Patent Application
20160221228
The present invention relates to the field of production of topographically surface structured cellulose patches or elements and devices such as patches or elements, generally speaking devices, also 3D devices, made using such a method or using elements made using such a method.
Wound dressings are designed to support the wounded region, protect it from infection, and, in certain cases, actively promote wound healing by creating a favorable environment for cell growth.
The response to wounding, defined as a breakage of bodily tissue, involves an inflammation phase, a migratory phase and a remodeling phase. The inflammation phase is the acute response to a wound and its purpose is to quickly seal the wound and produce chemical factors that employ cells to migrate into the wound and start the wound healing process. During the migratory phase, cells rapidly migrate into the wound and start laying down provisional extracellular matrix that will be the base of the healed tissue. During the remodeling stage, the newly created tissue slowly matures into its permanent form. Standard wound dressings facilitate wound healing by: 1. mechanically holding the wound edges together to allow easier cell migration; 2. mechanically sealing the wound to prevent contamination by pathogens; 3. in some advanced dressings providing an environment that actively promotes faster wound healing, usually by exposing the wounded tissue to a hydrated gel. Improved materials for these applications would be desirable.
In plastic surgery commercial silicone implants (e.g. breasts, calf, buttock, chest, biceps) often fail due to foreign body reaction and scar-tissue encapsulation. Also for such applications improved materials/coatings would be desirable.
In cosmetics disposable flat and unstructured cellulose masks are currently sold (e.g. face, hands and feet) as masks that enhance skin hydration, as well as the absorption of metabolic waste products and the release of nutrients or other compounds to the skins
It is therefore an object of the present invention to propose new methods for the manufacturing of materials for these applications, in particular topographically surface structured patches or coating, most particularly cellulose patches or elements and devices such as patches or elements, generally speaking including devices, also 3D devices, made using such a method or using elements made using such a method.
One important element of the proposed invention is the description of a simple and cost-effective procedure to transfer micro or nano-structured topographical features on the surface of bacterial cellulose. The process can be performed with minimal costs and laboratory equipment and does not require trained personnel. The process can be fully automated and exploits the self-assembly of cellulose by bacterial strains such as (but not only) Acetobacter Xylinum or Gluconabacter Xylinum. The protocol is schematized in
The process of Self Assembled Biolithography (SAB) is based on a simple technique which takes inspiration from soft lithography (SL). However, while for SL the molding of the target elastomer requires trained personnel and several passages, thus resulting in a time consuming and delicate procedure, the SAB is fully based on the natural process of cellulose production by bacteria. SAB interferes with this process locally directing the assembly and polymerization of cellulose fibers, and thus resulting in a high-fidelity negative replica of the surface geometry presented by the mold.
The natural porosity of bacterial cellulose can be controlled modulating the growth conditions of bacterial (Oxygen (partial) pressure, glucose concentration, bacterial strain) or can be modified on already assembled cellulose meshes with post-production chemical treatments. However, to our knowledge, the transfer of regular topographic arrays on the surface of contiguous cellulose matrixes of sufficient thickness to lead to a product that can be handled and commercially used has never been achieved.
The number of applications of bacterial cellulose is countless, and several items made of bacterial cellulose are currently available on the market for cosmetic or medical applications. Additionally, bacterial cellulose has been proven to have superior mechanical properties and to be virtually inert upon implantation in animals. FDA approval of this material for implantable biomedical devices will further increase the number of medical applications profiting from bacterial cellulose. The proposed invention adds a totally independent, critical, physical parameter to control the interaction between human tissues and bacterial cellulose. Controlling the surface topography of the cellulose, the adhesion, proliferation, differentiation, migration and apoptosis of cells can be modulated or directly induced. Therefore, the cost-effective engineering of the material surface may provide a full range of novel properties that improve the biocompatibility and effectiveness of biomedical devices featuring bacterial cellulose.
So a Self-Assembled Biolithography (SAB) is proposed as a new protocol to introduce pre-defined geometries in the micron and submicron scale on the surface of bacterial cellulose. The protocol applies the principles of elastomer-based soft lithography and allows for a cost-effective replica molding upon the generation of a cellulose network. In particular, a mold of an oxygen permeable material, preferably an oxygen permeable polymeric material, and most preferably a siloxane, e.g. (polydimethylsiloxane) PDMS mold is created via standard soft-lithography on a molding mask. The molding mask featuring the target geometry can be made of any thermoplastic polymer and is generally (but not exclusively) obtained via standard hot embossing. The PDMS mold is then placed at the interface with the bacteria growth media and represents a gas-permeable scaffold fostering the polymerization of bacterial cellulose. SAB yields a patch of cellulose featuring a negative replica of the surface geometry presented on the PDMS mold, and therefore equal to the initial molding mask. The surface-structured bacterial cellulose patch can be finally peeled from the PDMS mold without further passages. Both the PDMS mold and the original molding mask can be used for several cycles without deterioration. The SAB procedure allows a high-fidelity transfer of any texture in the micron and submicron range from a PDMS mold to bacterial cellulose and does not require any further action by the operator. In all, we invented a new method to couple the bio-chemical properties of bacterial cellulose to the control of cell-material interaction with interface textures. We envision the exploitation of this new technology to produce improved biological coatings, wound dressings, and/or cosmetics with enhanced interaction with human tissues.
The invention more specifically proposes a Self-Assembled Biolithography (SAB) protocol including the following steps: A mold, preferably a PDMS mold, is created, preferably via standard soft-lithography casting PDMS on a plastic mask (upper row). The mask featuring surface topography can be made of a thermoplastic polymer and is generally (but not exclusively) obtained via standard hot embossing. SAB using the mold as a structuring template using a cellulose producing strand in a growth medium on the topographically structured surface and allowing for oxygen to pass through the mold produces a flat cellulose patch with a negative replica of the surface geometry presented by the (PDMS) mold. The mold is simply placed at interface with the growth media and represents a gas-permeable scaffold inducing the polymerization of bacterial cellulose. The cellulose patch can be finally peeled from the PDMS mold without further passages.
As such there have been several attempts to surface texture cellulose structures. It has for example been proposed to use existing cellulose material and to emboss a surface structure mechanically. However using this technique leads to significant density changes in the resulting cellulose material, leads to destruction of the cellulose structure on a molecular level, resulting in insufficient homogeneity. In addition to that, in this case there is only the surface structure, while in the proposed approach there is not only the surface structure but also, due to the self assembled growth, a particular orientation of the cellulose fibres in this surface structure which is associated with and related to the surface structure. This has additional effects which cannot be achieved by embossing techniques.
Other approaches have tried to self assemble cellulose on a structure, however in all these cases no contiguous layers could be produced but only gridlike structures and in addition to that, due to the fact that they were grown on templates without allowing oxygen to be present on the interface between the template and the place where cellulose is generated, the resulting cellulose structures are inherently extremely thin and cannot be handled. So all these approaches did not lead to products which can actually be commercially used. More generally speaking, the present invention relates to a method for the self assembled production of a topographically surface structured cellulose element. The method involves a first step with the following elements:
In a subsequent second step the element is removed from said mold.
The term “topographically surface structured” according to the invention is in particular to be understood as follows:
The surface is provided with a topography, i.e. a 3D surface profile, which is a regular array of geometric features in at least one direction of the surface plane. This regular array of geometric features is preferably defined by the following parameters:
This notion of “topographically surface structured” is thus different from a normal surface roughness (what is obtained by simply growing cellulose), which is a random and not regular structure, and which roughness is characterized as the deviation of a surface from a perfectly flat profile. In this definition of “topographically surface structured” two surfaces can have similar roughness but display different topographic features, or feature arrangement.
The main practical difference between a topographically engineered cellulose surface and a native rough cellulose is that in the first case essentially ALL cells interacting with the surface will receive the same rationally-designed topographical signal, in the second case each cell (depending on the specific region of interaction) will receive a different set of signals. For this reason a coordinated cell response (such as the one required for wound healing or for the inhibition of inflammatory reaction) can ONLY be obtained using a topographically engineered surface.
One of the key and unexpected properties of the resultant structures is the finding, that they are stable upon dehydration/rehydration so there is a shape memory, the shape is preserved if an element is dried (and the topographically structured surface collapses) and subsequently wetted again (which leads to a swelling and a regeneration of the topographically structured surface). This simplifies and extends storage and makes the structures less prone to degradation.
As medical device, surface structured cellulose can be used for obtaining the following effects, alone or in combination:
According to a first preferred embodiment, the element has a thickness in the range of 0.5-10 mm or even up to 15 mm.
Preferably, the mold has a diffusivity to oxygen of at least 10−6 cm2/s. Preferentially, the mold, which can be a two-dimensional or three-dimensional mold, is made of siloxane, preferably PDMS, preferably produced in that a topographically complementary structured mask element is used as a template for a liquid applied or injected substrate material, preferably in a soft lithography process, optionally followed by a cross-linking and/or polymerization step, further optionally followed by a surface treatment step, preferably a plasma treatment step on the topographical surface.
The first surface can, according to yet another preferred embodiment, have a topographical structure in the form of a two-dimensional array of pillars, indentations, in the form of an array of ridges/grooves or in the form of a two-dimensional crossing structure or honeycomb patterned structure. So also the negative of a regular pillar structure, a two-dimensional array of indentations, is possible.
Generally speaking, the width of the positive structures, in particular of the ridges and/or of the negative structures, in particular of the grooves, is in the range of 0.5-100 μm, wherein preferably the width of the ridges is in the range of 0.5-5 μm and the width of the grooves is in the range of 0.5-5 μm, preferably both widths being essentially equal.
Even more preferably, the ridges have a height h of at least 0.4 μm, preferably in the range of 0.5-5 μm or in the range of 0.5-2 μm, more preferably in the range of 1-2 μm.
Generally speaking, preferably the (essentially all identical) pillars preferably all and regularly have a round, oval or polygonal cross section, preferably a regular polygonal, most preferably triangular, square, pentagonal or hexagonal cross-section.
The two-dimensional array of pillars can have a periodicity in one dimension in the surface plane, preferably in two or three different directions in the surface plane in the range of 5-50 μm, preferably in the range of 7-15 μm. Preferred is in case of e.g. hexagonal pillars an arrangement where there are three such periodicity directions skewed at 0°, 60° and at 120°. Preferably the periodicity is the same along the different directions in the surface plane.
The individual pillars can have a lateral extension in the range of 2-20 μm, preferably in the range of 4-10 μm.
Further preferably the individual pillars can have a height in the range of 0.2-5 μm, preferably in the range of 0.5-2 μm.
Preferred are regular two-dimensional arrays of hexagonal pillars, where preferably the periodicity is twice the lateral extension of the individual pillars.
When talking about pillars as detailed above in terms of dimensions this preferably also includes corresponding negative structures, so structures where there are no regular pillars but regular indentations having the shape of these pillars.
In the second step the mold with the element on its topographically surface structured first surface can be immersed into a liquid, and the element can be removed, preferably peeled off, from said first surface in said liquid, wherein said liquid preferably can be a NaOH solution, preferably with a concentration in the range of 0.5-2M, preferably around 0.5M. According to yet another preferred embodiment, after the second step the element is heat-treated, preferably by keeping it at a temperature above room temperature in a liquid, preferably in a NaOH solution, for a time span of more than 10 minutes, preferably of more than 60 minutes, most preferably at a temperature above 60° C.
The invention furthermore relates to a topographically surface structured cellulose element being in the form of a contiguous layer with a thickness in the range of 0.5-5 mm, preferably produced or producible using a method as outlined above, and having a topographical surface structure with a height in the range of 0.5-2 μm (micrometer), and in case of a groove/ridge topographical structure α periodicity of the structure in the range of 0.5-100 μm (micrometer).
This topographically surface structured cellulose element can, in particular for healing patch applications, be a patch, and this patch is generally bi-dimensional, so it extends as a flat layer in two dimensions and each dimension is larger than 1 mm, preferably larger than 2 mm, and it can be as large as 5-20 cm. The 2D shape of such a patch can be depending on the needs, e.g. square, circle, triangle et (see also
Furthermore the invention relates to the use of such an element as a patch for wound healing and/or cosmetic applications, as a coating for an implant or plastic surgery structure.
Possible is also the use of such a patch, made e.g. as a 3D element, e.g. in the form of a pouch, for covering an implant or for forming an implant as such.
In this sense the present invention also relates to a topographically surface structured element according to claim 12, wherein it has the form of a three-dimensional structure, with or without support element, and wherein preferably it has the form of a cover, bag, coating, pouch or pocket into which an implantable object can be put or to cover an implantable object, wherein preferably the implantable object is selected from the following group: cardiovascular device, in particular a pacemaker; cosmetic implant, preferably in the form of a breast implant, cuff implant, pectoral implant, biceps implant, buttock implant, gluteal implant; orthopedic prosthesis; a sensor and/or electrical stimulation device; draining system, preferably a catheter; pump or tubing system; ophthalmological device; hearing device; bionic device.
The following uses of such 3D coatings/pouches/covers/pockets/bags are thus generally possible:
commercial silicone implants (e.g. breasts, calf, buttock, chest, biceps) fails due to foreign body reaction and scar-tissue encapsulation. Cellulose prevents both these reactions. Enveloping of these implants in micro-patterned cellulose not only prevents negative reactions by the human body, but also improves a positive cellular reaction in the surrounding tissues. Micro-patterned cellulose would add market value to existing implants, by drastically reducing their failure rate.
disposable cellulose masks are currently sold (e.g. face, hands and feet) in cosmetics as masks that enhance skin hydration, as well as the absorption of metabolic waste products and the release of nutrients or other compounds to the skins. Commercial cellulose masks do not feature any surface topography. Micro-patterned topography would enhance i) mass transport with the skin by increasing the surface in contact with water or other liquids, ii) cellular response by apical guidance on the epithelial tissue. SAB can be adopted to develop micro-patterned cellulose cosmetic masks: SAB would increase performances of cosmetic masks and ultimately add market value to them.
3) Patch and wound healing:
cellulose is a fully biocompatible material that is ideal for contact with damaged tissues (artificial skin for burn treatments, occlusive dressing for chronic wounds such as diabetic ulcers, common friction wounds, etc. . . . ). Micro-patterned topography stimulates cellular response, in particular of human dermal fibroblasts (HDF), in terms of spreading, proliferation, differentiation and directional migration, which ultimately enhance the efficiency of wound healing. SAB can be used to fabricate micro-patterned cellulose patches for the handling of wounds.
Further preferred embodiments of the invention are outlined in the appended claims.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
PDMS molds were made of Polydimethylsiloxane (PDMS, Dow Corning, USA) at 1:10 mixing ratio. The mixed PDMS was degassed in a vacuum chamber for 10 minutes to remove trapped air and poured at 500 μm thickness onto a micropatterned cyclic olefin copolymer (COC) mask consisting of parallel grooves with 2 μm period, 1 μm groove width and 0.6 μm groove depth. Subsequently, the PDMS was briefly degassed for a second time and cured for 4 hours at 60° C. The cured PDMS molds were separated from the mold with tweezers and cut into squares of 1 cm2 with a scalpel. Blank molds were similarly created by pouring PDMS onto flat COC substrates for comparison purposes. Subsequently, all patches were left in ethanol overnight to dissolve any uncrosslinked material. The molds were then treated with oxygen plasma to increase the hydrophilicity of the surface. A process time of 120 seconds at 10 W was chosen after testing a range of intervals from 30 to 150 seconds as the one yielding the lowest contact angle (20.2±0.5°).
The mold 2 has grooves 6 with a width f and ridges 5 with a width e. This shall be illustrated in somewhat more detail in the context of
Within
Possible geometrical shapes of topographical arrays on healing/dressing patches are illustrated in
However, more complex geometries can be realized to interact with circular or complex-shaped wounds or burns. These geometries refer to the macroscopic pattern while at the micro-scale or submicron scale the topographical features may retain the same or similar feature size and periodicity. Visual examples are given below for the case of a longitudinal wound A), of a circular wound B) or of a complex shaped wound C) in
Growth of the Cellulose Patch on the Mold:
Materials:
Acetobacter Xylum (ATCC - strain 700178)1
1
A. Xylinum belongs to the family of bacteria that ferment carbohydrates to vinegar and is commonly found in soil and decaying fruit. It is peculiar for its cellulose production. Also other bacteria can be used having similar properties.
Procedure:
The resulting solution is autoclaved for 30 minutes at 121° C. After cooling down to room temperature, 50 ml of a filtered Glucose solution (50% in distilled water) is added.
Set-up of the bioreactor:
For a 10 cm Petri dish.
These values can be influenced by increasing the oxygen (partial) pressure across the mold.
Harvesting of the cellulose patch:
This procedure yields a semi-transparent cellulose patch (in the visible spectrum). To improve transparency (up to 90% of incident light) longer incubation in ethanol (up to 1 week) can be used.
As pointed out above, pillar structures are preferred, but also the corresponding negative structure is possible, i.e. a structure where there is a regular two-dimensional array of indentations, and in which the indentations are all essentially identical. In
In vitro vivo studies of patches of mice:
Animal tests were performed on Male C57BL/6J mice with a body weight of 25 to 35 g (n=18) in order to assess:
1. Wound healing performance of cellulose patches with topographically engineered surface.
2. Inflammatory and revascularization process in presence of cellulose
To study the revascularization process the modified dorsal skin fold chamber (MDSC) was used. Briefly, for chamber implantation, two symmetrical titanium frames were mounted on a dorsal skin fold of the animal. One skin layer was then completely removed in a circular area of 15 mm in diameter, and the remaining layers (consisting of striated skin muscle, subcutaneous tissue and skin) were covered with a glass cover slip incorporated into one of the titanium frames. Before skin grafting, a recovery period of 3 days was allowed. Then, skin and most parts of the hypodermal fat layer were carefully removed in a circular area of 7 mm in diameter from the back of the chamber in order to create an artificial full-thickness wound. The defect on the back of the chamber was then covered with bacterial cellulose substrates and covered with a glass cover slip incorporated into the other titanium frame. Surface-structured bacterial cellulose substrates were placed with gratings directed towards the wound bed. Additionally, to investigate the biocompatibility of bacterial cellulose substrates, all animals received a replica of the same bacterial cellulose substrate in a skin pocket in the groin.
The overall results have shown minimal inflammation in the wound bed in the presence of bacterial cellulose patch. This was as low as with a full skin graft of the same animal (used as control).
Therefore bacterial cellulose is confirmed as an ideal material for wound treatment and implantation. Additionally, insights from quantitative histological collagen formation analysis and distribution revealed that the group treated with topographically engineered cellulose had (after 21 days) already re-established a dense, homogenously distributed layer of collagen fibers. Therefore the performance of topographically engineered cellulose patches was superior to the one of identical non-structured patches.
3D cellulose structures are obtained by introducing 3D silicone molds with the bacterial culture. The silicone molds feature surface topography, an example is illustrated in
The silicone mold with surface topography is placed in the bacterial culture so to allow for:
complete wetting of the silicone mold external surface
The placement of the silicone mold can be helped with a bioreactor consisting of two chambers, for air and bacteria in medium, respectively. Oxygen circulation within the air chamber can be facilitated by leaving the chamber open or by controlling the oxygen flow in it, by using e.g. a pump or a gas bottle with a system of valves.
A schematic representation of a bioreactor configuration is illustrated in
After the culturing time, a cellulose layer is formed at the mold interface. The cellulose pouch/cover/pocket features surface topography on its internal surface and can easily be removed, washed, processed and sterilized as previously described for the flat cellulose patches. The cellulose pocket is eventually flipped inside-out in order to feature surface topography on its external surface. The target object can eventually be inserted within the cellulose pocket. The enclosing of the object can be optimized by suturing the open side of the cellulose pocket.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13185146.1 | Sep 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/069882 | 9/18/2014 | WO | 00 |
