H]thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The [3H]thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took up more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.     

How light dosimetry influences the efficacy of photodynamic therapy with 5-aminolaevulinic acid for ablation of high-grade dysplasia in Barrett’s esophagus

Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is a novel treatment for high-grade dysplasia (HGD) in Barrett’s esophagus (BE). Our aim was to evaluate the effectiveness of differing light doses. Patients with HGD in BE received oral ALA (60 mg/kg) activated by low (500 J/cm), medium (750 J/cm), high (1,000 J/cm), or highest (1,000 J/cm ×2) light dose at 635 nm. Follow-up was by regular endoscopy with quadrantic biopsies. Twenty-four patients were treated. Successful eradication of HGD was significantly correlated with light dose (log rank, p < 0.01). Six of eight patients (75%) treated with the highest light dose, one of two treated with high dose (50%), two of nine (22%) receiving medium light dose, and zero of five receiving low light dose had successful eradication of HGD (median follow-up 45 months, range 1–78 months). No skin photosensitivity or esophageal strictures occurred. The efficacy of ALA-PDT for eradication of HGD in BE is closely related to the light dose used. With a drug dose of 60 mg/kg and light at 635 nm, we recommend a minimum light dose of 1,000 J/cm of esophagus. This dose appears safe.     

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.     

Differential Uptake of Volatile Agents into Brain Tissue In Vitro: Measurement and Application of a Diffusion Model to Determine Concentration Profiles in Brain Slices

Background: The rate of onset of drug actions in experiments with brain slices in vitro can vary widely. One factor that influences the rate is access to tissue sites of action. To study the effects of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, also termed 2N in the literature) on physiologic processes under defined tissue concentrations, the authors performed electrophysiologic measurements of the effects of F6 and halothane, measured the uptake of these agents into brain tissue, and performed computational modeling to determine concentration-depth profiles during drug application.

Methods: Hippocampal brain slices 500 [mu]m thick were prepared from adult rats. Evoked population responses in the CA1 region were obtained using extracellular recordings and electrical stimulation of the Schaffer collateral pathway. F6 (24 [mu]m) and halothane (270 [mu]m) were applied via superfusion for 40 min. Uptake of drug into tissue slices was measured using gas chromatography. Computational modeling was used to obtain estimates of drug diffusion coefficients in brain tissue and to calculate tissue concentration as a function of time and depth during drug application.

Results: Halothane reduced the amplitude of the evoked population spike and reduced the population excitatory postsynaptic potential slope. F6 had no effect on either measure. Uptake experiments yielded a diffusion coefficient of 0.1 x 10-6 cm2/s for F6 and 0.8 x 10-6 cm2/s for halothane. After 40 min of drug application, the concentration reached at tissue depths from which physiologic signals were obtained, approximately the top 200 [mu]m of the slice, was estimated to be 58% of the final equilibrium value for F6 and 93% for halothane.     

Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.

We evaluated the effect of low intensity-pulsed ultrasound stimulation on rat chondrocytes in vitro using two different 1.0-MHz ultrasound signals with spatial and temporal average intensities of 50 or 120 mW/cm2. The pulses had a duration of 200 microseconds and were repeated every millisecond, with corresponding average peak-pressure amplitudes of 230 or 360 kPa, respectively. Cells were stimulated one, three, or five times for 10 minutes each day starting the third day after plating. One group of cells was exposed to sham ultrasound as a control. The cultures were evaluated for cell proliferation (by [3H]thymidine incorporation and DNA measurement), steady-state mRNA levels of alpha1(I) and alpha1(II) procollagens and aggrecan (by Northern blotting), and proteoglycan synthesis (by [35S]sulfate incorporation). The results revealed that ultrasound causes increases in the level of aggrecan mRNA (p < 0.05) and in proteoglycan synthesis (p < 0.03) after three and five treatments. Expression of mRNA for alpha1(II) procollagen increased over time, but ultrasound had no stimulatory effect. Expression of mRNA for alpha1(I) procollagen was initially low and remained unchanged with time. Although cell proliferation increased with time in both groups, there was no statistically significant difference between the cultures treated with ultrasound and the controls (p = 0.1). The in vitro results support our previous in vivo findings that low-intensity ultrasound stimulates aggrecan mRNA expression and proteoglycan synthesis by chondrocytes, which may explain the role of ultrasound in advancing endochondral ossification, increasing the mechanical strength of fractures, and facilitating fracture repair.