text:
Epithelial cells are attached to microcarriers to form a transplantation material for treatment of skin wounds. Preferably, keratinocyte-covered microcarriers are prepared using microcarriers having a diameter of 50 to 500 μm, and obtaining a coverage of keratinocytes of 30 to 100% of the maximum coverage. An optimum coverage is between 50 and 80% and preferably between 60 and 70%. Keratinocytes are selected from autologous cells, allogenic cells and a combination of these cells. The microcarriers may be pre-coated with eukaryotic cells or an extracellular matrix protein such as collagen before keratinocytes are attached. The keratinocyte-covered microcarriers may be combined with a cryoprotective substance for storage at +3¯ C. to -196¯ C. prior to use. Keratinocytes are attached to the microcarriers by pre-culturing the keratinocytes, and then culturing the keratinocytes in the presence of the microcarriers while intermittently stirring. A serum free culture medium may be used. Initial culturing in the presence of the microcarriers may be in the smallest amount of medium required to prevent the keratinocytes from dying, and after keratinocyte attachment, adding medium to provide a volume for optimum growth. The keratinocyte-covered microcarriers are applied to a skin wound and the wound is covered with a protective dressing. A pharmaceutical formulation and kit are prepared containing the keratinocyte-covered microcarriers may.
1. A biomaterial suitable for the treatment of skin wounds, comprising keratinocytes attached to microcarriers, wherein said microcarriers exhibit an average coverage between 50to 80% of the maximum coverage possible with keratinocytes.
2. The biomaterial according to claim 1, wherein said keratinocytes, are selected from the group consisting of autologous cells, allogenic cells and a combination of autologous cells and allogenic cells.
3. The biomaterial according to claim 1, wherein said keratinocytes are a combination of 10-30% autologous cells and 70-90% allogenic cells.
4. The biomaterial according to claim 1, wherein said microcarriers are biocompatible materials.
5. The biomaterial according to claim 4, wherein said biocompatible materials are selected from the group consisting of collagen, dextran, gelatin and silicon.
6. The biomaterial according to claim 1, wherein said microcarriers have diameter of 50 to 500 μm.
7. The biomaterial according to claim 1, wherein said microcarriers are pre-coated with a coating selected from the group consisting of eukaryotic cells or components thereof and extracellular matrix proteins, before said keratinocytes are attached.
8. The biomaterial according to claim 1, further comprising cryoprotective substances such that said biomaterial can be stored at a temperature between +3¯ C. to -196¯ C. prior to use.
9. The biomaterial according to claim 8, wherein said cryoprotective substances are selected from the group consisting of dimethyl sulfoxide and glycerol.
10. The biomaterial according to claims 1, wherein said keratinocytes are allogenic cells.
11. A method for preparing a biomaterial suitable for the treatment of skin wounds, comprising the steps of:
pre-culturing keratinocytes in a cell culture medium in the absence of microcarriers, then culturing the keratinocytes in the presence of microcarriers while intermittently stirring said keratinocytes, to produce keratinocyte covered microcarriers, and
isolating any keratinocyte covered microcarriers when said microcarriers are 50 to 80% covered with keratinocytes.
12. The method according to claim 11, wherein said keratinocytes are cultured in the presence of said microcarriers by stirring for 2 minutes at 40 rpm then culturing for 30 minutes without stirring.
13. The method according to claim 11, wherein said culturing of the keratinocytes in the presence of microcarriers is initially carried out in the smallest amount of cell culture medium required to prevent the keratinocytes from dying and after said keratinocytes are attached to said microcarriers additional cell culture medium is added to adjust the volume to a volume required for optimum growth.
14. The method according to claim 13, wherein said amount of cell culture medium is adjusted to the volume required for optimum growth after 6 to 8 hours.
15. The method according to claim 11, further comprising the addition of pituitary extract or fetal calf serum to said cell culture medium.
16. The method according to claim 11, further comprising forming a pharmaceutical formulation of said isolated keratinocyte-covered microcarriers, wherein said pharmaceutical formulation does not dehydrate said keratinocytes.
17. The method according to claim 16, wherein said pharmaceutical formulation comprises a hydrogel.
18. The method according to claim 17, wherein said hydrogel is selected from the group consisting of methyl cellulose, alginates, and agarose.
19. The method according to claim 11, further comprising precoating said microcarriers with a coating selected from the group consisting of epithelial cells or collagen before culturing said cells in the presence of said microcarriers.
20. The method according to claim 11, wherein said keratinocytes are cultured in serum free medium.
21. A method for treating a skin wound comprising the steps of:
applying a biomaterial comprising keratinocytes attached to microcarriers to said skin wound, wherein said microcarriers exhibit an average coverage between 50 to 80% of the maximum coverage possible with keratinocytes, and then covering said skin wound with a protective dressing.
22. The method according to claim 21, wherein said keratinocytes are selected from the group consisting of autologous cells, allogenic cells or a combination of autologous cells and allogenic cells.
23. The method according to claim 21, wherein said keratinocytes are a combination of 10-30% autologous cells and 70-90% allogenic cells.
24. The method according to claim 21, wherein said microcarriers, are biocompatible materials.
25. The method according to claim 21, wherein said microcarriers, have a diameter of 50 to 500 μm.
26. The method according to claim 25, wherein said microcarriers are pre-coated with a coating selected from the group consisting of eukaryotic cells or components thereof and extracellular matrix proteins, before said keratinocytes are attached.
27. The method according to claim 21, wherein said biomaterial is stored prior to use at a temperature between+3¯ C. to +10¯ C.
28. The method according to claim 21, further comprising the addition of cryoprotective substances such that said biomaterial can be stored at a temperature between +3¯ C. to -196¯ C. prior to use.
29. The method according to claim 28, wherein said cryoprotective substances are selected from the group consisting of dimethyl sulfoxide and glycerol.
30. The method according to claim 21, wherein said biomaterial is a mixture of stored and freshly prepared biomaterial.
31. The method according to claim 21, wherein said keratinocytes are allogenic cells.
32. The method according to claim 21, wherein said skin wound is selected from the group consisting of burn wounds, diabetic wounds, and senile ulcers.
33. The method according to claim 21, wherein said biocompatible materials are selected from the group consisting of collagen, dextran, gelatin and silicon.
34. A kit for treating a skin wound comprising a biomaterial containing keratinocytes attached to microcarriers, wherein said microcarriers exhibit an average coverage between 50 to 80% of the maximum coverage possible with keratinocytes, and a protective dressing selected from the group consisting of a fatty gauze dressing, a hydrophobic polymer dressing and a hydroactive dressing.
35. The kit according to claim 34, wherein said biomaterial and said protective dressing are in separate packages.
36. The kit according to claim 34, wherein said keratinocytes are allogenic cells.
This application is a 371 of PCT/EP95/04164, filed Oct. 24, 1995.
1. Field of the Invention
The subject-matter of the invention is a new biomaterial which contains epithelial cells on microcarriers (MCs), as well as the preparation and use of said biomaterial for preparing a medicament.
2. Description of the Prior Art
Skin consists of the epidermis, the basement membrane and the dermis. The epidermis forms the outer coat. The dermis consists essentially of fibroblasts with additional substances such as, e.g., collagen. The basement membrane essentially contains keratinocytes which are non-differentiated but proliferating. The epidermis essentially contains differentiated keratinocytes wherein the upper layer is horny. It is true that while the degree of differentiation of the cells is increasing, the ability of the cells to proliferate is decreasing. Accordingly, the non-differentiated keratinocytes exhibit the highest proliferation potency.
Burn wounds and slow-healing wounds (ulcers) usually are treated with skin sheets consisting of autologous keratinocytes (classical method). To this end, a piece of skin is cut off from the patient, the keratinocytes are cultured in vitro, and the expanded skin sheets so obtained are transplanted [Gallico, G. G., et al., New Engl. J. Med. 311 (1984) 448-451 (1); Cuono, C., et al., Lancet (1986) 1123-1124 (2); De Luca, M., et al., Burns 15 (5) (1989) 303-309 (3); Gallico, G. G., Clinics in Plastic Surgery 17 (3) (1990) 519-526 (4); De Luca, M., and Cancedda, R., Burns 18 (1) (1992) 5-9 (5)]. The transfer of thin skin sheets from cell culture flasks to, e.g., Vaseline gauze as well as the fixing thereof onto the wound surface is problematical. The covering of wounds on parts of the body that are difficult to reach is another problem. Besides this, only keratinocytes can be transferred by means of such classical skin sheets. Skin sheets from keratinocytes involve the drawback that these cells are almost completely differentiated when the sheets are being formed, which means that the sheets practically no longer contain any keratinocytes capable of proliferation. Thus, these sheets constitute a transplantation material which cannot grow, or continue growing, after transplantation. Epidermal transplants (classical skin sheets) with allogenic keratinocytes have been carried out in a number of clinics without any immunological rejection reactions being observed (Gboyse, S. T., et al., Plast. Reconst. Surg. 91 (1993) 632 (17); Burt, A. M., et al., Br. Med. J. 298 (1989) 915 (18); Hickerson, W. L., et al., Burns 20/1 (1994) 52 (19)).
From WO 92/06179 (6) and EP-A 0 242 270 (7) biomaterials are known which contain epithelial cells within a collagen gel. In this context, epithelial cells (fibroblasts or keratinocytes) are embedded in gel layers. It is known that such gels may also contain keratinocytes. However, the keratinocytes in the gel are immobilized and are thus incapable of proliferating and differentiating to an appreciable extend after transplantation. Accordingly, keratinocytes from such gels are not able to form a basement membrane and an epidermis. Another drawback of this biomaterial is, in particular, the low rate of migration of the epithelial cells from the biomaterial into the wound. Furthermore, the handling and fixing of these biomaterials is very difficult and laborious.
In WO 90/02796 (8) there is described a three-dimensional cell culture system on the basis of, for instance, cellulose, polyamide, polyester, on which epithelial cells can grow. In this context, an artificial tissue is obtained which is covered with epithelial cells (fibroblasts or keratinocytes) and which is said to be suitable for transplantation. These artificial transplantation materials, too, do not exhibit any advantage over the skin sheets applied in the classical method.
The object of the invention is to provide a biomaterial which does not exhibit the drawbacks described and which can be prepared in an easy manner and in large quantities. The subject-matter of the invention is the use of a biomaterial which contains epithelial cells adherently attached to microcarriers, for preparing a transplantation material for the treatment of skin wounds. The present invention makes it possible to:
1. transfer non-differentiated and, thus, potent keratinocytes that are capable of proliferation;
2. form a basement membrane and an epidermis on the wound after transplantation (the non-differentiated keratinocytes, after transplantation, separate from the microcarriers and proliferate and differentiate).
It was heretofore unknown that non-differentiated keratinocytes which are capable of proliferation, can be cultured in vitro. The method according to the present invention, makes it possible to produce such keratinocytes. Thus, the present method is susceptible of industrial application directly in vitro. Non-differentiated keratinocytes produce components which are different from those produced by differentiated keratinocytes (which, according to the prior art, have heretofore been the only ones obtained by culturing in vitro). An example of such a component is collagen IV. Accordingly, the invention offers a method of producing substances, such as collagen IV, by culturing non-differentiated keratinocytes. The non-differentiated keratinocytes can be selected by using immune staining with the markers Ki67, keratin 10 and keratin 14 (Slivka, S. R., et al., J. Invest. Dermatol. 100 (1993) 4046 (15); Regnier, M., et al., Skin Pharmacol. 3 (1990) 70-85 (16)).
FIG. 1 shows Cytodex 3Û (crosslinked dextran matrix with a denatured collagen layer) microcarriers with a 30% coverage with keratinocytes (example 2).
FIG. 2 shows the same system with 80% coverage.
FIG. 3 shows the migration of keratinocytes on the artificial dermis (Example 5)
As epithelial cells, all epithelial cells, such as, for instance, keratinocytes, fibroblasts and endothelial cells, are suitable. Such cells preferably are proliferating cells, and most preferably, non-confluent cells which have the properties of basal cells. CellGenesys is engineering epidermal keratinocytes to have "universal properties" so that they can be used in a full thickness skin (epidermis and dermis) graft without causing a donor-host immune response. (CellGenesys, research collaboration with Advanced Tissue Sciences, SCRIP No. 1961, 27 September 1994, p. 24).
By "microcarriers" there are to be understood small solid particles which are capable of being suspended in a slowly stirred, aqueous medium. Such microcarriers are used for the cultivation of eukaryotic cells, wherein the cells grow on the surface of these particles, in the form of a confluent layer. Microcarriers of this kind and their application in cell cultures are described, for instance, in Advances in Applied Microbiology 31 (1986) 139-179.
As microcarriers there may be used all microcarriers which consist of a biocompatible material (such as, e.g., collagen, polymers from hyaluronic acid or hyaluronane esters (e.g. ethyl or benzyl esters) (Andreassi, L., et al., Wounds 3 (1991) 116-126 (28); Myers, S. R., et al., Abstract No. 85 in 5th Annual Meeting of the European Tissue Repair Society (August 1995), Padua (29)) (e.g. HYAFF-11 from FIDIA Advanced Biopolymers, Abano T., Italy), fibrin binding polypeptides, dextran, gelatin, silicone, etc.) or of a suitable mixture of several biocompatible materials. There may be used, for example, Cytodex3Û (crosslinked dextran matrix with a denatured collagen layer, 100-230 μm) (Pharmacia AB, Sweden) based on dextran Cellgen (Koken Co., Japan), a reconstituted and crosslinked collagen from bovine skin, 200-500 μm, or Cultispher-GÛ (macroporous gelatin microcarrier) (Hyclone, Greiner, Germany) based on gelatin.
The size of the microcarriers is not critical. Usually, however, the carriers have a diameter of 50 to 500 μm. Microcarriers of a diameter below 50 μm are so small that they usually cannot be sufficiently covered with cells growing on them. Microcarriers of a size higher than 500 μm are so large that, on the one hand, a high coverage with cells is difficult to achieve; and on the other hand, these particles are not optimal with regard to transplantations because the detachment of the epithelial cells and spreading on the wound does not proceed satisfactorily in the case of such particles.
Surprisingly, it has been found that for an epidermal graft according to the invention there need not be used autologous cells as are applied in the known methods, but rather allogenic cells alone or mixtures of autologous and allogenic cells are also suitable. This offers the advantage that it is not necessary to prepare for each patient, in a time-consuming cultivation, a completely autologous transplantation material. The allogenic transplantation material can be standardized and will be available then in large amounts and at any time. This is especially important in the case of use for the treatment of large-area burns which must be treated as quickly as possible.
Allogenic keratinocytes, after transplantation, are essentially not rejected. However, it is not these keratinocytes themselves which form the new skin. The essential function of the non-differentiated allogenic keratinocytes is to stimulate those keratinocytes of the transplant recipient which are still present (these non-differentiated keratinocytes will form other components than those formed by differentiated keratinocytes). They secrete a large number of hormones (vitamin D, steroids, and others), cytokines (interleukin 1, 3, 6, PDGF, bFGF, TGF-α, TGF-β, and others) as well as matrix elements (laminine, type IV/-VII collagen, fibronectin), which effect an accelerated epithelization extending from the margin of the wound as well as of remaining areas of cutaneous appendage (which still contain residues of intact keratinocytes) (Brown, H., "Wound healing research through the ages", wound healing: biochemical and clinical aspects, Saunders, Philadelphia, page 5, editors: Cohen, I. K. Diegelman R. F., and Lindblad, W. J., 1992 (14)). In burns of the IIb degree, for instance, where residues of the patient's own keratinocytes are still present, using an allogenic transplant or a mixed transplant comprising allogenic and autologous keratinocytes, will be sufficient. The allogenic keratinocytes stimulate the remaining keratinocytes of the recipient and themselves die off after some time. In high degree buns (third degree), practically no keratinocytes will be present any longer. In such cases, a transplantation of autologous keratinocytes is necessary.
The time until the start of treatment can be considerably shortened also by using a biomaterial containing a reduced portion of autologous cells. In a preferred embodiment, the biomaterial may contain 10 to 30% of autologous cells. As far as the remainder of 70 to 90% is concerned, one may resort to stored allogenic biomaterial available in large amounts. By this the time for preparing a sufficient amount of transplantation material can be shortened because only a small amount of autologous cells of the cultivation need be obtained.
In addition to this, the biomaterial according to the invention offers new possibilities of preparing individual transplantation materials. For example, it is possible to prepare the transplantation material immediately before transplantation. This can also be done by mixing biomaterial according to the invention containing autologous and/or allogenic epithelial cells and/or various epithelial cells (e.g. keratinocytes or fibroblasts). By this the specific requirements of the wound to be treated (e.g. degree of burn, burn wound or type of wound) can be taken into account optimally.
This individual adaptation of the composition of the biomaterial to the transplantation problem is impossible to achieve with the prior art transplantation materials.
In order to prepare the biomaterial, the autologous and/or allogenic epithelial cells are isolated, in a manner known to one skilled in the art [Rheinwald, J. G., and Green, H., Cell 6 (1975) 331-334 (9); Limat, A., et al., J. Invest. Dermatol. 92 (1989) 758-762 (10)], from human or animal material and, suitably, are first pre-cultured adherently. Preferably after the first or the second passage, the cells are cultured further together with the microcarriers. This is accomplished either in a stirred culture (spinner culture or fermenter) or in a quiescent culture (e.g. cell culture flasks), wherein the cells spread out from the bottom of the cell culture flasks onto the microcarriers. It is also preferred to incubate beads, which have been allowed to swell in water, with the cells over a period of 2 to 5 hours, preferably 3 to 4 hours, and while doing so, to stir the culture briefly (for 3 to 5 minutes) once an hour.
In a preferred embodiment, the microcarriers can be pre-coated in order to improve the adhesion of the cells and enhance the extent of coverage of the microcarriers. For the coating, for instance proteins of the extracellular matrix, such as fibronectin or collagen, or whole eukaryotic cells (e.g. fibroblasts incapable of proliferation) or components thereof (e.g. membrane fractions) may be used.
Preferably, prior to cultivation, the microcarriers are coated with epithelial cells incapable of proliferation, such as, e.g., fibroblasts. Suitable fibroblasts are, for example, killed mouse fibroblasts from the cell line 3T3. Such a pre-coating according to the methods known to one skilled in the art is described in DE 26 51 685, for instance. It is also preferred to pre-coat the microcarriers with substances which improve the adhesion of the cells (e.g. collagen). Suitably, the pre-coating is carried out after swelling of the microcarriers and autoclaving for sterilization, but before incubation with the epithelial cells. To this end, the microcarriers are incubated with an aqueous solution of such an adhesion molecule or the cell suspension (the latter, suitably, in cell culture medium). Thereafter, the coated microcarriers are washed [preferably with PBS (phosphate buffered saline) NaCl: 8000 mg/l, KCl: 200 mg/l, Na2 HPO2 H2 O:1440 mg/l, KH2 PO4 :200 mg/l].
In order to coat the microcarriers with the epithelial cells, preferably the cultivation is carried out in serum-free medium, optionally with pituitary extract (bovine) or fetal calf serum being added.
In a preferred embodiment, growth- and cell differentiation-promoting factors, respectively, such as keratinocyte growth factor (KGF), nerve growth factor (NGF) or platelet-derived growth factor (PDGF), are added in order to enhance the extent of coverage.
After the desired extent of coverage has been attained, the cells are isolated. This can be accomplished, for instance, by sedimentation or centrifugation at low g-values (e.g. 10 to 50 g).
The cells isolated in this manner either are applied directly in a suitable galenic formulation for treatment of wounds or are preserved until treatment. For preservation, ordinarily cryopreservation is suitable. Cryopreservation is carried out preferably with the addition of cryoprotective substances, such as dimethyl sulfoxide (DMSO) or glycerol, at a temperature of +3¯ C. to -196¯ C., preferably for long-term storage (more than 6 hours). In the case of short-term storage (less than 6 hours), preservation can be carried out at a temperature of +3¯ C. to +10¯ C. without the addition of cryoprotective substances.
In order to prepare a biomaterial containing autologous and allogenic cells attached to microcarriers, preferably autologous epidermal cells are first isolated and cultured in the presence of the microcarriers. The cultivation is carried out for a sufficient time until the amount of autologous biomaterial desired as a proportion in the mixture has been attained. Then, allogenic biomaterial (which contains allogenic epithelial cells adherently attached to the microcarriers) is added and the epidermal graft according to the invention is prepared, as a mixture. In this context, it is preferred to employ cryopreserved allogenic biomaterial or allogenic biomaterial that has been stored at a temperature between +3¯ C. and +10¯ C.
Preferably, the proportion of autologous biomaterial in the mixture is between 10 and 30%. It is also possible to culture autologous and allogenic cells together in the presence of microcarriers. In such case, one will obtain a transplantation material in which both autologous and allogenic cells are attached to the individual microcarriers.
In a further preferred embodiment, a mixture is used which consists of microcarriers wherein each individual microcarrier particle is covered with one type of epithelial cells only, such as, e.g., fibroblasts or keratinocytes. It is also possible to use microcarriers on which several types of epithelial cells (preferably, fibroblasts and keratinocytes (fetal or adult)) are adherently attached. The ratio of mixing of the differently covered microcarriers or the ratio of mixing of the individual cells on one microcarrier may actually be any ratio desired and may vary depending on the intended use (e.g. burn wounds, senile ulcers, diabetic ulcers). In this context it is preferred to apply, for diabetic wounds, only fibroblasts, or fibroblasts in excess, and for burn wounds, only keratinocytes, or keratinocytes in excess over the other epithelial cells in the transplantation material.
The extent of coverage of the microcarriers with epithelial cells can be varied widely. However, the lower the coverage, the lower the efficacy of the transplant. Hence, in practice, it is advantageous to apply a coverage as high as possible.
The preferred coverage is between 30 and 100%. Surprisingly, it has been found, however, that a coverage between 50 and 80%, preferably between 60 and 70%, is optimum.
By coverage there is to be understood the average amount of cells adherently attached to the microcarrier, in reference to complete coverage.
This amount can be determined either visually under the microscope or after detachment of the cells from the microcarriers (for instance, by treatment with trypsin) and staining. Suitably, the complete coverage can be determined most easily visually under the microscope. Typical pictures of a 30% and 80% coverage of the microcarriers according to the invention are shown in FIGS. 1 and 2.
For therapeutic application, the preserved biomaterial (optionally mixed with fresh biomaterial which may also be autologous) is used.
To this end, the biomaterial is thawed, optionally the cryoprotective substance is removed by washing with medium, and the biomaterial is mixed with a preparation which constitutes the galenic medium. It is essential for the galenic formulation that the material used should not dehydrate the cells. It has been found that hydrogels, preferably based on methyl cellulose, are particularly suitable for this purpose. Examples of suitable hydrogels are as follows:
methyl cellulose (preferably about 4%, non-ionogenic cellulose derivatives, i.e., modified D-glucoglycans in which the hydroxyl groups of the cellulose are substituted with methyl groups) (Keresztes, A., Kedvessy, G., Pharmacia 20 (1965) 371 (20); Sarkar, N., J. Appl. Polymer Sci. 24 (1979) 1073) (21);
alginates (salts of alginic acid, e.g., sodium-, calcium-, magnesium- or ammonium alginate, e.g., Kelco-AlginatesÛ of the firm of Kelco) (Sellerie, R., et al., Boll. chim. farmaceut. Milano 119 (1980) 41 (22); Daigo, K., et al., Yakuri to Chiryo 11 (1983) 401) (23);
agarose (polysaccharide from agarobiose and neo-agarobiose) (Langford, J. J., Dambach, A. E., Soap. Chem. Specialties 45/5 (1969) 231) (24).
Type I collagen and type m collagen in medium may also be suitable.
It is also possible to use methyl cellulose dissolved in culture medium. The viscosity must be such that the material is easy to spread and that the cells can separate after transplantation and are capable of migration in order to form, e.g., as keratinocytes a basement membrane and an epidermis.
The basis of the galenic medium suitably is essentially the culture medium. It is, however, advantageous to add further substances which are compatible with the skin and with which the viscosity of the biomaterial can be altered. Optimizing the viscosity is especially advantageous for optimizing the handling of the biomaterial in applying it to the wounds, and for adhesion to the wounds. Suitable skin-compatible substances for altering the degree of viscosity are, for instance, thickening agents, auxiliary emulsifying agents, hydrocolloids or gelling agents, such as, e.g., gelatin, pectin, alginates, cellulose derivatives, Aqualose and polysaccharides.
Suitably, these substances are admixed to the galenic medium at such concentration that the microcarrier preparation is easy to apply.
In a preferred embodiment, the galenic medium may further contain substances that additionally improve wound healing, such as, e.g., NGF, TGF-β, PDGF, KGF and/or fibronectin or analogs (Scharffetter-Kochanek, K., et al., J. Invest. Dermatol. 98 (1992) 3-11 (30); Lange, T. S., et al., J. Exp. Dermatol. 4 (1995) 130-137 (31); WO 92/09200 (32) (for fibronectin peptides); WO 90/07577 (33); WO 91/17765 (36)), which improve the adhesion of the epithelial cells on the wound bed, especially after the cells have become detached from the carrier. Preferably, the medicament contains between 0.03 g and 0.1 g of fibronectin or analogs per gram of galenic medium. It is further preferred that the galenic medium should contain viable cells having the properties of macrophages, e.g., macrophages or Langerhans' cells for disinfecting the wounds. It is preferred that the medicament should contain about 10 to 30% of such cells.
The medicament according to the invention, as an epidermal graft, is suitable for use in the treatment of, for instance, burn wounds, diabetic wounds, senile ulcers and other slow-healing wounds. The medicament according to the invention is also suitable for speeding up the healing of normal wounds. A further use is in plastic surgery.
An essential advantage of the biomaterial according to the invention lies in the fact that, unlike the known epidermal grafts, it is flexible in all directions and easy to adapt to the body surface even on parts which are problematical (e.g. skinfolds, ear, eyelids).
For application, suitably, the medicament according to the invention is placed on the wounds using an appropriate dressing depending on the type of wound to be treated [for example: a) fatty gauze dressing, e.g., GRASSOLINÛ of the firm of Hartmann, Germany, b) hydrophobic polymer dressing, e.g., EPI-LOCKÛ of the firm of Merck & Co., Missouri/USA, c) hydroactive dressing, e.g., DUODERMÛ [gel formula dressing] of the firm of Conva Tec, Princeton/USA d) MITRAFLEX (microporous polyurethane membrane)], in order to maintain its effectiveness.
In a preferred embodiment, a mixture of autologous and allogenic epithelial cells, in particular keratinocytes, is used.
As keratinocytes, preferably epidermal keratinocytes (e.g. foreskin keratinocytes) are used.
In order to prepare a biomaterial based, in particular, on keratinocytes it is specifically preferred to pre-coat the microcarriers, as described above. Intermittent stirring is also preferred in the culturing of the cells in the presence of the microcarriers. To this end, the culture is preferably stirred for a few minutes (e.g. 1 to 5 minutes) at a speed between 10 and 100 rpm and subsequently cultured further for an extended period of time (about 20 to 40 minutes), without stirring. The intermittent stirring conditions are maintained over the entire period of culturing. It has been found that it is specifically advantageous for the culturing if stirring is carried out for about 2 minutes at about 40 rpm, followed by a rest period of about 30 minutes. These conditions may, of course, be varied to a certain extent. In this context care must be taken that stirring is carried out for a period as short as possible, yet attaining a thorough mixing of the medium, so that subsequently, culturing can be carried out as long as possible without stirring and with the cell growth and the adhesion of the cells to the microcarriers being impaired as little as possible.
In order to achieve a high coverage of the microcarriers in culturing, in particular, keratinocytes, it is further preferred to carry out the culturing first, in a first culturing phase, in an amount of culture medium as low as possible. This amount must be just high enough to ensure that the cells do not die to a considerable extent and are capable, at the same time, of binding to the surface of the microcarriers. It is only after the keratinocytes are adherently attached to the microcarriers to a considerable extent (usually after 6 to 8 hours) that, in a second culturing phase, further medium is added. The total amount of medium corresponds to that amount of medium which is optimally applied for the coverage of the microcarriers using uncritically growing epithelial cells, such as fibroblasts. For microcarriers which are known as carriers for adherent cells, this amount is usually specified by the manufacturer. In this context, the optimum amount of medium is 100 ml for 300 mg of CytodexÛ (crosslinked dextran (matrix) and for 100 mg of CultispherÛ (macroporous gelatin microcarrier). For culturing the keratinocytes, in the first culturing stage, only about 40 to 60% of this optimum volume of cell culture medium is added.
The examples and publications provided below shall illustrate the invention further, the protective scope of which is established by the patent claims. The methods described are to be understood as examples which describe the subject matter of the invention, even after modifications have been made.
Isolation and cultivation of human keratinocytes
1) Epidermal keratinocytes
Epidermal cells (keratinocytes) are isolated enzymatically from pieces of skin and foreskin, respectively, and are placed in the cell culture.
Isolation is carried out according to methods known in the art [Rheinwald, J. G., and Green, H., Cell 6 (1975) 331-334 (9); Limat, A., et al., J. Invest. Dermatol. 92 (1989) 758-762 (10); Rheinwald, J. G., Methods Cell. Biol. 21A (1980) 229-254 (12)].
The cultivation and proliferation of the keratinocytes until use for coverage of the microcarriers (MCs) is carried out in a static culture (cell culture flasks or cell factories) with serum-free medium.
2) Cells of the outer root sheath (ORS keratinocytes) from hair follicles
The establishment of primary cultures of ORS cells and expansion (cultivation) thereof in vitro is accomplished according to methods known in the art [Limat, A., et al., J. Invest. Dermatol. 92 (1989) 758-762 (10); Limat, A., and Noser, F., J. Invest. Dermatol. 87 (1986) 485-488 (13)].
Preparation of the microcarriers and cultivation of keratinocytes on microcarriers (MCs) (coverage of the MCs)
2.1 Preparation of the MCs for the cultivation
a) Cytodex 3Û (Pharmacia--crosslinked dextran matrix with a denatured collagen layer)
The MCs are weighed and allowed to swell in phosphate-buffered saline without calcium ions and magnesium ions (PBS -/-) overnight at room temperature (to be stirred up occasionally at the beginning). The PBS is decanted and the MCs are additionally washed twice with PBS; 50 to 100 ml PBS per gram of MC are applied.
The suspension is autoclaved (115¯ C., 15 psi, 15 min.) and thereafter the MCs are placed in culture medium (GIBCO, serum-free Keratinocyte-SFM). The MCs are washed once with culture medium and kept in the same medium at 4¯ C.
b) CultispherÛ (Hyclone--macroporous gelatin microcarrier)
The MCs are weighed and allowed to swell in phosphate-buffered saline without calcium ions and magnesium ions (PBS -/-) overnight at room temperature (to be stirred up occasionally at the beginning). The PBS is decanted and the MCs are additionally washed twice with PBS; 50 to 100 ml PBS per gram of MC are applied.
Pre-coating with collagen
The suspension is autoclaved (115¯ C., 15 psi 15 min.). Subsequently, the MCs are stirred overnight at 37¯ C. in a solution of collagen S type 1 (3 mg collagen in 10 ml of 0.1% CH3 COOH); 10 ml collagen solution per 100 mg MC are applied. The supernatant is discarded, the MCs are washed once with PBS-/- and placed in culture medium (GIBCO, serum-free keratinocyte-SFM). After single washing (with culture medium), the MCs are kept in culture medium at 4¯ C.
2.2 Cultivation of the keratinocytes on the MCs
Subconfluent (about 70%) serum-free cultured keratinocytes (in cell culture flasks) of the first or the second passage are released from the bottom of the culture vessel using a trypsin/EDTA solution (5 ml per cell culture flask [175 cm2 culture area] of a 0.05% trypsin/0.02% EDTA solution in PBS-/-, period of action: about 5 min. at 37¯ C). The release through trypsinization is stopped with 3 ml FCS (fetal calf serum). This suspension of individual cells is removed by centrifugation (10 min., at 180 g). The cell pellet is thoroughly resuspended in culture medium and cell counting is carried out using trypan blue.
Seeding with the MCs is carried out in a spinner flask (effective volume 50 ml). To about 1æ107 cells are added 300 mg Cytodex 3-MC and 100 mg Cultispher respectively (about 10 cells per carrier) and the liquid volume is adjusted to 30 ml with serum-free culture medium.
Stirring in the spinner flask is carried out intermittently: 2 min. at 40 rpm, followed by 30 minutes of cultivation without stirring. These stirring conditions are maintained during the whole period of cultivation.
After 6 to 8 hours, medium is added until a volume of 100 ml is attained (possibly, the culture is transferred to a spinner flask having an effective volume of 250 ml). During the further period of cultivation, the medium is changed twice a day (about every 12 hours) according to the following procedure: The carriers settle on the bottom of the flask, spent medium is sucked off to the highest possible extent and replaced by fresh medium.
During the period of cultivation, the coverage of the MCs is evaluated microscopically and documented photographically. In the case of the Cytodex 3 MCs, the cells are evaluated without staining. In the case of the Cultispher MCs, the keratinocytes are stained with the Hoechst dye 33342 (2'-[4-Ethoxyphenyl]-5-[4-1-piperazinyl]-2,5'-bi-1H-benzimidazole) beforehand. Examples using cells on Cytodex 3 MCs:
FIG. 1: About 30% coverage.
FIG. 2: About 80% coverage.
After the desired coverage has been attained, excess medium (after the settling of the carriers) is sucked off and the remaining suspension is removed by centrifugation at 10 to 50 g for 10 min. A galenic substance is admixed to the pellet and the whole thing is applied in this form, or the pellet is frozen with the addition of a cryoprotectant for use at a later date. The table given below shows the extent of keratinocyte coverage of the microcarriers in dependence upon the optimizing process features:
intermittent stirring
increasing the volume of the medium after 6 to 8 hours
______________________________________Volume Stirring Coverage______________________________________constant continuously 0 constant intermittently 20% increase 50% continuously 20% increase 50% intermittently 80%______________________________________
It can be seen from Table 1 that each of these optimizing features contributes to enhancing the coverage. The combination of intermittent stirring and increase in volume leads to a decisive improvement of the coverage.
2.3 Fast cultivation of keratinocytes (Kz) on MCs
The MCs are incubated at a concentration of 1.2æ105 MC/ml of culture medium with keratinocytes (cultured serum-free or with serum) at a concentration of 1æ106 Kz/ml of culture medium. The ratio of MC:Kz is at least 1:10. The incubation takes place in a 13 ml centrifuge tube in an incubation cabinet (37¯ C., 5% CO2, 95% rH) for at least 2 hours, not more than 6 hours, preferably for 3 hours. The suspension is to be shaken carefully once an hour for about 20 seconds. After that period, the majority of the Kz have adhered to the MC.
After 3 to 4 hours an aliquot is taken from the suspension and the coverage is examined microscopically.
______________________________________ Number of covered MCs, Coverage density of the MC in % individual MCs, in______________________________________ %Cytodex 3 50-70 70-90 Cellgen 70-90 70-90 Biosilon 50-70 40-70 Cultispher 0-5 0-5 Collasphere 0-5 0-5______________________________________
Galenic medium
Keratinocytes alone (without beads) were mixed with several substances (but always with medium) and examined for viability. The results are as follows:
in gelatin: low viability;
in a number of ready-to-use wound gels/ointments: low or poor viability
in Nifucin-GelÛ of the firm of Apogepha (nitrofurazone and adjuvant): good viability.
Determination of the coverage of the microcarriers:
The coverage of the microcarriers (determining the number of cells) is determined by means of protein determination. The protein determination is carried out according to the BCA method (test kit of the firm of Pierce, Catalogue No.: 23225).
Preparing the samples:
1 ml of a homogeneous suspension (microcarriers covered with cells in medium) is transferred to an Eppendorf vessel and removed by centrifugation in the Eppendorf centrifuge (2 min. at 10,000 rpm). The supernatant is discarded. The pellet is resuspended in 200 μl CAT lysis buffer (containing MOPS, NaCl and Triton X-100, pH 6.5, product of the firm of Boehringer Mannheim GmbH from CAT-ELISA-Kit, Catalogue No.: 1363727), resuspended and incubated at room temperature for 1 h.
Preparing the standards (standard series):
Keratinocytes from a confluent static culture are brought into suspension using a trypsin EDTA solution (0.05% trypsin/0.02% EDTA in PBS (Ca/MG-free). After the cell count (trypan blue method), 5 samples comprising in each case 1æ105, 2.5æ105, 5æ105, 1æ106, 2æ106 cells are prepared from this cell suspension. The samples are removed by centrifugation (Eppendorf centrifuge, 2 min. at 10,000 rpm) and the pellets are resuspended in each case in 200 μl CAT lysis buffer (see above) and incubated at room temperature for 1 h. Subsequently, the protein determination of the standards and the samples is carried out according to the BCA method (see above). The OD values of the standards (x axis) are plotted in a diagram against the cell counts in the standards (y axis). The OD values of the samples are read with the help of this standard curve in cell count/ml.
Calculating the extent of coverage:
a) According to theoretical calculations, the microcarriers, at a coverage of 100%, carry, on an average, 40 keratinocytes/microcarrier.
b) The number of microcarriers employed in the samples is known:
4æ104 microcarriers/ml sample.
c) The maximum possible number of cells in the sample (at a 100% coverage of the microcarriers) is: 1.6æ106 keratinocytes/ml sample
d) The cell count/ml in the samples read from the standard curve (protein determination) is compared with the maximum possible number of cells (see above: c) and, from this, the extent of coverage of the microcarriers with keratinocytes, in percent, is calculated.
In vivo assay
The migration of the keratinocytes from the MCs was examined on a slightly modified "Air-Liquid-Interface" model (Noser, F. K., and Limat, A., In Vitro Cell & Dev. Biol. 23 (8) (1987) 541-545 (34)). Collagen gels having inactivated fibroblasts embedded therein were used as the artificial dermis (Bell, E., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 1274-1278 (35)). Into these gels in each case 200 ul of a MC keratinocyte suspension were seeded and cultivated, in a submersed manner, with a FCS-containing culture medium for a period of 24 to 48 hours. Subsequently, the gels were placed on a fine Teflon net and the same was disposed on a Teflon ring. This arrangement was placed in Petri dishes and cultivated for a period of up to three weeks. The serum-containing culture medium was exchanged completely every other day and the volume was measured such that it extended as far as to the lower edge of the Teflon net. For carrying out the histological or immunohistochemical examinations, the gels were lifted off from the Teflon net and were either fixed in a 4%-formalin solution and embedded in paraffin or subjected to cryoconservation. The histological sections of these preparations were dyed histologically with haematoxylin and eosin, or immunohistochemically with antibodies against CK-5, CK-10, mip (Ki67), and collagen IV. It was shown that migration of the keratinocytes takes place from the MCs onto the artificial dermis (FIG. 3), that the development of stratified dermis occurs, and that keratinocytes having basal properties are present in the preparation.
In vivo tests
Swine is the animal species suitable for the in vivo tests (Kangesu, T., et al., Br. J. Plastic Surgery 46 (1993) 401-409 (25); de Vries, H., et al., Wound Rep. Reg. 1 (1993) 244-252 (26); Breuing, K., et al., J. Surg. Res. 52 (1992) 50-58 (27)).
MC: microcarriers without any coating
MCG: carrier gel with 4% methyl cellulose (without any microcarriers)
Kzy+MC: microcarriers (beads) coated with keratinocytes
The skin wounds are divided into five groups (group 1: control, without any applied substance; group 2: MC; group 3: MCG; group 4: MC+MCG; group 5: MC+MCG+Kzy). In order to be able to evaluate the tissue reactions occurring in each group, a number of seven preparations must be evaluated histologically. As the time course of the reaction is also important, the animals are examined after 3, 7 and 14 days. The points in time 3 and 7 days are covered by biopsies whilst after 14 days, the animals are sacrificed. Since as much as 30 skin wounds can be inflicted on each animal ((25), (26), (27)) there results a number of animals of n=5. On each animal 23 (3 times 7+2 reserves) skin wounds are inflicted.
Sedation with Ketamin/Azaperon im; initial anesthesia with Propofol iv; prolonged anesthesia: Isofluran, oxygen, nitrous oxide (intubation).
Under absolutely sterile conditions, the skin areas are cleaned, freed from fat and disinfected, skin wounds of standardized sizes and depths are inflicted (with a pattern and a dermatome; size: 1.5æ1.5 cm; depth: 3 mm), the substances to be tested are applied on the wounds, the individual wounds are covered by watertight and airtight vinyl chambers (these chambers allow separate observation of the individual wounds; such wound chambers are also used in clinics, for the covering of skin wounds; manufacturer: P.A. Medical Co., Columbia, USA).
Postoperative pain therapy:
As the skin wounds located on the backs of the animals, and the wound chambers placed thereon, cause pain and/or feelings of discomfort (e.g. itching) in the animals at least in the first postoperative phase, there is carried out routinely a therapy with Metamizol for a period of 3 days.
List of references
(1) Gallico, G. G., et al., New Engl. J. Med. 311 (1984) 448-451
(2) Cuono, C., et al., Lancet (1986) 1123-1124
(3) De Luca, M., et al., Burns 15 (5) (1989) 303-309
(4) Gallico, G. G., Clinics in Plastic Surgery 17 (3) (1990) 519-526
(5) De Luca, M., and Cancedda, R., Burns 18 (1) (1992) 5-9
(9) Rheinwald, J. G., and Green, H., Cell 6 (1975) 331-334
(10) Limat, A., et al., J. Invest. Dermatol. 92 (1989) 758-762
(12) Rheinwald, J. G., Methods Cell. Biol. 21A (1980) 229-254
(13) Limat, A., and Noser, F., J. Invest. Dermatol. 87 (1986) 485-488
(14) Brown, H., "Wound healing research through the ages", wound healing: biochemical and clinical aspects, Saunders, Philadelphia, page 5, editors: Cohen, I. K. Diegelman, R. F., and Lindblad, W. J., 1992
(15) Slivka, S. R., et al., J. Invest. Dermatol. 100 (1993) 40-46
(16) Regnier, M., et al., Skin Pharmacol. 3 (1990) 70-85
(17) Gboyse, S. T., et al., Plast. Reconst. Surg. 91 (1993) 632
(18) Burt, A. M., et al., Br. Med. J. 298 (1989) 915
(19) Hickerson, W. L., et al., Bums 2011 (1994) 52
(20) Keresztes, A., Kedvessy, G., Pharmacia 20 (1965) 371
(21) Sarkar, N., J. Appl. Polymer Sci. 24 (1979) 1073)
(22) Sellerie, R., et al., Boll. chim. farmaceut. Milano 119 (1980) 41
(23) Daigo, K., et al., Yakuri to Chiryo 11 (1983) 401
(24) Langford, J. J., Dambach, A. E., Soap. Chem. Specialties 45/5 (1969) 231
(25) Kangesu, T., et al., Br. J. Plastic Surgery 46 (1993) 401-409
(26) de Vries, H., et al., Wound Rep. Reg. 1 (1993) 244-252
(27) Breuing, K., et al., J. Surg. Res. 52 (1992) 50-58
(28) Andreassi, L., et al., Wounds 3 (1991) 116-126
(29) Myers, S. R., et al., Abstract No. 85 in 5th Annual Meeting of the European Tissue Repair Society (August 1995), Padua
(30) Scharffetter-Kochanek, K., et al., J. Invest. Dermatol. 98 (1992) 3-11
(31) Lange, T. S., et al., J. Exp. Dermatol. 4 (1995) 130-137
(34) Noser, F. K., and Limat, A., In Vitro Cell & Dev. Biol. 23 (8) (1987) 541-545
(35) Bell, E., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 1274-1278
Patent Citations
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text:
Moving old Altimas will take some quick footwork
- UAW troops air demands at convention rather than cast blame
- The latest tech is great -- until you have to replace it
- That vroom-vroom … is it real or digital?
- Porsche boss Mueller, 62, says he's young enough to be VW Group CEO
- Why March 30-31 might be the greatest two days of deals at FCA dealerships
NASHVILLE -- The next couple of weeks will be tense ones for Nissan retailers, owing to the expected appeal of the 2013 Altima. The nervousness will be over whether Nissan engineers and planners did too good a job redesigning the car.
Nissan is eager to clear out the remaining inventory of 2012 Altimas to make way for a new Altima that is being pitched as superior on several fronts -- fuel economy, electronic features, seating comfort and noise levels. Automakers are always eager to do this when a redesigned model comes to market, of course. But here's the twist: Except for the low-volume base model, the new and improved Altima is roughly the same price as the outgoing model.
Nissan shoppers will find a 2013 2.5 SV Altima that gets 38 mpg highway and NASA-influenced seat designs for $40 more than last year's model. The 2.5 S model is actually $210 cheaper now than it was in 2012.
So the challenge won't be selling the new one; it will be clearing out the old ones this month as they sit there, new and old, side by side.
Nissan has been doing what it must to clear the pipes as the redesigned car arrives in showrooms, with incentives, higher fleet sales and a green light to dealers to move more Altimas into their own rental car inventories. Action Nissan in Nashville last week advertised 2012 Altimas with leases of $99 a month.
Colin Dodge, chairman of Nissan Americas, acknowledged a few weeks ago that Altima fleet sales were going to be high to empty the lots of 2012s. But fleet sales "will drop immediately when the new Altima is launched," Dodge told a group of international analysts.
There was nothing wrong with the old Altima. Sales stayed strong even as Nissan began releasing ads showing off the new and improved one in spring. Nissan had one U.S. factory running overtime to continue pumping out old Altimas as a second U.S. factory began shipping the first new Altimas. That means there are still a fair number of 2012s on the market as dealers roll out the 2013s.
Nissan isn't worried in the least about the '13. Its only challenge for the next few weeks will be helping customers see the value in an old model as a better model arrives looking like a bargain.
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Charles Baudelaire
Charles Pierre Baudelaire was a French poet who produced notable work as an essayist, art critic, and pioneering translator of Edgar Allan Poe. His most famous work, Les Fleurs du mal (The Flowers of Evil), expresses the changing nature of beauty in modern, industrializing Paris during the 19th century. Baudelaire's highly original style of prose-poetry influenced a whole generation of poets including Paul Verlaine, Arthur Rimbaud and Stéphane Mallarmé among many others. He is credited with coining the term "modernity" (modernité) to designate the fleeting, ephemeral experience of life in an urban metropolis, and the responsibility art has to capture that ... more »
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Charles Baudelaire Poems
Get Drunk
Always be drunk. That's it! The great imperative! In order not to feel
Be Drunk
You have to be always drunk. That's all there is to it--it's the only way. So as not to feel the horrible burden of time that breaks your back and bends you to the earth, you have to be continually drunk.
Anywhere Out of the World
This life is a hospital where every patient is possessed with the desire to change beds; one man would like to suffer in front of the stove, and another believes that he would recover his health beside the window. It always seems to me that I should feel well in the place where I am not, and this question of removal is one which I discuss incessantly with my soul.
They are alike, prim scholar and perfervid lover: When comes the season of decay, they both decide Upon sweet, husky cats to be the household pride; Cats choose, like them, to sit, and like them, shudder.
At One O'Clock In The Morning
Alone, at last! Not a sound to be heard but the rumbling of some belated and decrepit cabs. For a few hours we shall have silence, if not repose. At last the tyranny of the human face has disappeared,
Soon we will plunge ourselves into cold shadows, And all of summer's stunning afternoons will be gone. I already hear the dead thuds of logs below Falling on the cobblestones and the lawn.
Her Hair
O fleece, that down the neck waves to the nape! O curls! O perfume nonchalant and rare! O ecstasy! To fill this alcove shape With memories that in these tresses sleep,
Looking from outside into an open window one never sees as much as when one looks through a closed window. There is nothing more profound, more mysterious, more pregnant, more insidious, more dazzling than a window lighted by a single candle. What one can see out in the sunlight is always less interesting than what goes on behind a windowpane. In that black or luminous square life lives, life dreams, life suffers. Across the ocean of roofs I can see a middle-aged woman, her face already lined, who is forever bending over something and who never goes out. Out of her face, her dress, and her gestures, our of practically nothing at all, I have made up this woman's story, or rather legend, and sometimes I tell it to myself and weep.
The Albatross
Often to pass the time on board, the crew will catch an albatross, one of those big birds which nonchalently chaperone a ship across the bitter fathoms of the sea.
The Carcass
Remember that object we saw, dear soul, In the sweetness of a summer morn: At a bend of the path a loathsome carrion On a bed with pebbles strewn,
It is not given to every man to take a bath of multitude; enjoying a crowd is an art; and only he can relish a debauch of vitality at the expense of the human species, on whom, in his cradle, a fairy has bestowed the love of masks and masquerading, the hate of home, and the passion for roaming. Multitude, solitude: identical terms, and interchangeable by the active and fertile poet. The man who is unable to people his solitude is equally unable to be alone in a bustling crowd.
Nature is a temple whose living colonnades Breathe forth a mystic speech in fitful sighs; Man wanders among symbols in those glades Where all things watch him with familiar eyes.
Au Lecteur
La sottise, l'erreur, le péché, la lésine, Occupent nos esprits et travaillent nos corps, Et nous alimentons nos aimables remords, Comme les mendiants nourrissent leur vermine.
The Vampire
You that, like a dagger's thrust, Have entered my complaining heart, You that, stronger than a host Of demons, came, wild yet prepared;
Quotationsmore quotations »
''What is exhilarating in bad taste is the aristocratic pleasure of giving offense.''Charles Baudelaire (1821-1867), French poet. Squibs, Intimate Journals, sct. 18 (1887), trans. by Christopher Isherwood (1930), rev. Don Bachardy (198...
''Hugo, like a priest, always has his head bowedbowed so low that he can see nothing except his own navel.''Charles Baudelaire (1821-1867), French poet. "Squibs," sect. 22, Intimate Journals (1887), trans. by Christopher Isherwood (1930), revised by Don Bach...
''It is the hour to be drunken! To escape being the martyred slaves of time, be ceaselessly drunk. On wine, on poetry, or on virtue, as you wish.''Charles Baudelaire (1821-1867), French poet. Complete Works, vol. 1, "Shorter Prose Poems," ed. Yves-Gérard le Dantec; rev. Claude Pichois (1953). Eni...
''We all have the republican spirit in our veins, like syphilis in our bones. We are democratized and venerealized.''Charles Baudelaire (1821-1867), French poet. Sur la Belgique, epilogue, Complete Works, vol. 2, ed. Yves-Gérard le Dantec, rev. by Claude Pichois (197...
''Whether you come from heaven or hell, what does it matter, O Beauty!''Charles Baudelaire (1821-1867), French. Flowers of Evil, "Hymn to Beauty," (1860).
Comments about Charles Baudelairemore comments »
Shobana Gomes
His poems tread on the catchy-ness of his phrases. Love it.
Lucianne Fasolo
Ah, Baudelaire. The forefather of modern poetry, and also of the French Symbolism. He had a unique style, his poems were flawless and beautiful. And he's one of my favorite poets ever, :)
ari anna arena
Why no 'Le Balcon'? A bella poesia.
Geneva Henderson
This is one of those cool poems. It makes you think. Personally I love Baudelaire. He has a beautiful rythem and sound that enchants me and inspires me.
Maya Angelou
(4 April 1928 - 28 May 2014)
Robert Frost
(March 26, 1874 – January 29, 1963)
Emily Dickinson
(10 December 1830 – 15 May 1886)
William Shakespeare
(26 April 1564 - 23 April 1616)
Pablo Neruda
(12 July 1904 – 23 September 1973)
Edgar Allan Poe
(19 January 1809 - 7 October 1849)
Langston Hughes
(1 February 1902 – 22 May 1967)
Walt Whitman
(31 May 1819 - 26 March 1892)
William Wordsworth
John Keats
(31 October 1795 – 23 February 1821)
Get Drunk
Always be drunk.
That's it!
The great imperative!
In order not to feel
Time's horrid fardel
bruise your shoulders,
grinding you into the earth,
Get drunk and stay that way.
On what?
On wine, poetry, virtue, whatever.
But get drunk.
And if you sometimes happen to wake up
on the porches of a palace,
in the green grass of a ditch,
in the dismal loneliness of your own room,
your drunkenness gone or disappearing,
ask the wind,
the wave,
the star,
the bird,
the clock,
ask everything that flees,
everything that groans
or rolls
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text:
Got a minute?
Got a minute?
It's common knowledge that your morning cup of coffee, with that dose of caffeine, helps you wake up. Therefore, it is not a big stretch to think that coffee also helps us think through decisions, because we are more awake to do so. However, that cup of coffee does so much more than that. In fact, it just might help you behave in more ethical ways.
Money is only one of the many reasons we work. Some people find incredible satisfaction in their careers, even when the pay is average is or low. The following are some of the most meaningful jobs that will not make you a millionaire, but might prove that money doesn't buy happiness.
Occupational psychologist Professor Cary Cooper has a lot to say about the benefits of working from home. He cites some laws that may protect workers, and discusses the psychology behind why remote workers are not always trusted to use their time well. Here are a few snippets of advice to people who want to succeed at working from home.
Educational psychology is the study of how people learn, but the field's potential to help improve cognitive abilities isn't limited to students. Workers can use these techniques to improve memory and do better in their jobs.
If there is one thing some people will never agree upon, it is which pet is superior: dogs or cats. These strong opinions are the result of innate personality differences in cat lovers vs. dog lovers. And, of course, our personalities inform our best career choices.
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text:
Young Thujopsis dolabrata 'Pendula' plant is staked
for upright habit.
That's a beauty. I really like it.
Any weeping Thujopsis in the USA yet?
Nope! :(
Hello Blue Yew
I like that weeping form where do you buy this ?
it's a beauty.
I got it as a normal Thujopsis dolabrata but its
a true weeping form there is a T dolabrata 'Pendula'
in Ireland Brook Hall Arboretum.However the true
name for T dolabrata 'Pendula' might be 'Decumebens'


text:
The Microsoft Windows XP Reloaded marketing campaign cranks up this week with the launch of Windows Media Player (WMP) 10, MSN Music, and the partner-made Portable Media Center devices. These releases follow the shipment of XP Service Pack 2 (SP2) and XP Tablet PC Edition 2005, both of which became available in early August and precede a host of other XP Reloaded releases that many have pegged for October. Chief among those second-stage releases, of course, is the next version of XP Media Center Edition (MCE), a product veiled in much secrecy.
With a public beta release earlier this summer and many public pronouncements from Microsoft about its design, WMP 10 is largely a known quantity. But MSN Music, Microsoft's first serious foray into online music services in the United States, has been the cause of much speculation. And although some people have suggested that the service will quickly relegate Apple Computer's successful iTunes to the dustbin of history, I'm not sure that's going to be the case.
More important, perhaps, is what's the difference? The online music market is currently tiny, only occupying about 2 percent of the $1.7 billion music business in the United States, and much, much less worldwide; even the most positive outlooks see online music occupying just 12 percent of the overall music business in the United States by 2007. As David Card, the research director of Jupiter Research, told the "New York Times" this week, "for Microsoft, \[entering the online music business\] is like Slate, not Xbox. It's a pretty small opportunity right now."
So why bother at all? My guess is that Apple's success in online music was eye-opening for the software giant, as was the failure of the PC industry's usual strategy, in which a host of Microsoft partners rally around a common format--Microsoft Windows Media Audio (WMA) in this case--and split up the market. Despite the emergence of WMA-based challengers such as BuyMusic.com, Musicmatch, Napster, Wal-Mart, and others, Apple continues to dominate online music, and the company appears to be reforming itself along consumer electronics lines as a result. However, if Apple is ultimately successful, Microsoft's wider plans for Digital Rights Management (DRM) and its Windows Media formats will fail, because consumers, hardware and software partners, and services will all want to use Apple's formats. Thus, although sales from online music might accurately be equated to an accounting error in the Microsoft Windows Division, the software giant needs to jump-start acceptance of its own formats if it expects to be a future player in more lucrative markets, such as digital movie delivery, High Definition (HD) film projection, and the like.
In any event, MSN Music will go live this week, alongside the new WMP version. The company is also launching a new "Plays for Sure" branding campaign designed to educate users about whether music purchased from MSN Music and all the WMA-compatible online music stores is compatible with a wide range of more than 70 portable audio players. The message here is clear: Apple's iPod might be successful, but it's only natively compatible with iTunes, an online service that offers relatively low-quality songs for sale. If you use a WMA-compatible solution, the software giant says, you'll have plenty of choices, both on the music-store side and on the portable-player side. By choosing WMA, you'll know that the music "plays for sure" on whatever device you choose. Assuming, of course, it's not an iPod.
Consumers will ultimately decide whether MSN Music or any of the other digital media-related products and services that Microsoft and its partners unleash over the new few months are good enough to merit purchase. But at this still-early stage of the game, Apple is ensconced in the driver's seat, and it's unclear whether anything Microsoft can do--no matter how good it is--will change that for the short term. In the long term, Microsoft's "network effect"--in which simply bundling WMP in Windows and setting the Microsoft Internet Explorer (IE) home page to MSN.com garners millions of users--might actually be enough to tip the scales. But that will take years and likely another Windows version. In the meantime, Microsoft is reloading XP with a host of new updates, products, and services. Let's see whether any consumers take the plunge.


text:
10/23/2012 (2 years ago)
Catholic Online (www.catholic.org)
Blessed Pope John Paul II gave the reason why it is no minor matter whether a person is loved or not loved: "Man cannot live without love. He remains a being incomprehensible to himself. His life is senseless if love is not revealed to him, if he does not encounter love, if he does not experience it and make it his own, if he does not participate intimately in it"
Catholic Online (www.catholic.org)
10/23/2012 (2 years ago)
Published in Marriage & Family
Keywords: John D. Meehan, marriage, family life, love
HOOKSET, NH (Catholic Online) - This is a repeat worth reflecting upon again. Some time ago, on a visit to the United States, Blessed Mother Teresa of Calcutta spoke of the glutted bellies and emaciated hearts of the people living in the United States: "In the Third World, there is often a famine of the stomach due to the lack of food, but the people are rich in love. They share what little they have with one another. In developed nations like yours (America) there is an abundance of food. But there is often a famine of the heart due to a lack of love. The victims of this famine of love are the new poor. And who are these poor people? They are the people sitting next to you."
Blessed Pope John Paul II gave the reason why it is no minor matter whether a person is loved or not loved: "Man cannot live without love. He remains a being incomprehensible to himself. His life is senseless if love is not revealed to him, if he does not encounter love, if he does not experience it and make it his own, if he does not participate intimately in it"
There is, then, an integral connection between a love-being and his or her becoming loved or loveable: "Love, then, seems to be both a state of being and [a condition] of becoming. In fact, love poems and love songs continue to attest always to the inner urge of the human heart for a love that lasts, longing for an everlasting love, a hunger for eternal love. Love is . . . a journey, an ongoing exodus out of the closed, inward-looking self towards its liberation through self-giving, and, thus, towards authentic self-discovery and, indeed, the discovery of God" (Pope Benedict XVI)
In 1965, the Fathers of Vatican Council II addressed the lamentable condition of the closed, loveless, inward-looking self: "Many, it is true, fail to see the dramatic nature of this state of affairs in all its clarity for their vision is in fact blurred by materialism, or they are prevented from even thinking about it by the wretchedness of their plight. Others delude themselves that they have found peace in a world-view now fashionable.
There are still others whose hopes are set on a genuine and total emancipation of humankind through human effort alone and look forward to some future earthly paradise where all the desires of their hearts will be fulfilled.
Nor is it unusual to find people who, having lost faith in life, extol the kind of foolhardiness which would empty life of all significance in itself and invest it with a meaning of their own devising.
Nonetheless, in the face of modem developments there is a growing body of people who are asking the most fundamental of all questions or are raising them with greater clarity: What is humanity? What is the meaning of suffering, evil, death, which have not been eliminated by all this progress? What is the purpose of these achievements, purchased at so high a price? What can people contribute to society? What can they expect from it? What happens after this earthly life is ended?" (Pastoral Constitution on the Church, no. 10).
As mentioned, CONSCIOUS SPIRITUAL EXISTENCE emerges from a COMPOSITE WHOLE BEING comprised of BODY, SOUL, AND SPIRIT. Human nature, then, is a spirit-filled organic entity. This is true because human beings are an image of God: "Man is the summit of the Creator's work, as the inspired account [in the Book of Genesis] expresses by clearly distinguishing the creation of man from that of the other creatures: "God created man in his own image, in the image of God he created him; male and female he created them." Man occupies a unique place in creation: (1) he is "in the image of God"; (2) in his own nature he unites the spiritual and material worlds; (3) he is created "male and female"; (4) God established him in his friendship" (Catechism, no. 343).
Reality? This is reality! The Catholic Church expounds on this truth of human existence: "Of all visible creatures only man is "able to know and love his Creator." He is "the only creature on earth that God has willed for its own sake," and he alone is called to share, by knowledge and love, in God's own life. It was for this end that he was created, and this is the fundamental reason for his dignity" (Catechism, no. 356).
Thus, the Catholic Church emphasizes the integral unity of human nature: The human person, created in the image of God, is a being at once corporeal and spiritual. The biblical account expresses this reality. It affirms that "then the Lord God formed man of dust from the ground, and breathed into his nostrils the breath of life; and man became a living being." Man, WHOLE AND ENTIRE, is therefore willed by God" (Catechism, no. 362).
Again, the Catholic Church stresses that HUMAN NATURE is CONSCIOUS SPIRITUAL EXISTENCE: :In sacred scripture, the term "soul" often refers to human life or the entire human person. But "soul" also refers to the innermost aspect of man, that which is of greatest value in him, that by which he is most especially in God's image: "soul" signifies the spiritual principle in man (Catechism, no. 363).
Lest it be ignored or misunderstood, the human body is an integral element of CONSCIOUS SPIRITUAL EXISTENCE. The body is a SPIRITUALIZED MATERIAL ENTITY precisely because it is animated by a spiritual soul: "Man, though made of body and soul, is [an integrated] unity. Through his very bodily condition he sums up in himself the elements of the material world. Through him, they are thus brought to their highest perfection and can raise their voice in praise given freely to the Creator. For this reason man may not despise his bodily life. Rather he is obliged to regard his body as good and to hold it in honor since God has created it and will raise it up on the last day" (Catechism, no. 364).
Since it is an "out-looking" physical structure, the body demonstrates that human beings exist for the Other (God) and for others: neighbors and the natural order of things. Indeed, the most intrinsic meaning of the human body is discovered in lived love-relationships. Practically speaking, then, the vocation of love begins with and through the human body.
At the same time, the human body is a "fence," as it were, separating one person from another. No one, for example, can look inside another person. The materiality of the human body hides everyone's inner self and his or her interior life. Better said, except for God, no person can look into the inmost being of another person: "Man judges by appearances, but God looks into the heart" (1 Sm 16:7).
In other words, the human body is a defined "boundary," so to speak, that sets each person apart and aside from every other person. The body, then, prevents anyone from seeing or touching the loves found in another person's heart. Furthermore, in a curious way, the human body causes every person to be a stranger to every other person.
Nevertheless, there is still a third dimension to the physicality of human nature. The human body serves as a "connector" between one person and another person. Everyone first meets another person through the human body. In fact, it is only through the body that a person can see and communicate with another person. Moreover, it is through the body that every person sees his or her "outer self" and, thereby, can approach other outward-looking persons.
In the postures and gestures expressed by the human body, every person can be seen in a somewhat visible way. Stated differently, one person can see another person in the way his or her body acts or reacts. Through the external movements of the human body, it is possible, on occasion for one person to see what the interior life of the other person might be like.
Nonetheless, the human body is still a solid "fence," a definite "boundary," yet a "connector" for relationships and communication. Unlike animals, however, the physicality of human nature is not lived by the bodily senses alone. The human person, a spirit-filled being, can rise above and go beyond the limitations of the sensory life. Thus, everyone seeks to be conscious of and, thus, deepen their lived love-relationships with God, others, self, and the world - even if it is at a distance.
"As defined in sacred scripture, the Catholic Church adheres to the COMPOSITE WHOLE of human nature: "BODY, SOUL, AND SPIRIT" (2Th 5:23): The unity of soul and body is so profound that one has to consider the soul to be the "form" of the body, that is, it is because of its spiritual soul that the body, made of matter, becomes a living, human body; spirit and matter. In man, there are not two natures united, but rather the union of spirit, soul, and body forms a single nature"
(Catechism, no. 365).
"When speaking of the integration and completeness of human nature, the Catholic Church returns always to the Creator-Spirit, God: "The Church teaches that every spiritual soul is created immediately by God - it is not "produced" by the parents - and, also, that the spiritual soul is immortal. The spiritual soul does not perish when it separates from the body at death, and it will be reunited with the body at the final Resurrection" (Catechism, no. 366).
Given the fantasies and fallacies bandied about by the modern media, to secure the integrity of human nature and, thus, the identity of the human person, the Catholic Church imparts this instruction: "Sometimes, the soul is distinguished from the spirit. Saint Paul, for instance, prays that God may sanctify his people "wholly," with "body, soul, and spirit," kept sound and blameless at the Lord's coming. The Church teaches that this distinction does not introduce a duality into the soul. "Spirit" signifies that, from Creation, man is ordered to a supernatural end and that his soul can gratuitously be raised beyond all it deserves to communion with God" (Catechism, no. 367).
Now, the "Who am I?" question, can be answered - with two words: being and becoming! BEING is who I am: a spirit-filled, composite entity created by God. BECOMING is how I choose freely to mature in the vocation of love given at the very moment of conception in my mother's womb. Therefore, the following presentations will continue to explain the vocation of love in terms that pertain directly to: the subjective order. "Male and female he created them."
John D. Meehan has been involved in the lay apostolate of the Catholic Church since the close of the Second Vatican Council. He resides in New Hampshire with his lovely wife Elizabeth.
St. Matthew
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St. Isidore the Farmer
By Hannah Raissa Marfil (NEWS CONSORTIUM)
The AAA Foundation for Traffic Safety analyzed about 1,700 takes of footage involving teenage drivers and the moments just before they crash. The videos are horrifying and disturbing, as the common cause of the accidents is distracted driving. MUNTINLUPA, PHILIPPINES ... continue reading
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Linnea Johansson became worried after seeing her usually emotional 3-year-old son do his best not to cry, saying he wouldn't cry because Spiderman doesn't. As a mother bothered by how gender stereotypes are affecting her child and a fan of comic books who believes that ... continue reading
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An upsetting video of an older woman ranting, giving disgusting suggestions about a misbehaving child she witnessed at a grocery store has gone viral. Identified as Kathleen Smith from Tomball, Texas, she suggests the mother could have done something so the child ... continue reading
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Couples should learn to relate with one another, in order to conquer challenges they cannot fully anticipate, according to David B. Hawkins from crosswalk.com. Some people assess that the individual person gets along more with others than their spouse. Hawkins ... continue reading
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Heather Barwick is a gay marriage advocate who was raised by two lesbian mothers. However, she recently published an open letter entitled "Dear Gay Community: Your Kids Are Hurting" in the Federalist. She stated she loves her two mothers and the gay community, but now ... continue reading
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A new campaign, in Ontario, directly asks people who they will help: the perpetrators or the victims. The video shows the way bystanders are being "accomplices" and also guilty of the sexual assault when they opt out from doing something and keep their silence. ... continue reading
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The intelligence developed by babies from stable breast-feeding carries on into their adulthood and makes them earn more money, according to research. MUNTINLUPA, PHILIPPINES (Catholic Online) - Although there are existing studies that establish correlation between ... continue reading
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Houston Gamer
Video games with Willie Jefferson
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Wheels down, Houston: Call of Duty Ghosts arrives
"Remember, Remember the 5th of November …" No, I'm NOT channeling "V" from "V for Vendetta," but today is the launch day for "Call of Duty: Ghosts." There's no doubt in my mind that MILLIONS of gamers worldwide will be clogging up the Interweb with headshots, dog fights, backstabs and the like. Oh, and calling […] [Read More]
Sony confirms PlayStation 4 launch lineup (Nov. 15th)
Let me be the first to say – there are so many nuggets in Sony's PlayStation 4 FAQ that it's very easy to overlook the wealth of information packed into it. This gem is one of the best. Sony actually noted the PS4′s launch lineup in the middle of it. Here it is: Sony Computer […] [Read More]
This weekend – Wii Party U and the GameStick
M4d Ski11z: THIS WEEKEND, I'll be tossing it up with "Wii Party U" for the Nintendo Wii U and the PlayJam Gamestick. You don't know what the Gamestick is? It's a small HDMI device that's packed with an Android OS. It comes with a controller and the ability to download Android-flavored games. Of course, I'm […] [Read More]
THIS WEEKEND – BEYOND: Two Souls, GTA V
This weekend, M4d Ski11z steps into the role of a paranormal in the upcoming thriller, "BEYOND: Two Souls." More about his weekend plans on the jump. [Read More]
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Houston Gamer's M4d Ski11z got a chance to quiz Julie Uhrman, co-founder of the OUYA microconsole. Check out her interview with Ski11z on the jump. [Read More]
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Microsoft's Major Nelson unboxes the Xbox One. What do you think about the new additions, including the headset? Have your say. [Read More]
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Posted May. 14, 2012 at 2:43 p.m.
Raleigh, N.C. — Scientists at N.C. State, private company HexaTech and researchers in Japan find way to produce semiconductors in LEDs that don't absorb infection-killing UV light waves....
LED lighting could be infection killer, NC researchers find
HexaTech graphic explains its chip focus Image 1 of 2 · Next Image…
Raleigh, N.C. — Scientists at N.C. State, private company HexaTech and researchers in Japan find way to produce semiconductors in LEDs that don't absorb infection-killing UV light waves....
Editor's Blog
The latest blog posts from our WRAL TechWire and WRAL editors. Read more articles…
I am surprised to see Hexatech graphics still out of date with modern measurements of the thermal conductivity of GaN. NCSU Professor John Muth's group has published some of the best numbers - see C. Mion, J. F. Muth, E. A. Preble, and D. Hanser, "Accurate dependence of gallium nitride thermal conductivity on dislocation density," Appl. Phys. Lett. 89, 092123 (2006). Also, we have commented in detail on such matters at http://kymatech.com/news/85-kyma-discusses-the-thermal-conductivity-of-their-gallium-nitride-materials.
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Whether active duty, reservist, or guardsman, you trained in your MOS or rate to a level of world-class proficiency.
Career/Life Coaching
Job #1 : Has anyone asked you, "Forget what you know how to do, or what training you have or do not have, and tell me what it is you would like to wake up each morning and do, every day, that would result in you laying your head down at night feeling great about the day you just had?" This question and others are an important part of life coaching and career/life counseling. Let's begin the conversation!
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GALLIANO, LA -- The Greater Lafourche Port Commission announced that the latest phase of Slip C dredging is complete, carving out a new expanse of waterfront property where there once was just open water. The Slip C Additional Hydraulic Dredging Project, which followed the initial Slip C dredging project in early 2012, creates a new slip in the Northern Expansion area of Port Fourchon.
The new Slip C is a 400-acre development which will provide 16,300 linear ft of new waterfront space in Port Fourchon. Once the adjacent property is filled and developed, companies will be able to lease up to 280 acres of waterfront property along the new slip. A fully developed Slip C is estimated to take five to eight years to construct, but over half of the Slip C waterfront property is already promised to companies who have signed leases or rights of first refusal.
"With continued investment in development projects like these," said Port Director Chett Chiasson, "we will be able to provide the basic infrastructure needed to serve our current tenants and attract future tenants, which ultimately means creating jobs and revenue for our community and the surrounding area."
Board President Larry Griffin is excited to see how quickly the port is growing. "People believe in our port and the way we do business. We're leasing out property faster than we can build it. It's amazing to see the port grow before our eyes."
And all of that growth takes a mixture of initiative, cooperation, time, and money. The Port Commission has historically re-invested money generated from the port into new port development projects, planning decades in advance, seeking grant funding, and working with private and public stakeholders to construct projects that best suit both business and environment. To date, the Slip C dredging project has placed approximately half of dredged material to fill in new property and half in a mitigation area that both builds additional habitat areas for wildlife and acts as a natural barrier to protect Port Fourchon from storm surge.
"Port Fourchon is in the forefront of the push to use dredge spoils beneficially," Port Director Chett Chiasson said. "Not only do we work to dredge channels at depths that port businesses need, but we also use the dredged materials to create new waterfront property and new marshlands as part of our mitigation. We make room for industry and at the same time enhance our environment with a balance that few other places can achieve."
The dredging of Slip C, which was funded by port-generated revenues and LA DOTD's Port Priority Program, was completed in early December, and bulkhead projects are anticipated to follow in 2013.
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Stadtarbeit: Open Call for Social Design Projects
Stadtarbeit is a format that offers designers the opportunity of participating in the festival with projects from the field of social design. The submitted projects [more] should demonstrate outstanding qualities in addressing social themes and are to be selected by a jury.
We are looking for outstanding social design projects beeing realized during VIENNA DESIGN WEEK 2015. Follow the link bellow for any further information.
Stadtarbeit: Open Call for Social Design Projects
© Sandra Jedlicka
MOCHI & KIM+HEEP: "Heimweh/Fernweh"
Die Kollaboration des Koreanisch-Deutschen Gestalter-Duos KIM+HEEP und den Machern des zeitgenössisch-japanischen Restaurants MOCHI untersucht damit auch die positiven Potentiale einer kulturellen Globalisierung. Dabei ist eben nicht der Einheitsbrei, sondern die gegenseitige angstfreie Inspiration und Rekombination gemeint.
Heimweh/Fernweh – Der Beginn
4. März 2015
18:30, stilwerk Wien, 1.OG
Das Festival
VIENNA DESIGN WEEK is Austria´s largest design festival, with a variaty of locations and events in Vienna. Last year it delighted over 34.200 visitors. The festival, curated by Lilli Hollein, will enter ...
Festival Headquarter
Concealed behind the high-spray fountain on Schwarzenbergplatz, the noble Palais Schwarzenberg has in recent years been something of a sleeping beauty. Before the historical palace and gardens will be ...
Focus District
Every year, next to several substantive focuses, the VIENNA DESIGN WEEK also chooses a geographical focus and declares it as its so called focus district. The focus district will be hosting the festival ...
Host Country
In its aspiration to provide a multifaceted view into the creativity of other European countries, the VIENNA DESIGN WEEK puts the focus on a different country every year. Although design is becoming increasingly ...
Every year five designers and design teams will receive a free ticket to take part in the festival and to present themselves in the category Social Design. Stadtarbeit [City Work] sees its task not in ...
Programme Partners - featured by Vienna Design Week
Every year design studios from home and abroad, museums, galleries and business enterprises accept our invitation to come to the festival and contribute as programme partners of the VIENNA DESIGN WEEK ...
Every year, the "Passionswege" provide a framework for bringing Austrian and international designers together with Viennese producers and businesses to share and exchange their knowledge in order to ...
The VIENNA DESIGN WEEK Laboratory is an impulse project supporting interdisciplinary projects at the interface of product design and communication design and makes these design processes accessible. The ...
The VIENNA DESIGN WEEK Debut offers two universities each year the chance of presenting a cross-section of their work or a special project from their design departments. Hence soon-to-be designers are ...
The VIENNA DESIGN WEEK has set itself the aim to be a festival for the public, for all the Viennese and all who take pleasure in design, people who are entertained, informed and astonished by it – and ...


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Smell of paint makes 4 Disneyland employees feel sickby Leah Zanolla
Mar 29, 2014
The smell of paint caused four Disneyland employees to be transferred to a hospital for evaluation Friday, March 28. Anaheim Fire & Rescue were called to the park around 1:30pm for a "possible hazardous material situation," which later turned out to be paint fumes. The fire department said no park guests were affected, nor were park operations.
Anaheim Fire Rescue later tweeted: The odor paint caused 4 employees to feel nausea. Not effecting park guests or operations. The employees were taken to area hospitals.
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Runners dressed in costume for the inaugural running of the Avengers Superheroes Half Marathon this weekend in Anaheim.
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- Succumbing to the Charms of Hong Kong Disneyland - Not surprisingly, the park has many things in common with the other two "Magic Kingdom" style parks I have visited, and it also has many features that make it unique.
- Disneyland announces Diamond Celebration Merchandise Event - The Disneyland Resort has announced a merchandise event to take place May 20 and 21, just before the Diamond Celebration kicks off.
- Save up to 20% on Premium Rooms at the Disneyland Resort - Save up to 20% on Premium rooms at the hotels of the Disneyland Resort select weeknights this Spring.
- Spring Time Roundup 2015 at Big Thunder Ranch - Check out Spring Time Roundup at Big Thunder Ranch where you can meet the Easter Bunny, Mickey and Minnie in their finest Spring outfits and Disney rabbits including bunny Pluto!
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- Menu for the re-themed Smokejumpers Grill at California Adventure - This was formerly the Taste Pilots' Grill.
- Smokejumpers Grill now open at California Adventure - Smokejumpers Grill is now open at California Adventure in the former Taste Pilots' Grill location.
- Five Guests who turn into Disney Villains
- Driving to Disney - do you drive overnight or during the day?
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- Smokejumpers Grill opens March 20 at California Adventure - As part of the re-theming of Condor Flats to Grizzly Peak Airfield, Taste Pilots Grill re-opens March 20th as Smokejumpers Grill.
- St. Patrick's Day at Disneyland's Downtown Disney - For St. Patrick's Day, several of the restaurants in Downtown Disney will have special menu items. Entertainment will be offered near the WonderGround Gallery.
- Disneyland's Innoventions closes March 31st - Disneyland has announced that Innoventions will be closing on March 31, 2015.
- Walt Disney and The Little Golden Books
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