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PubMed2006

The clinical impact of histopathologic response assessment by residual tumor cell quantification in esophageal squamous cell carcinomas.

BACKGROUND: The objectives of this study were to investigate histomorphologic features as a response classification after neoadjuvant radiochemotherapy (RTx/CTx) and to correlate the results with clinical outcome parameters (e.g., postoperative morbidity and mortality, recurrence, and survival) in patients with locally advanced esophageal squamous cell carcinoma (ESCC). METHODS: Three hundred eleven patients with histologically proven, locally advanced, intrathoracic ESCC (clinical T3 or T4, N0-N+, M0) located at or above the level of the tracheal bifurcation underwent preoperative, combined, simultaneous RTx/CTx followed by esophagectomy. Response to RTx/CTx was classified by the quantification of residual tumor cells. A histopathologic response was defined as <10% residual tumor cells found within the specimen compared with a histopathologic nonresponse, which was characterized by >10% residual tumor cells. RESULTS: A histopathologic response was correlated significantly with complete tumor resection status (R0 resection) (P .0001), histopathologic tumor (ypT) category (P <.0001), lymph node involvement (P <.0001), lymphatic vessel invasion (P <.001), and survival (P <.0001). A multivariate Cox regression analysis revealed that histopathologic response classification according to the percentage of residual tumor cells was an independent prognostic factor (P <.0001). Nonresponders had greater postoperative pulmonary morbidity (P = .01), a greater 30-day mortality rate (P = .02), and a dismal survival rate compared to histopathologic responders (P <.0001). CONCLUSIONS: Histopathologic response evaluation based on the quantification of residual tumor cells provided meaningful information for the assessment of outcomes among patients with ESCC who have underwent neoadjuvant RTx/CTx. The current results indicated that histopathologic responders may represent a subgroup of patients who benefit from neoadjuvant therapy followed by surgery.

Aged↗
PubMed1981

Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups.

The distribution of monoaminergic cell bodies in the brainstem of the cat has been examined with Falck-Hillarp fluorescence histochemical technique. Quantitative determinations indicate that the cat brainstem contains about 60,300 indolaminergic (IA) cells. The majority of these (about 46,700, or 77.5%) are located within raphe nuclei. The largest number is contained within nucleus raphe dorsalis (RD), accounting for around 24,300 IA cells, while raphe pallidus (RP) holds about 8,000 raphe centralis superior (RCS) 7,400, raphe magnus (RM) 2,400, raphe obscurus (RO) 2,300, linearis intermedius (LI) 2,100, and the raphe pontis (RPo) only some 280 IA cells. The IA cells represent, however, only part of the neuronal population of raphe nuclei, which, in addition, hold varying numbers of other medium-sized and small-sized neurons. Thus, quantification in Nissl-stained material indicate that the IA cells make up about 70% of the medium-sized cells in RD, 50% in RP, 35% in RCS and RO, 25% in LI, 15% in RM, and only 10% in RPo. The substantial numbers of small-sized perikarya observed in all raphe nuclei may represent interneurons. Significant numbers of IA cells were consistently located outside the raphe nuclei at all brainstem levels. In all, these amounted to approximately 13,600, or 22.5% of the total number of IA cells. Thus, IA cells occurred in the myelinated bundles, and sometimes in reticular formation, bordering the raphe nuclei; in the ventral brainstem forming a lateral extension from the ventral raphe (RP, RM, RPo, RCS, and LI) to the position of the rubrospinal bundle; in the periventricular gray and subjacent tegmentum of dorsal pons and caudal mesencephalon; in the locus coeruleus (LC) complex; around the motor trigeminal nucleus; caudal to the red nucleus; and in the interpeduncular and interfascicular nuclei. The wide distribution of IA cells leads to a considerable mixing with catecholaminergic (CA) cell groups. Our observations on CA cell distribution are essentially in accordance with previous reports. Quantifications indicate that the LC complex contains about 9,150 CA cells, unilaterally. A previously unnoticed group of scattered CA cells was found in relation to the vestibular nuclei and extending dorsally toward the deep cerebellar nuclei.

Animals↗
PubMed2002

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.

Cell Division↗
PubMed1990

Quantification of renal Na-K-ATPase activity by image analysing system.

The localisation of renal Na-K-ATPase activity along the rat nephron by a cytochemical method, and its quantification by an image analysis system, are described in this paper. Frozen kidney sections were exposed to a trapping agent, the lead ammoniac-citrate-acetate complex (LACA), and to all the substrates necessary to the enzyme activity. The absorbance of the histochemical reaction product (precipitated in situ), proportional to the enzymatic activity, was then measured through the analysis of the grey levels of the transmitted image of the kidney section. This method was both sufficiently sensitive and technically simple to permit measurements of the enzyme in large numbers of tubules and to determine its activity in each region of the nephron. The Na-K-ATPase activity has been determined in the proximal convoluted tubule (PCT), the medullary thick ascending limb of the Henle's loop (mTAL), and the distal convoluted tubules (DCT) of the rat nephron. The Na-K-ATPase distribution shows an activity per millimeter tubule length higher in the DCT than in the mTAL and the PCT: 1,406 +/- 33, 823 +/- 64, and 350 +/- 71 pmoles Pi/tubule mm/h, respectively. In conclusion, the described method allows the segmental quantification of Na-K-ATPase activity at a cellular level and offers a precise approach to the analysis of this enzyme along the length of nephrons.

Animals↗
PubMed1992

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Analog-Digital Conversion↗
PubMed1994

Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis.

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

Analog-Digital Conversion↗
PubMed2004

Quantification of EGFP expression on Molt-4 T cells using calibration standards.

BACKGROUND: Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. METHODS: Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0-9.0, temperatures of 37-80 degrees C, and osmolalities of 100-600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 microg and 0.4 microg) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. RESULTS: The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively constant from 37 degrees C to 65 degrees C, and from 100 to 600 mOsm/kg. After transfection, the expression of the populations with high and low EGFP expression averaged 8,098 +/- 584 MESF and 3,808 +/- 375 MESF respectively. No significant differences were observed after comparing the MESF values obtained by flow cytometry and the values obtained by Cytofluor technique (high: 8,791 +/- 492 MESF; low: 4,082 +/- 398 MESF). CONCLUSIONS: Our data demonstrate the feasibility of using calibration standards to quantify EGFP expression on cells. Our results emphasize the importance of monitoring the effects of environmental changes in the fluorescence intensity of both standards and samples when quantifying the expression of EGFP on living cells.

Calibration↗
PubMed2006

Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.

BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.

Antioxidants↗
PubMed1994

Quantification of silver-stained proteins resolved by two-dimensional electrophoresis: genetic variability as related to abundance and solubility in two maize lines.

Relative abundance and solubility of proteins from etiolated coleoptiles of maize were investigated using two-dimensional electrophoresis (2-D PAGE). Automatic quantification of silver-stained polypeptides on replicate 2-D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 micrograms to 135 micrograms per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2-D patterns (trichloroacetic acid-acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, either qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.

Analysis of Variance↗
PubMed2003

Investigation of a capillary electrophoretic approach for direct quantification of apolipoprotein A-I in serum.

In the present study a rapid, reproducible and robust capillary electrophoresis (CE) procedure for the quantification of apolipoprotein A-I (Apo A-I) in serum without pretreatment has been developed (total run time, 11 min). The coefficients of variation (CV; n = 10) for the relative peak area are 1.8% at a concentration of 145 mg/dL and 1.6% at 196 mg/dL; and for the inter-assay 8.9% at 161 mg/dL (10 consecutive days), i.e., similar to the CVs of a high-throughput immunonephelometric routine assay. The CV for the migration time is 0.4% (n = 20). The robustness of the CE approach was tested in patient samples with hemolysis, hyperbilirubinemia and hyperlipidemia. A comparison of 99 Apo A-I serum values with results of a fixed-time immunonephelometric routine assay showed a positive constant bias of 60% (mean) for the immunonephelometric values, no deviation from linearity, but significant deviations in several samples. Investigations on interferences in the CE analyses gave no evidence that CE failed. Our study shows that CE is amenable to a fast analysis and a reproducible and reliable quantification of Apo A-I level in sera of various clinical samples.

Apolipoprotein A-I↗
PubMed2005

Resolution of overlapping capillary electrophoresis peaks by using chemometric analysis: quantification of the components in compound reserpine tablets.

The application of multivariate curve resolution with alternating least squares (MCR-ALS) methods to second-order data from capillary electrophoresis with diode array detector (CE-DAD) is reported. Initial qualitative solutions obtained by evolving factor analysis (EFA) and pure-variable detection method can be further optimized by a simultaneous analysis of multiple electrophoresis run data with ALS regression. While unknown samples are analyzed simultaneously against the corresponding standards in different composition ratios, the exact amounts of common components in different CE runs can be determined by the traditional calibration curve method, and quantification can thus be achieved. The above methods are applied to the determination of the components in compound reserpine tablets in overlapping peaks from CE. The quantification results are compared with those of the first derivative of the electropherogram method and artificial neural network (ANN) method.

Electrophoresis, Capillary↗
PubMed2005

A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser-induced fluorescence detection.

Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC20) has been implicated in numerous contractile and motile events, e.g., smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC20 phosphorylation in minute samples is critical to determine the importance of LC20 phosphorylation in diverse physiological processes. We have developed a method for the separation and quantification of unphosphorylated, monophosphorylated, and diphosphorylated LC20 with a detection limit of 1 pg (50 amol). LC20 is initially isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to a polyvinlyidene difluoride (PVDF) membrane. The region of the membrane containing the LC20 band (identified by electrophoresis of purified LC20 in a neighboring lane) is cut out and fluorescently labeled with Alexa Fluor 488 C5 maleimide. The labeled LC20 is eluted from the membrane with detergent and subjected to capillary isoelectric focusing (CIEF) to separate unphosphorylated, mono-, and diphosphorylated LC20, which are detected and quantified by laser-induced fluorescence (LIF). A linear relationship between log(peak area) and log(LC20 amount) is observed over the range of 50 amol-150 fmol. Quantification of LC20 phosphorylation by CIEF with LIF detection was compared with three commonly used methods with much lower levels of sensitivity: urea/glycerol-PAGE with Western blotting, phosphorylation by [gamma-32P]ATP with Cerenkov counting, and phosphorylation by [gamma-32P]ATP followed by SDS-PAGE, autoradiography, and scanning densitometry. All four methods gave very similar quantitative results, the major difference being that the new method exhibits 3000-fold enhanced sensitivity. This method is therefore applicable to quantitative analysis of phosphorylation of minute quantities of LC20.

Animals↗
PubMed2006

2-Methoxy-2,4-diphenyl-3(2H)-furanone-labeled gelatin zymography and reverse zymography: a rapid real-time method for quantification of matrix metalloproteinases-2 and -9 and tissue inhibitors of metalloproteinases.

Measurement of matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases (TIMPs) by the techniques of zymography and reverse zymography provide useful information regarding the status of matrix accumulation or breakdown. This report describes the use of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), a fluorescent compound which can be used to label gelatin as a substrate for detection of the gelatin degrading MMP-2 and -9 by zymography. In addition, a modification of the zymographic technique by addition of excess MMPs enables the use of the MDPF-labeled gelatin substrate for the identification and quantification of TIMPs by reverse zymography. Both systems are real-time sensitive reliable quantification techniques, easily used for measurement of these MMPs and TIMPs in clinical, biological, and tissue culture samples.

Animals↗
PubMed2006

Rapid quantification of human complement component C4A and C4B genes by capillary gel electrophoresis.

Complement component 4 (C4) is an important plasma protein playing a major role in the human defense mechanism against infectious diseases and inflammatory processes. The C4A and C4B genes, encoding the two isoforms of complement 4, are located in the nuclear serine/threonine protein kinase-C4A or B gene-cytochrome 21-hydroxylase-tenascin X module (RP-C4-CYP21-TNX) and manifested by variable copy numbers among individuals between zero to six in the human diploid genome. Quantification of the C4A and C4B genes has great clinical importance since unbalanced production of C4A and C4B proteins might be associated with pathological immune processes. Albeit, high-throughput analysis methods for C4 gene dosage determination are not yet available. Here we present a novel combination of allele-specific PCR and CGE separation for rapid quantification of the C4A and C4B genes where a single-step, single-tube PCR reaction generates two allele-specific (C4A and C4B) and two control amplicons, followed by CGE analysis of the four fragments. The method presented in this paper enables automated and high-throughput gene dosage analysis of large sample cohorts.

Complement C4a↗
PubMed2003

Development of a real-time RT-PCR assay for the quantification of the most frequent MLL/AF9 fusion types resulting from translocation t(9;11)(p22;q23) in acute myeloid leukemia.

One strategy to predict clinical outcome in patients with acute myeloid leukemia (AML) is detection of minimal residual disease (MRD) after achievement of hematologic complete remission (CR). We established a real-time RT-PCR assay by use of TaqMan technology for the identification of MRD by quantification of the most frequent fusion transcripts resulting from t(9;11)(p22;q23). To achieve comparable PCR efficiencies between the different PCR assays, primers were chosen to obtain amplicons of nearly identical lengths. MLL/AF9 copy numbers were normalized to the housekeeping gene porphobilinogen deaminase (PBGD). The sensitivity of the assay, as determined at the cellular level, was comparable to that of qualitative single-round RT-PCR. Samples from eight patients with t(9;11)-positive AML were analyzed. At diagnosis and relapse, normalized copy numbers were positive and ranged from 490 to 5,558. Samples from two of seven patients collected at the time of CR became negative, whereas five cases still had positive normalized copy numbers with values between 5 and 5,286. The implications of MRD detection by MLL/AF9 fusion transcript quantification for the clinical management of t(9;11)-positive AML have to be determined in further studies.

Acute Disease↗
PubMed2001

Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)-positive acute myeloblastic leukemia by real-time RT-PCR.

We used a newly established real-time RT-PCR assay for the quantification of the leukemia-specific CBFB/MYH11 transcripts in inv(16)-positive acute myeloblastic leukemia. CBFB/MYH11 could be quantified over a five log range, with a detection limit of 10 molecules of a CBFB/MYH11 plasmid and a 1:10(5) dilution of RNA of the inv(16)-positive ME-1 cell line, respectively. The fusion transcripts were also quantified in 19 patients with acute myeloblastic leukemia and an inv(16) at initial diagnosis. The expression of CBFB/MYH11 varied over a two log range without correlation to clinical response or relapse rate. In nine patients, CBFB/MYH11 was also quantified during/after chemotherapy and autologous or allogeneic stem cell transplantation. All of these patients showed a similar decline of CBFB/MYH11 after intensive therapy. Six of these patients are in complete remission with a stable low-level or absent CBFB/MYH11 expression. Three patients relapsed, and their CBFB/MYH11 transcripts rose again to pretreatment levels. In two patients, this increase in CBFB/MYH11 could be detected by real-time PCR before hematological relapse. These data indicate that real-time RT-PCR can be used for the sensitive detection and quantification of CBFB/MYH11 transcripts in the follow-up of patients with inv(16)-positive AML.

Chromosome Inversion↗
PubMed2007

Quantification of various growth factors in different demineralized bone matrix preparations.

Besides autografts, allografts, and synthetic materials, demineralized bone matrix (DBM) is used for bone defect filling and treatment of non-unions. Different DBM formulations are introduced in clinic since years. However, little is known about the presents and quantities of growth factors in DBM. Aim of the present study was the quantification of eight growth factors important for bone healing in three different "off the shelf" DBM formulations, which are already in human use: DBX putty, Grafton DBM putty, and AlloMatrix putty. All three DBM formulations are produced from human donor tissue but they differ in the substitutes added. From each of the three products 10 different lots were analyzed. Protein was extracted from the samples with Guanidine HCL/EDTA method and human ELISA kits were used for growth factor quantification. Differences between the three different products were seen in total protein contend and the absolute growth factor values but also a large variability between the different lots was found. The order of the growth factors, however, is almost comparable between the materials. In the three investigated materials FGF basic and BMP-4 were not detectable in any analyzed sample. BMP-2 revealed the highest concentration extractable from the samples with approximately 3.6 microg/g tissue without a significant difference between the three DBM formulations. In DBX putty significantly more TGF-beta1 and FGFa were measurable compared to the two other DBMs. IGF-I revealed the significantly highest value in the AlloMatrix and PDGF in Grafton. No differences were accessed for VEGF. Due to the differences in the growth factor concentration between the individual samples, independently from the product formulation, further analyzes are required to optimize the clinical outcome of the used demineralized bone matrix.

Bone Matrix↗
PubMed1996

Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay.

Difficulties in the geometrical definition and measurement of resorption pits is a major problem for the quantitative analysis of bone resorption by isolated osteoclasts cultured on bone or dentin substrates. In this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically quantifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or dentin. A consistently high correlation between the formation of resorption pits and the release of antigenic collagen fragments was observed for isolated rabbit osteoclasts seeded at various densities and cultured for various periods on bovine, elephant, and human substrates. In a further support of the osteoclastic nature of the collagenolytic effects, a high consistency between pit formation and collagenolysis was also observed when the rabbit bone cells were cultured in the presence of very differently acting but typical inhibitors of pit formation, i.e., the carbonic anhydrase inhibitor acetazolamide, the cysteine proteinase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisphosphonate ibandronate (BM 21.0955). In conclusion, the ELISA represents a simple, precise, and objective way to dynamically monitor bone resorption in vitro through quantification of the collagenolytic activity of isolated osteoclasts.

Amino Acid Sequence↗