Biomedical subjects

H K Koerten

Publications and source records attributed to H K Koerten.

At least 37 records · Page 2Linked to original sources

PubMed1999

Bilayered biodegradable poly(ethylene glycol)/poly(butylene terephthalate) copolymer (Polyactive) as substrate for human fibroblasts and keratinocytes.

The purpose of this study was to find an optimal polymer matrix and to optimize the culture conditions for human keratinocytes and fibroblasts for the development of a human skin substitute. For this purpose porous, dense bilayers made of a block copolymer of poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT; Polyactivetrade mark) with a PEGT/PBT weight ratio of 55/45 and a PEG molecular weight (MW) of 300, 600, 1000, or 4000 Da were used. The best performance was achieved with PEGT/PBT copolymer with MW of PEG 300 D (300PEG55PBT45). When fibroblasts were seeded into the porous underlayer and cultured for 3 weeks in medium supplemented with 100 microg/mL ascorbic acid, all pores were filled with fibroblasts and with extracellular matrix, which was judged from the presence of collagen types I, III, and IV, and laminin. When seeded onto the dense top layer of the bilayered (cell free or fibroblast populated) copolymer matrix, human keratinocytes grew out into confluent sheets. After subsequent lifting to the air-liquid interface, a multilayered epithelium with a morphology corresponding to that of the native epidermis was formed. Some differences could still be observed: the expression and localization of some differentiation specific proteins was different and close to that seen in hyperproliferative epidermis; a basal lamina and anchoring zone were absent.

Biocompatible Materials↗

PubMed1999

The effect of methylated beta-cyclodextrins on the tight junctions of the rat nasal respiratory epithelium: electron microscopic and confocal laser scanning microscopic visualization studies.

The nasal absorption enhancer randomly methylated beta-cyclodextrin (RAMEB) is thought to increase the paracellular permeability of the nasal epithelium by opening of the tight junctions. The effects of RAMEB on the cytoskeleton of the rat nasal epithelium in vivo were determined by confocal laser scanning microscopy (CLSM). The effects on the tight junctions of the rat nasal epithelium were also investigated, using transmission electron microscopy (TEM) of thin sections. The effects of RAMEB were compared with those of the absorption enhancer sodium taurodihydrofusidate (STDHF). Fifteen minutes after nasal administration of 2% RAMEB in vivo, the distribution of cytoskeletal actin was comparable to the untreated control, suggesting that RAMEB does not cause opening of the tight junctions via cytoskeletal interactions. In contrast, administration of 1% STDHF resulted in changes in the actin staining. Furthermore, with TEM severe damage of the nasal epithelium was observed after treatment with 1% STDHF. Ultrastructural changes of the tight junctions were not apparent in TEM sections after treatment with 2% RAMEB. In conclusion, CLSM and TEM are suitable methods to visualize the effects of absorption enhancers on nasal epithelial morphology.

Actins↗

PubMed1999

Degradation of calcium phosphate ceramics.

Degradation of three types of sintered calcium phosphate ceramic spheres was investigated in vitro at low pH conditions (LPC) and in an in vivo model, that is, injection into a mouse peritoneal cavity. Degradation was observed under both conditions. The rate of degradation depended on the type of ceramic, with beta-TCP degrading faster than HA and HA degrading faster than FA. Degradation was characterized by dissolution of the necks and the formation of cracks and irregularities in the grains. Intraperitoneal injection of the spheres into a mouse peritoneal cavity led to the formation of foreign body granulomas in which degradation could be observed. The in vivo degradation pattern was similar to that observed in vitro, but longer implantation times resulted in a further degradation. Small fragments rich in Ca and P were present in inclusion bodies. Calcium phosphate crystals sometimes also were observed in mitochondria, many of which were subject to lysis. We observed that ceramic type and implantation period also were related to the number of dead cells in the granulomas. Furthermore, extracellular deposits were seen between cells and ceramic spheres. Ca and P and also Fe were detected in these deposits. The presence of Fe is indicative of a lysosomal origin and thus of exocytotic activity.

Animals↗

PubMed1999

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.

Cryoultramicrotomy↗

PubMed1998

Efficacy of micronized titanium dioxide-containing compounds in protection against UVB-induced immunosuppression in humans in vivo.

Micronized pigment-containing sunscreens may provide a good alternative to chemical sunscreens in protection against ultraviolet (UV) B-induced immunosuppression. The metal particles in these products are likely to remain on the skin surface where they can offer broadband protection for both the UVA and UVB regions. We have tested the protective capacity of three titanium dioxide (TiO2)-containing compounds in humans in vivo. The effect on sunburn cell formation has been investigated using transmission electron microscopy, while the mixed epidermal cell lymphocyte reaction (MECLR) has been used as a model for immunosuppression. Furthermore, the influence of titanium on the integrity of the stratum corneum barrier (intercellular lipids and desmosomes) has been examined using freeze fracture electron microscopy. We find that all three compounds protect against sunburn cell formation. The immunoprotection studies show that one of the three compounds does not prevent UVB-induced changes of the MECLR responses. Application of this compound without subsequent UVB irradiation also induces a significant decrease of the MECLR responses. Moreover, the same compound affects the intercellular lipid layers, and desmosomes cannot be detected. The deleterious effect of this compound is probably caused by an incomplete hydrolysis during the TiO2 synthesis. Our findings indicate that micronized pigment-containing compounds can offer good protection against short-term UVB-induced immunomodulation in humans in vivo. However, accurate screening of the synthesis of these compounds is a prerequisite for their safe use as sunscreening agents in human subjects.

Freeze Fracturing↗

PubMed1998

Variations in melanin formation by cultured melanocytes from different skin types.

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Culture Media↗

PubMed1998

Some characteristics of hydroxylapatite powder particles after plasma spraying.

We have studied the particles of hydroxylapatite (HA) powder, the particles after plasma spraying, their distribution on substrate surface and their condition after transfer through the plasma torch. Mean particle size of HA powders was as follows: HA-A: 3.8 microm, HA-B 88.2 microm. The area of HA coating after plasma spraying, when the torch had a constant position against the substrate surface, shows two characteristic zones: the central part of coating formed mainly from deformed particles and the marginal part of coating with small non-deformed particles. These small non-deformed particles can be found in all zones of the coating and together with greater non-deformed particles and partially deformed particles will unfavourably affect the adhesive and cohesive strength of the coating and its porosity. The maximum diameter of the molten (spherical) particles in the conditions of Ar + H2 plasma, output P = 24 kW was: DA = 25 microm (HA-A) and DB = 65 microm (HA-B). The intervals of dimensions in which most of molten particles occurred were HA-A: 0-15 microm (98%), HA-B: 5-35 microm (84%). From comparison of HA-A and HA-B powders it can be concluded that the transport of HA-A powder was not continuous, the amount of molten HA-A particles was considerably greater (90%) than that of HA-B powder (63%). Phase decomposition and also solubility of HA-A powder (at in vitro tests) was greater. If we consider transport of particles, their melting, splitting and spraying efficiency, the suitable size of HA powder particles for the given spraying conditions is somewhere between the size of HA-A and HA-B particles--let us say--in the interval from 20 to 60 microm.

Biocompatible Materials↗

PubMed1998

The effect of biological environment on the surface of titanium and plasma-sprayed layer of hydroxylapatite.

The solubility of titanium samples with different surface coatings, i.e., hydroxylapatite (HA) powders, a two-layer coating of ZrO5+HA on a titanium substrate in solution and of tooth implants after long-term functioning in the human organism, was studied. A minimum difference in solubility of titanium samples with different surface finishes (polished or grit blasted) was established. For the HA powders and coatings, the lowest solubility was observed with a coarse-grained HA-B powder and a coating made of that powder. Clinical tests of tooth implants after long implantation times were performed. A titanium implant (implantation 12 y), a titanium implant with a two-layer coating of ZrO5+HA-A (implantation time 4 y) and a titanium implant with a two-layer coating of (Al5O3+3% TiO2)+HA-A (implantation time 6 y) were studied. The results show that the titanium surface and HA-A layers were dissolved. Nevertheless, after 6 y implantation, total removal of HA-A coating from that part of implant set into the bone, was not observed.

Journal Article↗

PubMed1998

Cryo-electron diffraction as a tool to study local variations in the lipid organization of human stratum corneum.

The human skin provides the body with a barrier against transepidermal water loss and the penetration of harmful agents (e.g. microbes) from outside. This barrier function is produced mainly by the outermost, nonviable layer of the epidermis, the stratum corneum (s.c.). The s.c. consists of terminally differentiated corneocytes surrounded by a continuous intercellular lipid domain, which contains mostly ceramides, cholesterol and free fatty acids. Small- and wide-angle X-ray diffraction studies have elucidated the lamellar and lateral lipid organizations in these domains. However, these techniques require bulk quantities of SC, as a result of which local structure information on the lipids cannot be obtained. Insights to these local lipid arrangements are important when new transdermal drug delivery systems have to be developed. Therefore, the technique of electron diffraction arose as a tool to study the lateral packing of the lipids in the intercellular domains of SC, locally. In a previous study, the suitability of electron diffraction was demonstrated using a lipid model system that resembled the lipid composition of the SC. The spacings calculated from the electron diffraction patterns were in good agreement with the spacings revealed by wide-angle X-ray diffraction. The results presented here succeed this previous study. We improved the microscope settings and developed a new preparation method to study ex vivo human s.c. by cryo-electron diffraction. The method is based on the conventional tape-stripping method and offers the possibility to study depth-related changes in the lipid organization of human SC. Diffraction patterns of both hexagonal and orthorhombic lipid lattices have been recorded with spacings that resembled those found in human s.c. by wide-angle X-ray diffraction. After lipid extraction, such diffraction patterns could no longer be detected in the samples.

Epidermis↗

PubMed1998

Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

Adult↗

PubMed1998

Myosin V colocalizes with melanosomes and subcortical actin bundles not associated with stress fibers in human epidermal melanocytes.

Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells myosin V is expressed and treatment of melanocytes with the cyclic AMP-inducer 3-isobutyl-1-methylxanthine causes an induction of the myosin V message. In all cells myosin V colocalizes with actin bundles, concentrating in the subcortical cell zone. In the melanocyte it is closely associated with melanosomes. Quantitative analysis of myosin V labeling in melanocytes reveals a significantly higher (p < 0.005) presence of myosin V in the periphery of dendrites. These results suggest that myosin V is important in melanosome transport in human melanocytes. Possible roles in the other skin cells remain to be elucidated.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Study on the lipid organization of stratum corneum lipid models by (cryo-) electron diffraction.

The barrier function of the skin resides in the stratum corneum (SC). This outermost layer consists of protein-rich corneocytes and lipid-rich intercellular domains. These domains form the rate-limiting step for transepidermal water loss and the penetration of substances from the environment. To study the nature of the barrier function, stratum corneum lipid models have been examined with wide-angle X-ray diffraction. A disadvantage of this technique is that it requires bulk quantities of lipid and thus information on variations in the lateral packing cannot be obtained in the microm-range. To the best of our knowledge, this is the first study in which electron diffraction is applied on SC lipid model systems. Using this technique, local structural information was obtained about mixtures prepared from isolated pig ceramides, cholesterol, and long-chain free fatty acids. It appeared that addition of free fatty acids caused a transition from a hexagonal to an orthorhombic packing and that electron diffraction can be applied to distinguish between these two lattices. The results are in good agreement with wide-angle X-ray diffraction data and suggest that application of electron diffraction in skin studies can provide new information on the lipid organization in well-defined areas of the stratum corneum.

Animals↗

PubMed1997

Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics.

To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding.

Animals↗

PubMed1997

Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin.

Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can influence the quantity of stratum corneum that is removed by a piece of tape, such as the manner of tape stripping, the hydration of the skin, cohesion between cells, body site and interindividual differences. However, few data are available about the influence of furrows in the human epidermis on the tape-stripping technique. In this study, we investigated the efficacy of tape stripping in removing complete cell layers from the superficial part of the human stratum corneum. A histological section of skin that was tape-stripped 20 times clearly showed nonstripped skin in the furrows, indicating persistent incomplete tape stripping. Replicas of tape-stripped skin surface demonstrated that even after removing 40 tape strips the furrows were still present. We validated the tape-stripping method further with X-ray microanalysis in the mapping mode by scanning electron microscopy, using a TiO2-containing compound as a marker. TiO2 applied to the skin before the tape-stripping procedures was still present after the tenth tape strip, and was specifically located on the rims of the furrows. We emphasize that results from studies using the tape-stripping method have to be viewed from the perspective that cells on one tape strip of the stratum corneum may be derived from different layers, depending on the position of the tape strip in relation to the slope of the furrow, and such results should be interpreted with considerable caution.

Adhesives↗

PubMed1997

Melanogenesis in cultured melanocytes can be substantially influenced by L-tyrosine and L-cysteine.

We investigated the effect of varying concentration of 1-tyrosine and 1-cysteine in culture medium on melanin production by human skin melanocytes (skin phototype II/III). In addition to the analyses of dopa oxidase activity and total melanin, pheomelanin production in the cells was assessed by high-performance liquid chromatography determinations of pheomelanin degradation products, 3-aminotyrosine and 4-amino-3-hydroxyphenylalanine. As another marker for pheomelanin, melanosomal sulfur was determined by the use of X-ray microanalysis. With varying concentration of both amino acids, profound changes in the pigmentation patterns of the melanocytes were observed. A high concentration of 1-tyrosine (0.2 mM) was always connected with increased pigmentation. In combination with a low 1-cysteine content we saw an increase in tyrosinase activity and the highest melanin content. At high concentrations of both 1-tyrosine and 1-cysteine, the melanocytes showed reduced tyrosinase activity and they produced notably more pheomelanin. In case of the pheomelanin measurements by high-performance liquid chromatography and the sulfur detection with X-ray microanalysis, strongly increased concentrations were found when cells were maintained in high 1-tyrosine medium as compared with those grown with low 1-tyrosine. This was especially true for the combination with low 1-cysteine showing that the 1-tyrosine content of the medium strongly influences not only the eumelanin but also the pheomelanin production in the cultured melanocyte. It can be concluded that variations in the concentrations of 1-tyrosine and 1-cysteine in culture medium can be used to regulate the melanogenetic phenotype under in vitro conditions.

Cells, Cultured↗

PubMed1996

The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus.

We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.

Animals↗

PubMed1996

Impaired desquamation in the in vitro reconstructed human epidermis.

A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at the air-liquid interface shares many similarities with native tissue. However, in contrast to native epidermis, its stratum corneum consists of a larger number of compactly packed corneocyte layers, indicating that the processes involved in corneocyte desquamation are disturbed. Although the tri-lamellar appearance of desmosomes in viable cell layers of both types of epidermis is similar, abnormalities in the transformation of desmosomes into the corneosomal plug in the stratum corneum of reconstructed epidermis have been observed. In the native epidermis, desmosomes are transformed into corneosomes at the stratum granulosum/stratum corneum interface. This process is retarded in vitro, since desmosomal structures with preserved lamellar appearance are present in the lower parts of the stratum corneum. Moreover, the corneosome frequency in reconstructed epidermis is significantly higher than in native human skin. A comparison of reconstructed epidermis with hyperproliferative epidermis, such as UV-irradiated epidermis, psoriatic epidermis and recently healed burn-wounds treated with cultured epidermal autografts, has revealed that only the structure of the stratum corneum derived from psoriatic skin is similar to that of reconstructed epidermis. The stratum corneum organization in the UV-treated epidermis and in recently healed burn-wound is, however, close to that seen in native skin.

Adult↗

PubMed1996

The use of vibratome sections for the ruthenium tetroxide protocol: a key for optimal visualization of epidermal lipid bilayers of the entire human stratum corneum in transmission electron microscopy.

In this paper we describe a preparative procedure which allows maximal visualization of all lipid bilayers in the human stratum corneum (SC). We used 50-microns-thick vibratome sections of paraformaldehyde/glutaraldehyde-fixed human skin. The sections were post-fixed in 1% osmium tetroxide and 0.5% ruthenium tetroxide. The vibratome sections were dehydrated only in 70% ethanol in order to prevent dissolution of the lipids. Lipid bilayers, including the alternating electron-dense and electron-lucent lamellae, were visible between all cell layers of the SC. In addition, this preparative procedure also appeared to be excellent for the ultrastructural preservation of lamellar bodies.

Epidermis↗