Adsorption of Organic Dyes on Magnetic Iron Oxide Nanoparticles. Part II: Field-Induced Nanoparticle Agglomeration and Magnetic Separation.

This paper (part II) is devoted to the effect of molecular adsorption on the surface of magnetic iron oxide nanoparticles (IONP) on the enhancement of their (secondary) field-induced agglomeration and magnetic separation. Experimentally, we use Methylene Blue (MB) cationic dye adsorption on citrate-coated maghemite nanoparticles to provoke primary agglomeration of IONP in the absence of the field. The secondary agglomeration is manifested through the appearance of needlelike micron-sized agglomerates in the presence of an applied magnetic field. With the increasing amount of adsorbed MB molecules, the size of the field-induced agglomerates increases and the magnetic separation on a magnetized micropillar becomes more efficient. These effects are mainly governed by the ratio of magnetic-to-thermal energy α, suspension supersaturation Δ0, and Brownian diffusivity Deff of primary agglomerates. The three parameters (α, Δ0, and Deff) are implicitly related to the surface coverage θ of IONP by MB molecules through the hydrodynamic size of primary agglomerates exponentially increasing with θ. Experiments and developed theoretical models allow quantitative evaluation of the θ effect on the efficiency of the secondary agglomeration and magnetic separation.

Superhydrophobic polypyrene films to prevent Staphylococcus aureus and Pseudomonas aeruginosa biofilm adhesion on surfaces: high efficiency deciphered by fluorescence microscopy.

A blue luminescent and superhydrophobic coating based on an electropolymerized fluorinated-pyrene monomer and its planktonic bacteria and biofilm repellent properties are reported. Two different pathogenic bacterial strains (Gram-positive and Gram-negative) at two different incubation times (2 h planktonic bacterial and 24 h biofilm adhesion) were studied and monitored (analyzed) using multicolor scanning confocal fluorescence microscopy. The coating was proved to reduce bacterial adhesion by 65%. It is highly effective against biofilm attachment, with 90% reduction of bacteria surface coverage. This blue fluorescent surface provides a facile method to characterize the coating, observe the bacterial distribution and quantify the bacterial coverage rate by fluorescence imaging of different colors. Furthermore, the film does not show significant bacterial toxicity during the working incubation times.

Expression of MMP-2, 9 and 13 in newly formed bone after sinus augmentation using inorganic bovine bone in human.

BACKGROUND AND OBJECTIVE: The aim of the present study was to analyse the expression of MMP-2, MMP-9 and MMP-13 in newly formed bone following maxillary sinus augmentation using inorganic bovine bone substitute, because these MMPs play a major role in bone remodeling and bone resorption. MATERIAL AND METHODS: Deproteinized bovine bone (Bio-Oss(®)) was used to fill cavities after elevating the sinus mucosa. Twenty patients with edentulous posterior maxilla were treated with 20 sinus-augmentation procedures using a two-stage technique. Forty-nine Straumann(®) endosseous implants were used to complete the implant-prosthetic rehabilitation. One cylinder-shaped bone biopsy from each patient was taken from the augmented maxillary region using trephine burs at the second stage of surgery, 8 months after grafting. A biopsy was also taken as a control from the upper molar region from six different patients who did not undergo the sinus procedure. All biopsies were subjected to biochemical analysis and staining for TRAP. RESULTS: No implant losses or failures occurred. The large number of TRAP-positive multinucleated osteoclasts in resorption lacunae indicated that the resorption was very active in all grafts, in contrast with the control group. Zymography and western blot analysis demonstrated a significantly increased expression of MMP-2, MMP-9 and MMP-13 in the newly formed bone compared with controls (p < 0.05). CONCLUSION: The quantity of osteoclastic cells and the increased expression of proteolytic enzymes suggest that 8 months after grafting, inorganic bovine bone is slowly resorbing and is the site of important remodeling of the newly formed bone by means of resorption and synthesis.

Pertinent cell population to characterize periodontal disease.

The purpose of this in situ study is to quantify the inflammatory cell subsets and the area fraction (AA%) occupied by collagen fibers in human healthy and diseased (four different stages) gingival connective tissue in order to establish a possible correlation between periodontal disease resulting in collagen breakdown and specific inflammatory cell subsets. Paraffin gingival tissue sections from eight healthy controls (group 0), 10 patients with gingivitis (group 1), 10 patients with moderate periodontitis (group 2) and 10 patients with severe periodontitis (group 3) were immunohistochemically investigated using antibodies against CD-45+, CD-3+, CD-8+, CD-20+, CD-68+, and EMA+ (plasma cells). The AA% occupied by gingival collagen fibers significantly decreased from 54.12% in group (0) to 38.58% in group (1), to 31.87% in group (2), and to 25.46% in group (3). In progressive lesions of periodontal disease, CD-3(+) and CD-8+ cell numbers were increased in early stages within the connective tissue, while CD-20+ cell numbers were increased only in late stages. On the other hand, EMA+, CD-68+ and CD-45+ cell numbers were progressively increased from group (0) to group (3). We demonstrated that CD-68+ monocyte/macrophages, CD-45+ leukocyte common antigen and notably EMA+ plasma cells are pertinently correlated with the severity of periodontal disease and related collagen breakdown.

Possible involvement of gelatinase A (MMP2) and gelatinase B (MMP9) in toxic epidermal necrolysis or Stevens-Johnson syndrome.

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are considered to be drug-induced diseases, and are characterized by extensive mucocutaneous disorder and epidermal necrosis which result in the detachment of the epidermis. Inactive and active forms of metalloproteinases (MMP2 and MMP9) secreted by skin explants maintained in organ culture for 72 h and in blister fluid from two TEN and three SJS patients were investigated. Interestingly, lesional skin from both the TEN and the SJS patients cultured for 3 days in conditioned medium showed high levels of both 72 kDa progelatinase A and 66 kDa activated gelatinase A, and the 66 kDa activated form was not observed in cultures of skin from control individuals. Furthermore, indirect immunodetection showed the presence of MMP2 and MMP9 in TEN and SJS patients' skin. Increased gelatinase activity in the culture medium of TEN and SJS skin maintained in organ culture and in blister fluid indicates that these gelatinases may be responsible for the detachment of the epidermis in these drug-induced necrolyses.

Morphometric analysis of elastic skin fibres from patients with: cutis laxa, anetoderma, pseudoxanthoma elasticum, and Buschke-Ollendorff and Williams-Beuren syndromes.

Computed morphometric analysis of elastic skin fibres in patients with cutis laxa, anetoderma, Williams-Beuren syndrome, pseudoxanthoma elasticum (PXE), and Buschke-Ollendorff syndrome, all clinically ascertained, was performed and compared with data obtained from healthy individuals of the same age. The diameters, area fractions (AA%) and volume fractions (VV%) occupied by pre-elastic fibres and dermal elastic fibres were determined. Irrespective of age the diameter of dermal elastic fibres followed a Gaussian distribution for all groups studied. These diameters were taken into consideration for VV% determinations. Compared with data from skin of healthy subjects of similar age range, VV% of pre-elastic fibres was significantly decreased in patients with cutis laxa, anetoderma, Williams-Beuren syndrome, and PXE and undetectable in Buschke-Ollendorff patients. VV% of dermal elastic fibres was four- to fivefold increased in Buschke-Ollendorff syndrome, two- to threefold increased in PXE skin, four- to fivefold decreased in cutis laxa and anetoderma skin and about twofold decreased in Williams-Beuren skin. The diameter of oxytalan fibres was decreased in anetoderma and Williams-Beuren syndrome while oxytalan fibre diameter was unchanged in PXE and cutis laxa. The diameter of dermal elastic fibres was increased in PXE and Buschke-Ollendorff syndrome, but was decreased in anetoderma and Williams-Beuren syndrome and unchanged in cutis laxa. We demonstrated that cutis laxa, anetoderma, Williams-Beuren syndrome, PXE, and Buschke-Ollendorff syndrome could be easily differentiated by morphometric analysis of elastic skin fibres. Thus we propose that morphometric analyses together with skin biopsies are a valuable tool for distinguishing between inherited and/or acquired skin diseases known to display alterations of elastic fibres.

Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue?

BACKGROUND: Evidence of the role of matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in periodontal destruction is now well established. The purpose of this study was to quantify, in healthy and diseased upper gingival connective tissue, the area fraction (AA%) occupied by collagen fibers, the cell number belonging to inflammatory cell subsets, and the amounts of MMPs and TIMPs (tissue inhibitors of MMPs) in order to investigate the possible correlations, if any, between such molecules, collagen loss, and inflammatory cell subsets. METHODS: Gingival tissue specimens from 6 healthy controls (C) and 6 patients with severe periodontitis (P) were divided into 2 groups. The first group of specimens was frozen and used for the staining of collagen fibers by sirius red F3Ba and for immunohistochemistry with antibodies against CD8, CD4, CD22, CD68, and TIA-1 molecules. The second group was used for organ culture, zymography, Western blotting, and dot blotting. Morphometric and automated image analysis was performed for the evaluation of the area fraction occupied by collagen fibers, the number of inflammatory cell subsets and for enzymatic activities developed by MMPs, and the amounts of TIMPs expressed during periodontal disease. RESULTS: In group P, the area fraction of collagen fibers (33 +/- 10%) was significantly decreased (P < 0.0002) when compared to group C (60 +/- 7%), and was correlated with the number of all inflammatory cells and amounts of MMPs and TIMPs. In group P, there were significant increases of CD8+, CD22+, CD68+, and TIA-1+ cells, as well as increases in the amounts of MMP-1, MMP-2, MMP-3, MMP-9, and the active form of MMP-9. The active form of MMP-9 and the amount of TIMP-1 were positively correlated with the number of CD22+, CD68+, and TIA-1+ cells. CONCLUSIONS: The present study showed an imbalance between MMPs and TIMPs associated with the pathologic breakdown of the extracellular matrix during periodontitis. The active form of MMP-9 could be a marker for the clinical severity of periodontal disease.

Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth.

BACKGROUND: The control of human hair follicle growth and differentiation is dependent upon several well-identified factors, including androgens, cytokines, and growth factors. In humans, alopecia androgenetica is a common aging process thought to be regulated through complex genetic imbalances, which also involve several of these crucial identified factors (and probably others not yet characterized), alone or in combination. Among these factors, epidermal growth factor (EGF), as well as pro-inflammatory cytokines, play a pivotal role, as evidenced by their direct inhibitory effects on hair growth both in vitro and in vivo. Following such treatments, the in vitro growth of hair follicles was rapidly arrested and deleterious modifications of hair morphology were also observed. AIM: Because these cytokines act, at least partly, through the induction of matrix metalloproteinases (MMP), and because tissue remodeling occurs during the hair cycle, we attempted to identify and localize MMP in the human pilosebaceous unit. METHOD: We used zymography to observe human hair follicles in culture in vitro. RESULTS: We observed that human hair follicles in culture in vitro mainly and almost exclusively produce MMP-2 and MMP-9 gelatinolytic activities. Furthermore, after stimulation with EGF, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1alpha (IL-1alpha), MMP-9 production was strongly increased. Using immunohistochemistry, we then precisely localized MMP-9 in the lower part of the inner root sheath (Henle's layer) of control human anagen hair follicles. CONCLUSIONS: Cytokine- and EGF-induced upregulation of MMP-9 in the lower epithelial compartment of the human hair bulb is a major mechanism through which hair follicle involution, observed in alopecia, may occur.

Collagen fibers and inflammatory cells in healthy and diseased human gingival tissues: a comparative and quantitative study by immunohistochemistry and automated image analysis.

BACKGROUND: Periodontal disease is histologically characterized by the degradation of extracellular matrix components associated with a gingival infiltration of inflammatory cell populations. The purpose of this in situ study was to quantify inflammatory cell subsets and the area fraction (AA%) occupied by collagen fibers in healthy and diseased upper gingival connective tissue in order to investigate the association, if any, between collagen loss and inflammatory cell infiltrate. METHODS: Paraffin gingival tissue sections from 10 healthy controls (C), 9 patients with gingivitis (G), and 10 patients with severe chronic periodontitis (P) were immunohistochemically stained by antibodies against CD45, CD3, CD8, CD20, CD68, TIA-1, and GrB molecules, and the collagen fibers were stained using sirius red F3Ba. The quantitative evaluations of inflammatory cell numbers and the AA% occupied by collagen fibers were performed by morphometric and automated image analysis. RESULTS: In group P, CD45+, CD20+, CD68+, TIA-1+, and GrB+ cell numbers were significantly increased (P<0.05) when compared to both C and G groups. The present study revealed significant differences (P <0.01) between means of AA% observed in group C (63%), group G (46%), and group P (26%), and AA% of group G and group P was inversely correlated with the numbers of TIA-1+ cells (P<0.01) and GrB+ cells (P<0.01 and P<0.05, respectively). CONCLUSIONS: This study showed great differences in the number of the distinct inflammatory cell subsets according to the severity of the periodontal disease and suggested that activated cytotoxic cells could play a pivotal role in the loss of collagen fibers observed during these pathological states. During periodontitis, collagen loss was significantly correlated with all inflammatory cell subset numbers. Finally, the quantitative evaluation of the area fraction occupied by gingival collagen fibers may reflect the clinical severity of the periodontal disease.

Immunohistologic and morphometric analysis of cytotoxic T lymphocytes in gingivitis.

BACKGROUND: Gingivitis is an inflammatory phenomenon localized in gingival tissues and histologically characterized by an infiltration of several inflammatory cell populations. The purpose of this study was to characterize, localize, and quantify in situ inflammatory and cytotoxic T lymphocytes using immunolabeled gingival tissue sections in order to specify their implication during human gingivitis, since it is well known that such cells play an important role in the defense against bacterial elements. METHODS: Paraffin gingival tissue sections from 7 patients with gingivitis (G) and from 7 clinically and histologically healthy controls (C) were immunohistochemically stained by specific antibodies (anti-CD45, anti-CD3, anti-CD8, anti-CD20, anti-TIA-1, anti-GrB, and anti-CD68), allowing the quantification of inflammatory cells in upper gingival epithelium (Ep), in the basal epithelium layer (BEp), and in upper connective tissue (CT). Collagen fibers were stained by sirius red F3Ba in order to evaluate, by morphometric and automated image analysis, the surface occupied by collagen bundles and to histologically confirm the absence of pathology of the clinically selected healthy controls. RESULTS: In the gingivitis group, CD45+, CD3+, CD8+, TIA-1+, and GrB+ lymphocyte numbers were significantly increased in Ep (P<0.05); and CD45+, CD3+, and TIA-I+ lymphocyte numbers were significantly increased in BEp (P <0.05) compared respectively to Ep and BEp of group C. In Ep of group G, mean CD8+/CD3+ cell ratio was significantly increased (P<0.05) compared to BEp and CT, and 25% of TIA-1+ cytotoxic cells were activated GrB+ cells. CONCLUSIONS: The present study suggests that intraepithelial cytotoxic T lymphocytes play an important role during gingivitis and CD8 expression and that activation of TIA-1+ cytotoxic cells could be induced in Ep in response to epithelial environment. Thus, gingival epithelial tissue, which is generally only considered as a physical barrier, in fact contains numerous immune cell populations preventing the infiltration of pathogenic elements into the connective tissue. Particular clinical attention must be taken for the preservation of the epithelial tissue integrity.

Increased fibroblast elastase activity in acquired cutis laxa.

BACKGROUND: Acquired cutis laxa is a rare disease characterized by sagging skin, premature wrinkling and reduced skin elasticity. OBSERVATION: We report a 21-year-old woman, who presented with acquired cutis laxa on the face and the ear lobes. Urticarial papules had preceded for 6 years. There was no systemic involvement. Skin specimens were obtained from lax skin and urticarial papules, and from healthy controls. Histology showed only few perivascular lymphocytes in lax ear skin and a dense inflammatory infiltrate in urticarial skin. In both biopsies elastic fibres were decreased as demonstrated by computerized morphometric analyses. Elastase activities of fibroblasts in culture were evaluated. There was a 2- to 3-fold increase in elastase activity in urticarial skin fibroblasts, contrasting with a normal elastase activity in lax ear skin. CONCLUSION: Our findings suggest that the inflammatory cells could play a significant role in the destruction of elastic fibres.

[Congenital generalized cutis laxa: 5 cases]. / Cutis laxa généralisée congénitale: 5 cas.

BACKGROUND: Congenital cutis laxa is an exceptional condition. No large scale series has been reported in the French literature. We report 5 cases observed between 1993 and 1997. PATIENTS AND METHODS: Five children with a morphotype compatible with congenital generalized cutis laxa were examined. A family study, complete visceral workup and skin biopsy with standard histology, orceine coloration and histomorphometric analysis of the collagen and elastic fibers of the dermis were performed. Karyotype and copper metabolism (cupremia and ceruloplasminemia) were available in 3 children. RESULTS: The diagnosis was clinical and proven histologically by orceine coloration of skin biopsies in all cases. There were discrete ultrastructure anomalies in the pure cutaneous form expressed in case n(o) 1 with possible autosomal dominant inheritance. Cupremia and ceruloplasminemia were normal in the 3 children explored; this corresponds to absence of the Elhers-Danlos type IX phenotype. The karyotype was normal in 3/3 children, in agreement with the absence in these three children of marfanoid cutis laxa phenotype. Patients n(o) 2, 3, 4 and 5 had common features: probable autosomal recessive inheritance and severe prognosis. Patient n(o) 2 died at the age of 3 weeks and had severe pulmonary emphysema. This child's sister also had cutis laxa but with no visceral component (autosomal recessive inheritance with variable expression). Patients n(o) 3, 4 and 5 had a severe multiple malformative syndrome with facial dysmorphism, growth retardation, unexplained digestive disorders and psychomotor retardation. DISCUSSION: Our series of 5 patients and data in the literature confirm that primary cutis laxa is a heterogeneous group of conditions both clinically and genetically. The anomalies associated in patients n(o) 3, 4 and 5 were not directly related to anomalous elastic tissue as was also the case for the craniostenosis in patient n(o) 3 reported in other cases in the literature.

Congenital soft tissue dysplasias: a morphological and biochemical study.

The term congenital soft tissue dysplasias (CSTDs) regroups some localized malformations of covering soft tissues in children, presenting as various clinical entities, either recognized as particular syndromes (e.g., Parkes-Weber, Klippel-Trenaunay, Proteus) or, most often, appearing less stereotyped (e.g., segmental hypertrophy or gigantism, lymphedema, angiodysplasia, phakomatosis), with a common histopathological lesion, the hamartoma. The aim of this paper is to report a morphological and biochemical study of the extracellular matrix of skin and subcutaneous tissue in children with CSTD. For every patient, pathological tissues were compared with contralateral, symmetrical tissues, taken as controls. In all CSTDs, pathological samples were characterized by an increase in water and total glycosaminoglycan (GAG) content with a decrease in collagen content. Other results lead the authors to distinguish two main entities, segmental dysplasia (SeD) and neuroectodermal dysplasia (NeD). Elastic fiber content was increased in SeD and decreased in NeD. Hyaluronic acid (HA) and dermatan sulfate (DS) were increased in NeD, whereas in SeD, HA was decreased with an increase in the DS/HA ratio. Cultured fibroblasts from dysplastic skin had slower proliferation in vitro than fibroblasts from control skin, whereas their biosynthetic activity concerning collagen and GAGs was greater. The difference in the composition of extracellular matrix supports the clinical classification of CSTDs in two main groups: segmental dysplasia with or without gigantism and neuroectodermal dysplasia (in von Recklinghausen's disease and nevi).

Presence of gelatinase A and metalloelastase type protease at the plasma membrane of human skin fibroblasts. Influence of cytokines and growth factors on cell-associated metalloendopeptidase levels.

Gelatinase A and elastase type proteinase (Homsy, et al., 1988) present at plasma membranes of human skin fibroblasts (HSF) were separated by anion exchange chromatography on a DEAE Tris acryl M column. Elastase type proteinase (HSFE1) was able to convert 72 kDa progelatinase A to a lower 66 kDa M.W. active enzyme. Several cytokines (IL-1 beta, IL4, IL6), interferon gamma (IFN gamma) and tumor growth factor beta (TGF-beta) were studied for their ability to modify the levels of those plasma membrane associated proteinases. Among these mediators, only IL-1 beta was found to enhance the amounts of HSF membrane-bound HSFE1 and Gelatinase A.

Protective effect of oleoyl peptide conjugates against elastolysis by neutrophil elastase and kappa elastin-induced monocyte chemotaxis.

Elastin can impair the human neutrophil elastase (HNE) inhibitory capacity of elastase inhibitors. We synthesized oleoyl-alanyl-alanyl-prolyl-valine (Ol-Ala-Ala-Pro-Val-OH) (oleoyl peptide) and the amides (NH2 and NH-C3H7) of this peptide and studied their HNE-inhibitory potencies using succinyl-alanyl-alanyl-alanine-p-nitroanilide (Suc-Ala-Ala-Ala-pNA) or 3H-labeled elastin as substrates, as well as cryostat sections of rabbit skin as an ex vivo substrate. Using Suc-Ala-Ala-Ala-pNA, Ol-Ala-Ala-Pro-Val-OH had an IC50 of 3 microM. When the COOH terminal of the oleoyl peptide was derivatized to amide forms, the compound lost its ability to interact with HNE while keeping its elastin-protecting function: IC50 values for NH2 and NH-C3H7 derivatives were 22 and 17 microM, respectively. Also, the HNE-inhibitory capacity of Ol-Ala-Ala-Pro-Val-OH was only reduced 2-fold by using elastin as a substrate. This decrease was much lower than those determined with other HNE inhibitors of similar potency and could be accounted for by the ability of oleoyl peptide to bind to elastin. Cryostat sections of rabbit skin were also used as an ex vivo substrate for assessing the elastin-protecting property of Ol-Ala-Ala-Pro-Val-OH. Preincubating HNE and oleoyl peptide before application to tissue sections led to an IC50 of 8 microM, close to the value determined with elastin as a substrate. Treatment of sections with oleoyl peptide before adding HNE gave a lower IC50 (4 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

The consequences of aortic calcium overload following vitamin D3 plus nicotine treatment in young rats.

Treatment of young rats with vitamin D3 plus nicotine has been proposed as a model of cardiovascular calcium overload. This treatment produced a 20-35-fold increase in the calcium content of the aorta, a compliance vessel, and this increase was accompanied by a 1.6-fold elevation of pulse pressure. In aortic rings, the maximal inhibition by the endothelium-dependent vasodilator, carbachol, of vasoconstriction induced by noradrenaline decreased from 90% in controls to 61% in treated animals. There were significant correlations between aortic calcium content and pulse pressure and aortic calcium content and carbachol-induced relaxation. In conclusion, the vitamin D3 plus nicotine model may be useful for the study of the role of calcium overload in decreased arterial compliance coupled with endothelial injury.

Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils.

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.

Evolution of the elastic fiber network of the human uterine cervix before, during and after pregnancy. A quantitative evaluation by automated image analysis.

To assess the evolution of the elastic fiber network of the human uterine cervix before, during and after pregnancy, biopsy samples were obtained from 49 women. The high affinity of the polyphenolic compound (+)-catechin for elastin was used to stain the elastic fibers selectively, and enabled automated image analysis. In the human uterine cervix, the elastic fiber network is made up of: (1) fibers running parallel to the basement lamina of the epithelium, and (2) thinner, perpendicular fibers. Quantification using automated image analysis shows a decline in the cervical elastin content from a prepregnancy level of 1.33 +/- 0.08 (SEM) to 0.73 +/- 0.09% (Vv) at the end of pregnancy. In parallel with a constant decline, dissociation and disorganization of the fibers become more clearly evident as pregnancy progresses. However, by 5-7 weeks postpartum the elastic fiber network appears almost completely restructured. These changes support a role of elastin in the processes of cervical maturation and reconstruction during pregnancy and after delivery.

A selective histochemical method for the quantitative estimation of elastic fibers by computerized morphometric analysis. Effect of colchicin treatment.

A morphometric technique is reported that uses a new selective staining of the elastic system fibers in skin biopsy specimens to facilitate the quantitative evaluation of the volume fraction occupied by these elastic fibers in the tissue. The study of elastic fibers in the dermis of 30 patients, before and after six months of treatment with Colchicin, was carried out with a Quantimet 720 system. Preelastic (oxytalan and elaunin) fibers and mature elastic fibers were quantitated separately. Compared to the average volume fraction (surface occupied by the elastic fibers) before treatment with Colchicin (1.449 +/- 0.64%), the mean values after treatment were significantly increased (2.076 +/- 0.61%). The same results were found for the preelastic fibers: 0.807 +/- 0.51% before treatment and 1.025 +/- 0.54% after treatment. These results demonstrate the advantages of our monochromatic staining method for automatic quantitation of elastic fibers as well as the possibilities of the quantitative study of the elastic fibers in human dermis. This methodology should be applicable to other inherited or acquired diseases affecting skin elastic fibers as well as to other tissues containing elastic fibers.

Selective staining technique for identification of human skin elastic fibers.

The staining methods proposed for the histological detection of elastic fibers are not entirely selective for the elastic fiber system and stain other tissue components also as for example nuclei and collagenous components. We worked out several staining methods which enable us to obtain a selective staining of all the elastic and pre-elastic fibers with no staining of the background by using in the staining solution polyphenols like: pyrogallol, (+) catechin, tannic acid and others. As the background is colourless, our method is suitable for the quantitation of elastic system fibers by automated computerized image analysis.