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PubMed2001

Cell reactions with biomaterials: the microscopies.

The methods and results of optical microscopy that can be used to observe cell reactions to biomaterials are Interference Reflection Microscopy (IRM), Total Internal Reflection Fluorescence Microscopy (TIRFM), Surface Plasmon Resonance Microscopy (SPRM) and Förster Resonance Energy Transfer Microscopy (FRETM) and Standing Wave Fluorescence Microscopy. The last three are new developments, which have not yet been fully perfected. TIRFM and SPRM are evanescent wave methods. The physics of these methods depend upon optical phenomena at interfaces. All these methods give information on the dimensions of the gap between cell and the substratum to which it is adhering and thus are especially suited to work with biomaterials. IRM and FRETM can be used on opaque surfaces though image interpretation is especially difficult for IRM on a reflecting opaque surface. These methods are compared with several electron microscopical methods for studying cell adhesion to substrata. These methods all yield fairly consistent results and show that the cell to substratum distance on many materials is in the range 5 to 30 nm. The area of contact relative to the total projected area of the cell may vary from a few per cent to close to 100% depending on the cell type and substratum. These methods show that those discrete contact areas well known as focal contacts are frequently present. The results of FRETM suggest that the separation from the substratum even in a focal contact is about 5 nm.

Journal Article↗
PubMed1985

Diagnostic value of clinical examination, direct microscopy, and culture in the Gardnerella vaginalis syndrome.

In a general practice 467 women aged 15-49 years with vaginal discharge were consecutively examined in order to compare the diagnostic value in the Gardnerella vaginalis syndrome of the clinical examination, direct microscopy of the vaginal secretion, and culture. We found significant correlations between all relevant combinations of the diagnostic methods. The predictive value of a positive test for the clinical examination, the microscopy, and the clinical examination combined with microscopy, respectively, was 0.75, 0.89 and 0.90, whereas the predictive values of the negative test were 0.59, 0.72 and 0.61, respectively. Because even small numbers of Gardnerella vaginalis may yield growth on selective culture media, the clinical examination and the microscopy are often negative despite positive culture. To avoid overtreatment of a benign condition we therefore conclude that the presence of a characteristic clinical picture and positive microscopy constitute a safe basis for the diagnosis of the Gardnerella vaginalis syndrome, and that the diagnosis should be based on this combination or on the fulfilment of three of the following four criteria: 1) characteristic vaginal secretion; 2) vaginal pH greater than 4.5; 3) positive potassium hydroxide test or characteristic herring-brine smell, and 4) clue cells at microscopy. With the present methods culture for Gardnerella vaginalis should not routinely be performed, until the value of quantitated methods has been proved. Coexistence of other microorganisms rendered the diagnosis difficult. We recommend to treat the most predominant infection first.

Adolescent↗
PubMedDate not supplied

Effect of providing free sputum microscopy service to private practitioners on case notification to National Tuberculosis Control Program.

BACKGROUND: This study was undertaken to see whether providing free sputum microscopy services to private practitioners helps in case notification to the national tuberculosis control program. The knowledge, attitudes and practices of these practitioners regarding tuberculosis were also evaluated. METHODS: A questionnaire was administered to all the private practitioners practicing in a densely populated area of Karachi. They were asked to fill tuberculosis notification cards for the first three months and then for another three months when an incentive in the form of free sputum microscopy was provided to the practitioners. RESULTS: Although the majority of the practitioners knew that cough, fever and weight loss are the main symptoms of tuberculosis, less than half knew that blood in sputum, poor appetite and chest pain could also be associated with tuberculosis. Only 66% of the practitioners indicated sputum microscopy as the preferred diagnostic method for tuberculosis. Only 50% of the practitioners self treated the patients, while the remaining half referred their patients to specialists. Around 80% of the practitioners were aware of the four first-line anti-tuberculosis drugs. Less than half of the practitioners considered sputum microscopy as the most useful follow-up investigation in a patient with pulmonary tuberculosis. Generally, there was a poor response in case notification by private practitioners on provision of free sputum microscopy. CONCLUSION: An overwhelming majority of the practitioners had poor knowledge concerning the correct treatment practices in Tuberculosis. Providing sputum free microscopy does not significantly help in improving tuberculosis case notification. Strategies for public-private collaboration in tuberculosis control are needed.

Clinical Competence↗
PubMed1994

Body fluid microscopy in US laboratories. Data from two College of American Pathologists surveys, with practice recommendations.

Questionnaires addressing laboratory practices in cerebrospinal fluid and body fluid (serous, synovial) microscopy were distributed to participants in the Clinical Microscopy Survey of the College of American Pathologists, Northfield, Ill, in 1985 and 1989. In both Surveys, cell enumeration was performed primarily by hemocytometry, while nearly all respondents used Wright-stained microscopy. There was little formal quality control to assess the accuracy of counts or differential cell count. Less than 55% of laboratories used the cytocentrifuge. About half of respondents performed a differential cell count on every sample. Slides with atypical or malignant cells were usually (> 85%) reviewed by physicians without automatic referral to the cytopathology section. Only about half of respondents examined every synovial fluid specimen with polarized microscopy for crystals. Other than a modest increase in use of the cytocentrifuge, the 1989 Survey showed little interval change in practices. In 1989, there was equal dependence on Wright's and Papanicolaou's stains for an infrequent diagnosis of malignancy. The low rate of positives may have related to the high prevalence of wedge smears, a suboptimal technique. The Hematology and Clinical Microscopy Resource Committee of the College of American Pathologists makes recommendations for optimal laboratory handling of body fluid specimens for microscopy.

Body Fluids↗
PubMed1998

Scanning acoustic microscopy study of titanium-ceramic interface of dental restorations.

Failures that occur in titanium-ceramic restorations are of concern in clinical dentistry. The purpose of this study was to nondestructively characterize the internal cracks and nonadherent defects at the titanium-porcelain interface using scanning acoustic microscopy. Titanium samples coated with porcelain without a bonding agent, with sputter coated palladium or chromium as an oxygen diffusion barrier on the titanium, and with the use of a porcelain bonding agent (control group) were compared. The scanning acoustic microscopy analyses were correlated with four-point bending test results. The group that was initially coated with palladium had fewer interfacial defects and a higher load to failure than the control group, and the group that did not contain the bonding agent had a higher void area and a lower load to failure than the control group. The use of chromium produced no differences from the control group. Samples after a four-point bending test were also analyzed by scanning electron microscopy. The scanning electron microscopy was not able to characterize interfacial defects at the fractured titanium-ceramic interface for some of the samples. The validity of nondestructive analysis at the Ti-ceramic interface using scanning acoustic microscopy was demonstrated in this study.

Chromium↗
PubMed1994

Comparison of phase-contrast and fluorescence digital autofocus for scanning microscopy.

Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields. Autofocus could be performed directly with fluorescence images, but due to photobleaching and destructive fluorescence by-products, it is best to minimize fluorescence exposure for photosensitive specimens and live cells. This exposure problem could be completely avoided by using phase-contrast microscopy, implemented through the same optics as fluorescence microscopy. The purpose of this work was to evaluate functions for both phase-contrast and fluorescence autofocus and determine the suitability of phase-contrast autofocus for fluorescence microscopy. Eleven autofocus functions were independently evaluated for fluorescence and phase-contrast microscopy. The most suitable functions were then chosen from these and phase-contrast and fluorescence autofocus were compared on scans each comprising more than 1,000 microscope fields. Autofocus standard deviation (S.D.) of better than 100 nm was achieved for both phase contrast and fluorescence. There was a measurable difference between the best focus positions in the two modes, but the difference was constant enough to be measured and corrected, suggesting the possibility of using phase contrast to predict best focus in fluorescence microscopy. The scanning experiments also showed that autofocus can be performed at least as fast as 0.25 s/field without loss of precision.

3T3 Cells↗
PubMed2006

Spectral mapping tools from the earth sciences applied to spectral microscopy data.

BACKGROUND: Spectral imaging, originating from the field of earth remote sensing, is a powerful tool that is being increasingly used in a wide variety of applications for material identification. Several workers have used techniques like linear spectral unmixing (LSU) to discriminate materials in images derived from spectral microscopy. However, many spectral analysis algorithms rely on assumptions that are often violated in microscopy applications. This study explores algorithms originally developed as improvements on early earth imaging techniques that can be easily translated for use with spectral microscopy. METHODS: To best demonstrate the application of earth remote sensing spectral analysis tools to spectral microscopy data, earth imaging software was used to analyze data acquired with a Leica confocal microscope with mechanical spectral scanning. For this study, spectral training signatures (often referred to as endmembers) were selected with the ENVI (ITT Visual Information Solutions, Boulder, CO) "spectral hourglass" processing flow, a series of tools that use the spectrally over-determined nature of hyperspectral data to find the most spectrally pure (or spectrally unique) pixels within the data set. This set of endmember signatures was then used in the full range of mapping algorithms available in ENVI to determine locations, and in some cases subpixel abundances of endmembers. RESULTS: Mapping and abundance images showed a broad agreement between the spectral analysis algorithms, supported through visual assessment of output classification images and through statistical analysis of the distribution of pixels within each endmember class. CONCLUSIONS: The powerful spectral analysis algorithms available in COTS software, the result of decades of research in earth imaging, are easily translated to new sources of spectral data. Although the scale between earth imagery and spectral microscopy is radically different, the problem is the same: mapping material locations and abundances based on unique spectral signatures.

Algorithms↗
PubMed1988

Aspiration cytology of neuroblastoma: light microscopy with transmission and scanning electron microscopic correlations.

Fine-needle aspiration biopsies from three patients with neuroblastoma were studied by light microscopy, and the morphologic findings were correlated with those from transmission and scanning electron microscopy. Light microscopic examination of the aspiration smears from all three cases revealed small and large round cells with variable numbers of intertwining cytoplasmic processes. Transmission electron microscopy confirmed the light microscopic finding of cytoplasmic processes; in addition, it revealed the presence of other diagnostic morphologic features, including neurosecretory granules, microtubules, and synaptic cell junctions. Scanning electron microscopy demonstrated that the tumors were composed of a mixture of undifferentiated round cells and more differentiated cells with long cytoplasmic processes. The morphologic spectrum of these processes and their interrelationships with one another and with other cells could be studied in detail. These findings indicate that scanning electron microscopy may be used effectively in the morphologic evaluation and pathologic diagnosis of neuroblastoma.

Biopsy, Needle↗
PubMed1988

Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film.

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.

Animals↗
PubMed1997

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation.

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scanning electron microscopy. The knees were loaded at the patella with a simulated "quadriceps force" of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freeze-substitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as x 30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating a tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.

Animals↗
PubMed2001

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.

Adsorption↗
PubMed2001

The contributions of electron microscopy to the understanding and diagnosis of plasma cell dyscrasia-related renal lesions.

Electron microscopy has been crucial in the definition of many renal diseases. Ultrastructural evaluation has been instrumental in the characterization of many of the morphological manifestations of plasma cell dyscrasia-related renal lesions. Although it is recognized that there is controversy in regards to what the term multiple myeloma specifically refers, for the purposes of this article, myeloma and plasma cell dyscrasia are used interchangeably without consideration to perhaps significant conceptual differences that may exist between the two. Although distal nephron obstructive nephropathy ("myeloma kidney") was rather accurately defined solely on the basis of light microscopic findings and the association of AL-amyloidosis with underlying myeloma was confirmed using histochemical stains, the more subtle and intricate expressions of plasma cell dyscrasia-associated pathology required the electron microscope for proper characterization. The fibrillary nature of amyloid was discerned ultrastructurally, and detecting its characteristic ultrastructural features remains paramount to make a definitive diagnosis of amyloidosis when evaluating difficult cases. Pristine light- and heavy-chain deposits were objectively confirmed as immunomorphological correlates were depicted by immunofluorescence and at the ultrastructural level, substantiating the findings. Tubular interstitial alterations in these disorders other than cast nephropathy were firmly documented when careful ultrastructural studies were conducted experimentally and using clinical material. Likewise, electron microscopy has also played an important role in assessing vascular pathology in these conditions, especially when changes are focal, segmental, subtle, or such that they are easily confused with other pathological entities by light microscopy. Had it not been for the electron microscope, a clear definition of the immunomorphological scope of plasma cell dyscrasia-associated lesions, as understood currently, would not have been possible. Immunoelectron microscopy has provided a welcome added dimension, allowing a thorough characterization by expanding merely morphological data and providing exquisite immunomorphological correlations. This review highlights the role that electron microscopy has played and continues playing in the characterization of plasma cell dyscrasias-related renal lesions.

Amyloidosis↗
PubMed1990

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line.

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

Adenocarcinoma↗
PubMed1996

Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots.

Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations.

Biomechanical Phenomena↗
PubMed1998

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Evidence for aggregation in oxalate stone formation: atomic force and low voltage scanning electron microscopy.

PURPOSE: The aim of this investigation was to differentiate between aggregation and crystal growth by studying the structure of oxalate stones at high spatial resolution using recently developed microscopy techniques. MATERIALS AND METHODS: Sections from 6 complete human oxalate stones and 4 stone fragments were prepared by ultramicrotomy and examined by both low voltage scanning electron microscopy and atomic force microscopy. RESULTS: The scanning electron microscopy showed lamellar structures up to 10 microns. in size, consistent with previous results, and provided evidence that these structures were composed of smaller particles. The atomic force microscopy clearly showed arrays of the small particles, whose size varied between 500A and 2800A. CONCLUSION: Our images suggest that an ordered aggregation of small crystallites is responsible for oxalate stone formation.

Humans↗
PubMed1997

Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry.

Spermiogenesis and the spermatozoon of Echinostoma caproni (from experimentally infested laboratory mice) were investigated by several methods. Transmission electron microscopy shows that spermiogenesis consists of proximo-distal fusion of three processes followed by elongation of the spermatid. Scanning electron microscopy shows that the spermatozoon is a filiform cell, 235 microns in length, with a cylindrical anterior extremity and a broader posterior extremity. Epifluorescence microscopy, including immunocytochemistry of tubulin and labelling of nucleus with specific dyes, has provided valuable additional information. Migration of the nuclei from the common cytoplasmic mass of spermatids to the distal part of the elongating spermatids is visualized, and centrioles demonstrated in the proximal, anterior region, and the nucleus in the distal, posterior region of the spermatozoon. One axoneme has a distal extremity which in the mature spermatozoon extends 30 microns more distally than the other, with the result that the posterior part of the spermatozoon contains a single axoneme and nucleus. Immunocytochemistry experiments show that a region, 15 microns in length, not labelled by the anti-tubulin antibodies with certain fixation-permeabilization procedures, corresponds to a region which, by transmission electron microscopy, shows external ornamentation on the membrane. This region has a bilaterally asymmetric pattern (in TEM), forms angles or coils according to the fixation used, and marks the boundary between two distinct patterns of movement. Spermiogenesis and the spermatozoon in E. caproni correspond to the general pattern found in the digeneans, with the exception of this asymmetric region. It is emphasized that the use of various methods provides a better understanding of sperm structure than transmission electron microscopy alone, particularly in the case of long, filiform spermatozoa.

Animals↗
PubMed2000

Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded onto a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopy study.

Hyaline cartilage has only a limited capacity of regeneration, thus, lesions of articular cartilage can lead to early osteoarthrosis. Current concepts in conservative orthopedic therapy do not always lead to satisfying results. As one new attempt to facilitate cartilage repair, autologous transplantation of articular chondrocytes is investigated in different assays. This study was designed to create a resistible and stable cell-matrix-biocomposite with viable and biosynthetically active human chondrocytes, osteoblasts or fibroblasts. This biocomposite might serve as an implant to treat deep osteochondral defects in the knee. We collected cartilage, spongiosa and skin probes from healthy patients undergoing hip-surgery and enzymatically liberated the chondrocytes, seeded them into culture flasks and cultured them until confluent. The spongiosa and the skin samples were also placed in culture flasks and cells cultured until confluent. After 4-6 weeks, cells were trypsinized and grown on a type I/III collagen matrix (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland) for 7 days in standard Petri dishes and in a special perfusion chamber culture system. As controls, cells were seeded onto plastic surfaces. Then scaffolds were fixed and embedded for light microscopy and electron microscopy by routine methods. Light microscopically, chondrocytes grown on the surface of the scaffold form clusters or a dense layer of sometimes rather fibroblast-like and sometimes roundish, chondrocyte-like cells. Only a few cells grow deeper into the matrix. In transmission electron microscopy, the cells have a rather chondrocyte-like morphology which emphasizes the matrix-induced redifferentiation after dedifferentiation of chondrocytes in monolayer-culture in culture flasks. Chondrocytes on plastic surfaces have a spinocellular aspect with little signs of differentiation. Grown on Chondrogide, cells are more roundish and adhere firmly to the collagen fibrils of the scaffold. Osteoblasts grown on the collagen scaffold and examined by light microscopy form a thin cell-layer on the surface of the matrix with a reticular layer of dendritic cells underneath this sheet. Transmission electron micrographs show spinocellular and flat cells on the collagen fibrils. Scanning electron micrographs show large dendritic osteoblasts on plastic and a confluent layer of flattened, dendritic cells on the collagen scaffold. Fibroblasts form a thick multi-layer of typical spinocellular cells on the collagen matrix. Fibroblasts grown on plastic surfaces and examined by scanning electron microscopy also show a dense layer of fibroblast-like cells. For all three different types of cells no morphological differences could be seen when comparing cultivation in the perfusion culture system to cultivation in standard Petri dishes, although mechanical stress is believed to induce differentiation of chondrocytes. Especially the observed partially differentiated chondrocyte-matrix biocomposite might serve as an implant to treat deep cartilage defects, whereas osteoblasts and fibroblasts seem to be less suited.

Aged↗