Oxygen consumption in T-47D cells immobilized in alginate.

OBJECTIVES: Encapsulation or entrapment of cells is increasingly being used in a wide variety of scientific studies for tissue engineering and development of novel medical devices. The effect on cell metabolism of such systems is, in general, not well characterized. In this work, a simple system for monitoring respiration of cells embedded in 3-D alginate cultures was characterized. MATERIALS AND METHODS: T-47D cells were cultured in alginate gels. Oxygen concentration curves were recorded within cell-gel constructs using two different sensor systems, and cell viability and metabolic state were characterized using confocal microscopy and commercially available stains. RESULTS: At sufficient depth within constructs, recorded oxygen concentration curves were not significantly influenced by influx of oxygen through cell-gel layers and oxygen consumption rate could be calculated simply by dividing oxygen loss in the system per time, by the number of cells. This conclusion was supported by a 3-D numeric simulation. For the T-47D cells, the oxygen consumption rate was found to be 61 ± 6 fmol/cell/h, 3-4 times less than has previously been found for these cells, when grown exponentially in monolayer culture. CONCLUSIONS: The experimental set-up presented here may be varied in multiple ways by changing the cell-gel construct 3-D microenvironment, easily allowing investigation of a variety of factors on cell respiration.

Formulation and bacterial phototoxicity of curcumin loaded alginate foams for wound treatment applications: studies on curcumin and curcuminoides XLII.

Curcumin loaded alginate foams are proposed for application in antimicrobial photodynamic therapy of infected wounds. The drug loaded foams were formulated to provide a burst release of the photosensitizer when hydrated. The foams remained intact after hydration and would be possible to remove from the wound prior to irradiation without causing any tissue damage. The characterization of the prepared foams showed that both curcumin loaded and unloaded foams hydrated within 1 min and absorbed from 12 to 16 times their dry weight of a model physiological fluid. Curcumin, the model photosensitizer, has an extremely low solubility in water and may aggregate in aqueous environment. Cyclodextrins (CDs) and polyethylene glycol 400 (PEG 400) were therefore selected as solubilizers of curcumin in the foams to provide a burst release of the photosensitizer. Exposure to the prepared foams in combination with visible light irradiation (∼9.7 J/cm(2)) resulted in >6 log reduction of Entrococcus faecalis cells. However, curcumin mediated photokilling of Escherichia coli was ineffective when CDs were selected as solubilizer of curcumin in the foams. An 81% reduction in viable E. coli cells was detected after treatment with the foam containing PEG 400 as the only solubilizer of curcumin combined with visible light irradiation (∼29 J/cm(2)).

Evaluation of novel alginate foams as drug delivery systems in antimicrobial photodynamic therapy (aPDT) of infected wounds--an in vitro study: studies on curcumin and curcuminoides XL.

The aim of the present study was to incorporate a model water-insoluble photosensitizer, curcumin, in novel alginate foams, further to evaluate the suitability of the curcumin loaded foams in antimicrobial photodynamic therapy of infected wounds. Six foam formulations were prepared and characterized with respect to physical characteristics, in vitro release and storage- and photo-stability of curcumin. One formulation was sterilized (gamma-sterilization). The foams contained hydroxypropyl-beta-cyclodextrins or hydroxypropyl-gamma-cyclodextrins as solubilizers of curcumin. A reference foam without cyclodextrins was prepared with PEG 400 as the solubilizer. At a curcumin load of 0.153% (w/w), the water insoluble photosensitizer was uniformly distributed in the hydrophilic foams matrix. All foams were easy to handle, flexible and hydrated rapidly in a model physiological fluid. Release of curcumin in its monomeric form was demonstrated in vitro and found to be dependent on the type and amount of cyclodextrins in the formulation. Curcumin was stable during storage, but susceptible to photodegradation in the foams, especially when the formulations contain PEG 400 or hydroxypropyl-gamma-cyclodextrins. Curcumin did not degrade after gamma-sterilization, however a decrease in the in vitro release rate of curcumin and changes in the foams physical characteristics were detected.

Cell therapy using encapsulated cells producing endostatin.

Despite aggressive surgery and post-operative radiation and chemotherapy, the prognosis is poor for glioblastoma patients. Anti-angiogenic therapy with compounds such as endostatin could delay the onset of relapse. However, the short systemic half-life of this proteins as well as the blood-brain barrier makes the use of this therapy difficult for brain cancer patients. The aim of this project is to develop and implant genetically engineered producer cells secreting endostatin that are encapsulated in calcium cross-linked alginate gel beads. Encapsulation of cells within alginate gels has a potential as a sustained release system in addition to the fact that the encapsulation technology protects the cells from rejection by the immune system. Human embryonal kidney 293 cells have been transfected with the gene for endostatin. These cells have been encapsulated in calcium cross-linked alginate gels and optimized for the secretion of endostatin. Alginate gel beads implanted into rat brain have shown only a moderate loss in cell viability but extended endostatin release for periods of up to 12 months. Visualization of the anti-angiogenic effect on C6 rat glioma growth, tumor vasculature and microhemodynamics has been demonstrated by using intravital video microscopy. The data indicates that endostatin greatly affects tumor-associated microcirculation but does not appear to affect normal microcirculation. The local delivery of endostatin seems to specifically affect tumor-associated microvessels by reduction of the vessel density, diameter and functionality. Tumor cell migration and invasion was greatly reduced in the endostatin treated animals.

Controlling the size of alginate gel beads by use of a high electrostatic potential.

The effect of several parameters on the size of alginate beads produced by use of an electrostatic potential bead generator was examined. Parameters studied included needle diameter, electrostatic potential, alginate solution flow rate, gelling ion concentration and alginate concentration and viscosity, as well as alginate composition. Bead size was found to decrease with increasing electrostatic potential, but only down to a certain level. Minimum bead size was reached at between 2-4 kV/cm for the needles tested. The smallest alginate beads produced (using a needle with inner diameter 0.18 mm) had a mean diameter of approximately 300 microm. Bead size was also found to be dependent upon the flow rate of the fed alginate solution. Increasing the gelling ion concentration resulted in a moderate decrease in bead size. The concentration and viscosity of the alginate solution also had an effect on bead size as demonstrated by an increased bead diameter when the concentration or viscosity was increased. This effect was primarily an effect of the viscosity properties of the solution, which led to changes in the rate of droplet formation in the bead generator. Lowering the flow rate of the alginate solution could partly compensate for the increase in bead size with increased viscosity. For a constant droplet size, alginates with a low G block content (F(GG) approximately 0.20) resulted in approximately 30% smaller beads than alginates with a high G block content (F(GG) approximately 0.60). This is explained as a result of differences in the shrinking properties of the beads.

Aneuploidy in benign tumors and nonneoplastic lesions of musculoskeletal tissues.

BACKGROUND: Aneuploidy in DNA flow cytometry (FCM) of musculoskeletal tumors is generally considered to be a sign of malignancy. Previously, giant cell tumor of the bone has been reported to contain aneuploid (near-diploid) DNA stemlines. Otherwise, only spordic cases have been reported. The authors wanted to study the relationships among DNA FCM, histology, and clinical course of nonmalignant musculoskeletal lesions. METHODS: Twenty-eight histologically benign tumors and seven nonneoplastic lesions were subjected to DNA FCM: After tissue preparation mechanically and with ribonuclease and trypsin, the isolated nuclei were stained with propidium iodine using chicken and rainbow trout erythrocytes as controls. In the DNA FCM histograms, ploidy and cell cycle fractions were determined using a computerized mathematical model. The histologic diagnoses were made without knowledge of the DNA FCM results. RESULTS: Aneuploidy was found in eight lesions. A shoulder in the diploid peak, suggesting a diploid and a near-diploid population, was found in DNA histograms of a condensing osteitis of the clavicle (a benign inflammatory process) and of a giant cell tumor of bone. The latter lesion also had a tetraploid population. Six benign tumors--two enchondromas, one osteochondroma, one subcutaneous and one intramuscular lipoma, and a calcifying aponeurotic fibroma--showed clear aneuploidy with separate peaks. The S-phase fraction was less than 10% in all cases. The highest aneuploid population, DNA index = 1.70, in a subcutaneous lipoma, was small, with an undetectable S phase. Despite nonradical operations in seven lesions, no recurrences were observed during a median follow-up of 49 months (range, 28-73 months). CONCLUSIONS: Small aneuploid populations with low DNA synthetic activity may be compatible with a benign histologic picture and uneventful clinical course of the musculoskeletal lesion.

The clinical importance of DNA synthesis and aneuploidy in bone and soft tissue tumours.

DNA flow cytometric analysis including the analysis of S-phase fraction was performed on 46 bone and soft tissue tumours. Histologically, 24 lesions were benign, 11 were low grade and 11 were high grade malignant tumours. The percentages of aneuploidy in these groups were 29, 54 and 82, respectively. The S-phase fraction was smaller than 14% in all benign tumours. High S-phase fraction (> or = 14%) was found in 3 out of 11 low-grade tumours (27%) and in 9 out 11 high-grade tumours (82%). Seven of nine patients who died of metastatic disease during the average 5 year follow-up had aneuploid stemlines with S-phase fraction higher than 14%. Histologically, these were all high-grade tumours. Three patients with high S-phase in low-grade tumours survived. We conclude that high DNA synthetic activity in an aneuploid population, more than the DNA aneuploidy alone, has prognostic significance in musculoskeletal tumours.

The binding of cis-dichlorodiammineplatinum(II) to extracellular and intracellular compounds in relation to drug uptake and cytotoxicity in vitro.

The biological consequence of the binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to serum protein as well as to cellular components in general, was studied on human NHIK 3025 cells in vitro. As expected, we found that the cytotoxicity of cis-DDP was lost by binding to serum protein, and that protein-bound platinum was impermeable to the cells. As we have previously shown that electropermeabilisation may transiently increase the influx of cis-DDP, we applied this technique in an attempt to increase the efflux of cis-DDP or any other cytotoxic intermediates. Our data demonstrate that if cells are electropermeabilised shortly after treatment with cis-DDP, cell survival increased. This indicates that cis-DDP in an active form is released from the cells; furthermore, the plasma membrane represents a barrier against efflux, as it has also been shown to be against influx of active cis-DDP. Thus, our data are consistent with the idea that there must be an intracellular pool of either cis-DDP, or some biologically active intermediates, in cells treated with this drug. Additionally, our data indicate that the binding rate of cis-DDP to biological molecules is much quicker intracellularly than in the extracellular environment: We found the biological half-life at 37 degrees C to be about 2.1 h in human serum and about 11 min inside our cells.

In vitro modulating effect of the reversible protein synthesis inhibitor zilascorb (2H) on cis-diamminedichloroplatinum (II)-induced cytotoxicity.

Zilascorb (2H) (5,6-benzylidene-d1-L-ascorbic acid sodium salt), a derivative of deuterated benzaldehyde inducing reversible protein synthesis inhibition, was tested on human NHIK 3025 cells in combination with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). The inactivating effect of cis-DDP, measured as loss of colony forming ability, was found to increase when a non-toxic treatment with zilascorb (2H) was given simultaneously with, or shortly prior to, treatment with cis-DDP. No potentiating effect was seen when zilascorb (2H) treatment occurred after removal of cis-DDP. Furthermore, the data showed that the potentiating effect of zilascorb (2H) reached a maximum at concentrations of between 2 and 4 mM. The underlying mechanism for this potentiation is unknown, but could possibly be related to the effect of zilascorb (2H) on protein synthesis. Alternatively, it is also possible that generation of free radicals from the ascorbate moiety of the zilascorb (2H) molecule may increase cellular damage induced by cis-DDP, or inhibit repair or restitution of cis-DDP-induced damage. Also, in cells of a cis-DDP-resistant subline, NHIK 3025/DDP zilascorb (2H) was found to potentiate the effect of cis-DDP. The cis-DDP-resistant cells, however, were found to be partly cross-resistant to cytotoxic treatments with zilascorb (2H).

DNA flow cytometry in sperm cells from testicular cancer patients. Impact of different treatment modalities on spermatogenesis.

In 77 patients with testicular cancer, sperm cell density was evaluated before and after treatment together with the following parameters as recorded by DNA flow cytometry: the percentage of condensed and non-condensed haploid sperm cells, and the number of non-haploid cells. The patients received either abdominal radiotherapy, cisplatin-based combination chemotherapy (+/- surgery) or did not undergo antiproliferative treatment at all. Ejaculates with a low sperm cell density had high numbers of non-condensed haploid and non-haploid cells, whereas the percentage of condensed haploid cells was decreased. However, even 'azoospermic' semen samples (by light microscopy) contained haploid cells indicating ongoing sperm cell production. One year after radiotherapy there was a significant decrease in the sperm cell density and the number of condensed haploid cells. Chemotherapy led to a significant reduction of sperm cell density evaluated 1 year after treatment. Three years after the diagnosis of testicular cancer, sperm cell production had recovered in most patients. In patients who did not have radiotherapy or chemotherapy the median density of sperm cells 3 years after treatment significantly exceeded the corresponding figure from the pretreatment situation. A low pretreatment percentage of condensed haploid cells (less than or equal to 90%) was correlated with the lack of 1-year recovery of sperm cell production. It is too early to state whether longitudinal flow cytometric data from ejaculates in testicular cancer patients yield clinically valuable information, in addition to that obtained by light microscopy. From this preliminary observation it seems that DNA flow cytometry in ejaculates from testicular cancer patients may give valuable clinical information about sperm cell production following treatment.

Prediction of objective response to recombinant interferon-alpha with or without vinblastine in metastatic renal cell carcinoma.

Clinical, histopathological and DNA cytometric parameters were analyzed before treatment in 57 patients with metastatic renal cell carcinoma (RCC) with regard to their ability to predict objective response to treatment with interferon-alpha (IFN) with or without vinblastine (VB). No pretreatment factor could be identified which was correlated with response. Patients who had at least 30% size reduction of their indicator lesion(s) after 2 months and did not present with new visible metastases had a significantly higher chance to obtain objective response than those in whom this condition was not fulfilled. We suggest that interferon treatment (with or without VB) should be offered for 2 months to all patients with metastatic RCC who are to receive systemic therapy. If at least 30% tumour size reduction is observed at that time, the patient will most probably respond objectively. If the size reduction is less, IFN with or without VB is likely to be ineffective in terms of response achievement.

T2/T3 bladder carcinomas treated with definitive radiotherapy with emphasis on flow cytometric DNA ploidy values.

From 1972 through 1982, 124 patients with muscle invasive T2/T3 bladder carcinoma without evidence of distant metastases were treated with definitive radiotherapy (60 Gy in 6 weeks) to the pelvis at The Norwegian Radium Hospital. Sections from paraffin embedded biopsies taken prior to the start of treatment were prepared for DNA flow cytometry, and 121 histograms were considered interpretable for DNA ploidy values. Significantly improved survival was seen in patients with the following characteristic, univariate analysis: T2 tumor, macroscopically complete removal of the tumor prior to radiotherapy, clinically complete response to radiotherapy, normal serum creatinine, absence of hydronephrosis, tetraploid tumor, WHO performance status 0 or 1, age below 65 years. In a multivariate analysis the following pretreatment variables turned out with prognostic power in this order: normal serum creatinine, T2 tumor, tetraploidy, and absence of small vessel invasion. When including response to therapy, the following factors were significantly predictive of a beneficial survival: clinically complete response after radiotherapy, normal creatinine, T2 tumor and tetraploidy. Tetraploidy was associated with response to radiotherapy when compared to diploid tumors (p = 0.07). This relationship alone did not explain the better survival of patients with tetraploid tumors, and other factors concerned with "tumor aggressivity" may be additional explanatory parameters. DNA ploidy values provide valuable information in the pretreatment situation concerning the prognosis for patients with T2/T3 bladder carcinoma.

DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment.

Light microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age-matched males (control group). A computer-based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub-haploid, haploid (1c), and diploid (2c) and greater than 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and greater than 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.

Growth stimulation by giant-cell tumor of bone? A case report.

Overgrowth of the femur and tibia was observed in a 20-year-old man who had a giant cell tumor in his distal femur. Growth stimulation by the tumor is suggested to explain the overgrowth.

Oxygen- and temperature-dependent cytotoxic and radiosensitizing effects of cis-dichlorodiammineplatinum(II) on human NHIK 3025 cells in vitro.

The radiosensitizing effect of the chemotherapeutic drug cis-dichlorodiammineplatinum(II) (cis-DDP) was tested on human NHIK 3025 cells cultivated in vitro. cis-DDP was found to exert a radiomodifying effect under hypoxic but not under aerobic conditions. These results confirm that cis-DDP may act as a radiosensitizer of hypoxic cells; however, the radiosensitizing effect was seen only at concentrations of cis-DDP having a considerable cytotoxic activity, and for practical reasons concerning survival level the highest drug concentration that was investigated was 15 microM at 37 degrees C. The radiosensitizing effect was of a dose-modifying type and with a dose-modifying factor (DMF) of 1.2 at 15 microM in hypoxic cells. The radiosensitizing as well as the cytotoxic effect of cis-DDP was found to be strongly temperature dependent. Isoeffect doses of cis-DDP was reduced with a factor of 3 at 22 as compared to 37 degrees C. We also found that hypoxic cells were less sensitive to cis-DDP than cells treated in the presence of oxygen. To test the correlation between cytotoxicity and radiosensitization on the one hand and cellular uptake of cis-DDP on the other, cell-associated Pt was measured by atomic absorption spectroscopy. From these studies the cytotoxicity of cis-DDP at 22 and 37 degrees C under aerobic conditions was found to be the same as long as the amount of cell-associated Pt (i.e., the cellular uptake) was the same. However, whether the cells were treated under hypoxic or aerobic conditions, the cellular uptake of Pt was the same. While the radiosensitizing effect was present at 37 and at 40 degrees C, no such effect could be found at 22 degrees C. Since the cytotoxicity of cis-DDP as well as the drug uptake was reduced about three times at 22 as compared to 37 degrees C, we increased the concentration threefold, to 50 microM at 22 degrees C. Still no radiosensitizing effect was found at this temperature.

DNA flow cytometric values in bladder carcinoma biopsies obtained from fresh and paraffin-embedded material.

DNA-histograms were obtained by flow cytometry (FCM) of nuclei prepared from fresh bladder carcinoma biopsies and from 100 micron sections of paraffin-embedded material from the same biopsies. Linear regression analysis of ploidy values from fresh material versus those from paraffin blocks showed excellent correlation (R2 = 0.957). Owing to the high background, the paraffin-embedded material was less suited for analysis of S-phase fraction. It is concluded that DNA FCM of nuclei obtained from paraffin-embedded bladder carcinoma biopsies yields reliable results concerning ploidy, but does not permit evaluation of the S-phase fraction.

Flow cytometric DNA measurements in paraffin-embedded bladder carcinoma tissue before and after precystectomy radiotherapy.

From 1976 through 1983, 48 patients with T2/T3 bladder carcinomas were treated with pre-operative radiotherapy (46 Gy) and total cystectomy. DNA histograms were recorded by flow cytometry (FCM) from all pre-treatment paraffin-embedded tumour biopsies and, if still present after radiotherapy, from tumour tissue from the cystectomy specimen. Five patients were excluded from the study because no DNA histograms could be recorded due to extensive destruction of the cell nuclei during preparation. Thus, 43 patients are completely evaluable. Before the start of radiotherapy, 32 bladder carcinomas were interpreted as non-diploid, whereas 11 were diploid. Non-diploidy of the pre-treatment biopsy was associated with radiotherapy-induced stage reduction (p = 0.13). The survival rates in patients with diploid and non-diploid tumours were 65% and 45% respectively at 4 years (p = 0.13). Although not statistically significant, our results suggest that DNA-FCM may provide clinically relevant information about the tumour biology and response to radiotherapy concerning bladder carcinomas.

Increase in cis-dichlorodiammineplatinum (II) cytotoxicity upon reversible electropermeabilization of the plasma membrane in cultured human NHIK 3025 cells.

A series of brief electrical high-voltage discharges were given to cultured NHIK 3025 cells to render the plasma membrane transiently permeable to drugs. Using [14C]sucrose as an inert marker which normally does not cross plasma membranes, increased permeability could be demonstrated for no longer than 10 min following electrical treatment, indicating that the permeabilization was entirely reversible. The reversibility of the treatment was further demonstrated by a lack of effect on cell growth and colony-forming ability. When cells were given electrical discharges immediately before or during exposure to cis-dichlorodiammineplatinum(II)(cis-DDP) the cytotoxic drug effect increased. By using electrical discharges during a 2 hr drug treatment period the cytotoxicity was enhanced to an extent corresponding to at least a 3-fold increase in drug uptake relative to unpermeabilized cells. This increase in drug uptake was confirmed by direct measurements of the amount of cell-associated Pt by atomic absorbtion spectroscopy. The results suggest that uptake across the plasma membrane may be the rate-limiting factor in the cytotoxic effect of cis-DDP. Furthermore, the methodology applied in the present study may prove useful in assessing the influence of membrane permeability on the effect of other cytotoxic drugs.

Reduced cellular uptake of cis-dichlorodiammine-platinum by benzaldehyde.

Synchronized human NHIK 3025 cells were treated with cis-dichlorodiammineplatinum (cis-DDP) either alone or in combination with benzaldehyde as a 2 h pulse in G1-phase. After this pulse, the cells entered S-phase and the rate of DNA synthesis was measured by DNA-flow cytometric recordings of serial samples. After treatment with 10 microM cis-DDP alone, the rate of DNA synthesis was 38% of the control rate. If 3.2 mM benzaldehyde was present together with 10 microM cis-DDP, the rate of DNA synthesis was 56% of the control rate, this being similar to the rate measured following treatment of cells with 5 microM cis-DDP alone. Thus, the simultaneous presence of benzaldehyde with cis-DDP mitigates the inhibition of DNA synthesis induced by cis-DDP. However, when cells were electropermeabilized during the treatment pulse, benzaldehyde did not protect the cells from cis-DDP-induced cell inactivation. The protective effect of benzaldehyde thus seems to reside with the cell membrane and it seems that benzaldehyde, when present together with cis-DDP, partially inhibits the uptake of cis-DDP into cells. Atomic absorption spectroscopy confirmed that the simultaneous presence of 5 mM benzaldehyde halved the amount of cell-bound platinum from that measured following treatment with cis-DDP alone.

DNA flow cytometry in human testicular cancer.

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22-51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.