WO2016030481A1

WO2016030481A1 - Scaffold comprising buckled fibers

Info

Publication number: WO2016030481A1
Application number: PCT/EP2015/069687
Authority: WO
Inventors: Lorenzo Moroni; Roman Kurt TRUCKENMUELLER; Honglin Chen
Original assignee: Universiteit Maastricht
Current assignee: Universiteit Maastricht
Priority date: 2014-08-27
Filing date: 2015-08-27
Publication date: 2016-03-03
Anticipated expiration: 2017-02-27

Abstract

The invention relates to a method for making a buckled fiber, comprising a) depositing at least one fiber on a surface of a shrinkable material, which material is shrinkable in at least one direction, wherein the deposited fiber at least partially adheres to the shrinkable material; and b) shrinking the shrinkable material in at least one direction, thereby buckling the fiber. Furthermore, methods to make a buckled fiber mesh by buckling one or more layers comprising a multitude of fibers are also disclosed. A buckled fiber and a buckled fiber mesh as described can be used in a variety of applications, among which tissue engineering, filtering, catalytic materials and textiles.

Description

SCAFFOLD COMPRISING BUCKLED FIBERS

Field of the invention

The invention pertains to a method of making buckled fibers. The invention also pertains to a method of making a buckled fiber mesh, as well as to buckled fibers and buckled fiber mesh, and use of a buckled fiber and/or a buckled fiber mesh in various applications.

Background of the invention

Fibers are well-known. Usually, fibers are straight or relatively straight threadlike materials, commonly made of synthetic polymers or natural polymers such as cotton or silk. Also, biology abounds with natural fibers, such as for example collagen fibers or various peptide or polysaccharide fibers.

Natural biological fibers are usually not straight, but are often shaped in wave- like or sinusoidal patterns, or even in corkscrew-type/like or coil/spiral spring-type/like curls. There has been ongoing interest to create synthetic fibers with similar curves and/or curls, among for others tissue engineering purposes. However, existing techniques suffer of various drawbacks, which makes that no easily applicable method for the creation of curved fibers exist, or for the creation of fiber materials comprising a curve/wave/crimp or even curl pattern. Problems with existing methods include the difficulty of control over the curved pattern, the complicated process, higher cost and lower productivity. Furthermore, the available methods are only able to create a curved pattern on single fiber but show no answer to create curled pattern on a fiber mesh.

Caves (Adv. Mater. 2010, volume 22, issue 18, pages 2041 -2044) describes microcrimped collagen fibers, which are obtained by a membrane templating technique. Polyurethane membranes are crimped and used as a template to create extracellular cell matrix (ECM) mimicking materials. However, the prior fabrication of a template, which needs to be frozen at low temperature and fixated using glutaraldehyde vapor, makes that the crimp pattern cannot be easily adjusted. In addition, the process for making the crimped collagen is multistep and complicated. Varesano (Eur. Pol. J. 2007, volume 43, issue 7, pages 2792-2798) describes an electrospinning process for making polyamide nanofibers, which are formed into crimped filaments by electrospinning in a cylinder which comprises a tangential air flow. The air-flow drives the polymer jet into the electric field, thereby curling the filament to allow collection of curled filaments on the collector. This technique has as a drawback that only singular fibers can be curled, not whole fiber materials, and the equipment needed is highly specialized. Also, there is little control over the crimp pattern, and large amount of fibers still straight instead of curling.

Denver (Biomacromolecules 2010, volume 1 1 , issue 12, pages 3624 - 3629) describes self-crimping electrospun fibers made of poly(LLA-CL) by depositing the fibers on a rotating mandrel. The crimping is the result of residual stress present in the fibers due to the alignment process, and by the difference between operating temperature during electrospinning and the glass transition temperature of the polymer. A drawback of this technique is that careful selection of the polymer is required, because alignment of the fibers during electrospinning should result in sufficient stress to allow for spontaneous crimping of the fibers after electrospinning. In addition, there is limited control over the curled pattern, and only single fibers can be curled, not whole fiber materials.

Lin (Adv. Mater 2005, volume 17, issue 22, pages 2699 - 2703) describes electrospun composite fibers of polyacrylonitrile and polyurethane. Removal of the polyurethane results in nanofibers with a U-shaped cross-section, which curve. Also here, the curvature is difficult to control, and only single fibers and not whole fiber materials can be made. In addition, a high fraction of material is waste material.

WO 1992 - 07981 also describes curved fibers, made of an inorganic or an organic precursor fiber. Here, single fibers are crimped by a mechanical crimping means in combination with a heating or irradiation zone. However, only single fibers can be crimped, and control over the crimping pattern can only be achieved by adapting the mechanical means, which precludes flexibility over the crimping pattern.

Electrospun scaffolds have been widely used in tissue engineering (Biomaterials 1999, volume 26, issue 15, pages 2527-2536; Adv. Funct. Mater. 2009, volume 19, issue 18, pages 2863-2879). However, such scaffolds generally suffer from the problem that there is insufficient cellular infiltration due to fiber arrangement and density. In addition, they are microscopically too stiff. The present invention is directed to a method which overcomes at least the above drawbacks. Legend to the figures

Figure 1 : A Scheme describing the setup for buckling at least one fiber using ESP and a thermoshrinkable material. Figure 2: A fiber before (A) and after (B) buckling on a poly(lactic acid) (PLA) shrinkable material.

Figure 3: Buckling of fibers with different alignments relative to the direction of shrinkage. "Straight": before buckling; "curled": after buckling. From left to right: perpendicular, parallel, 1^st diagonal 45 °, 2^nd diagonal

45 °. X axis means the direction of shrinkage.

Figure 4: Diagram displaying the size differences in amplitude and wavelength for fibers buckled in different orientation. The number of stars indicates the statistical significance for a comparison between the parallel and diagonally oriented fibers: one star (^*) means p-value < 0.1 , two (^**) means p-value < 0.05, and three (^***) means p-value < 0.001.

Figure 5: Buckling pattern of fiber is changed by using a PLA frame, instead of a PLA film. The green arrow indicates the shrinking direction of the PLA frame.

Figure 6: Tayloring buckling patterns on fibers by sacrifying a second layer of polyvinyl alcohol) (PVA) fibers. (A) Illustration of fiber layer deposition; (B) SEM image of buckled Polyactive^®-poly(vinyl alchol) (PA-PVA) fibers before sacrifying PVA fibers; (C) SEM image of PA-fibers obtained by buckling PA-PVA fibers and subsequently sacrifying the PVA fibers by dissolution; (D) SEM image of buckled PA fibers without using a scrificial layer of PVA. ( E) enlarged curls (red circles) in PA-fibers obtained by the use of a sacrificial layer of PVA-fibers. Figure 7: Buckling of fiber layers of various thickness, with parallel (right) and

perpendicular (left) orientation relative to the X axis of shrinkage. Increasing time corresponds to increasing layer thickness. (E) and (K) indicate the transition from buckled fiber to buckled fiber mesh. Figure 8: Graph depicting the thickness of samples with fibers deposited perpendicular (blue line, diamonds) or parallel (red line, squares) to the axis of shrinkage, as a function of deposition time. In yellow the transition points (at 9 and 5 minutes respectively) are marked from where the buckling effect on fibers is translated into waves at a mesh multiscale level from curls at a single fiber level. Figure 9: Changing buckling patterns on fibers by using a sacrificial layer of PVA. (A) Depicts ESP blending; (B) SEM image of PA PVA fiber mesh before shrinking; (C) SEM image of PA PVA fiber mesh after shrinking; (D) SEM image of buckled PA/PVA fiber mesh after sacrificing PVA.

Figure 10: Fabricating buckled fiber mesh mimicking the structure of trachea by using PLA tubes. (A) ESP tube before shrinking; (B) ESP tube after shrinking.

Figure 1 1 : Bright field and DAPI images for two time points and respective conditions. The nuclei are in blue and the scale bar represent 100 urn. Images show that in a buckled fiber mesh with a wave pattern, there is a significantly higher cell infiltration at both day 2 and day 5, compared to flat or non-buckled scaffolds.

Figure 12: Results of the Luciferase assay with and without DNA normalization. (A) data presented without normalization to DNA; (B) data presented after normalization to DNA.

Figure 13: Fabricating buckled fiber meshes by using polystyrene films of shrinkable material. (A-B) show random and aligned fiber before shrinking the shrinkable material; (C-D) show random and aligned fiber after shrinking the shrinkable material.

Figure 14: Fabricating buckled fibers and buckled fiber mesh from PVA. (A and B) fibers with alignment parallel to the direction of shrinkage of shrinkable materials before and after shrink, respectively; (C and D) fibers with alignment diagonal to the direction of shrinkage of shrinkable materials before and after shrink, respectively; (E and F) fiber mesh with alignment parallel to the direction of shrinkage of shrinkable materials before and after shrink, respectively. Figure 15: (a) shows artery wall composed of curled collagen fibers (from

www.sciencephoto.com); (b) buckled fiber mesh fabricated by our method.

Figure 16: (a) SEM image of collagen from the dermis of the skin displaying a curled pattern (from http://www.pinterest.com/explore/sem/); (b) buckled fiber mesh fabricated by our method.

Figure 17: (a) SEM image of a ciliary body and (c) SEM image of the iris (from

htip://www.pinterest.com/search/pins/?q=ciliary), (b) and (d) SEM pictures of two different configurations of a buckled fiber mesh fabricated by our method.

Figure 18: The wettability of the fiber mesh before (black line) and after (red line) buckling. Wettability was analyzed by contact angle measurements. Y-axis is mean contact angle, X-axis is time.

Figure 19: Comparison of dynamic contact angle of fiber mesh before (A-E) and after (F-J) buckling.

Summary of the invention

The present invention relates to an easy and versatile method to create one or more buckled fibers and/or a buckled fiber mesh, which allows multimodal control over the buckling pattern of the fiber and/or mesh, and also is easy and fast to apply and cheap to use. The method comprises

a) depositing at least one fiber on a surface of a shrinkable material, wherin the shrinkable material is shrinkable in at least one direction, wherein the deposited fiber at least partially adheres to the shrinkable material; and b) shrinking the shrinkable material in at least one direction, thereby buckling the fiber.

In a preferred embodiment, the invention provides a method of making a scaffold comprising a buckled fiber, the method comprising:

a. depositing a biocompatible polymer fiber onto a thermoshrinkable material by electrospinning, wherein the depositing results in a construct of a fiber mesh of multiple layers of fibers, adhered to the thermoshrinkable material, wherein the fiber mesh has a thickness of at least 50 times the average diameter of the fiber,

b. heating the construct in order to shrink the thermoshrinkable material and c. cooling down the construct in order to obtain a scaffold comprising a buckled fiber, adhered to the thermoshrinkable material, and d. optionally, removing the scaffold comprising a buckled fiber from the thermoshrinkable material.

Detailed description of the invention

Micrometers

The term um is used herein to indicate micrometers (μηη).

Fibers

Fibers for use in the present invention can be of any material. Fibers are generally cylinder-shaped, with a substantially rounded cross-sectional area, such as circular or oval. However, fibers of different cross-sectional area, such as essentially square, essentially triangular or any other shape are contemplated to work in a similar fashion in the buckling method of the invention.

The length of the fiber is a substantial multiplication of its diameter. Usually, fibers have a length of at least 50 times the diameter, preferably at least 100 times the diameter, more preferably at least 200 times the diameter. Fibers for use in the present method may generally have a diameter of 10 nm to 100 um, preferably 50 nm to 20 um, more preferably 100 nm to 10 um. In addition, fibers may generally have a length of up to several meters, such as up to 10 meters. Preferably, fibers have a length between 100 um and 1 m, more preferably between 1 mm and 10 cm.

The diameter of a fiber, for the scope of the present invention, is the longest possible length in the average cross-sectional area of the fiber.

Fibers may be preformed, i.e., obtained in fibrous shape and potentially stored, before deposition on the surface of a shrinkable material, but fibers may also be formed during the process of deposition on the surface, or they may be formed while on the surface.

Preferably, fibers for use in the present invention are flexible to a certain degree, preferably under the conditions which induce the shrinking of the shrinkable material. As such, it is preferred that fibers to be buckled by the method of the present invention have a flexibility characterized by Young's modulus, linearity range, ultimate stress, and ultimate strain.

Fibers may have a Young's modulus of for instance 0.1 MPa to 10 MPa, preferably 0.6 MPa to 6 MPa, more preferably 1 MPa to 5 MPa. Fibers may have a linearity range of 0.1 -20%, preferably 0.5-15%, more preferably 1 -12%. Fibers may have an ultimate stress of 0.1 MPa to 4 MPa, preferably of 0.2 MPa to 2 MPa, more preferably of 0.3 MPa to 1 .5 MPa. Fibers may have an ultimate strain of 50% to 250%, preferably 100% to 200%, more preferably 1 10% to 180%.

These parameters can be determined as follows: the electrospun fiber or mesh is fixed in a standard clamp and it is aligned to the 500 N load cell of a Zwick materials- testing machine.

The specimens are preconditioned by a series of ten cycles until the strain of 3% with a strain rate of 0.1 %/s, in order to reduce the hysteresis. Then they were extended or bent at 0.3%/s until failure. With cross-sectional area and strain

measurements, a stress-strain curve representing the mechanical properties of the electrospun mesh can be obtained. From the stress-strain curves, the following parameters, which define the mechanical properties of the network, were obtained:

• Young's Modulus (E)

o The intrinsic stiffness of a linear material in tension or compression expressed as the ratio of stress to strain: E = stress/strain. It is defined as the slope of the linear region of the stress-strain curve;

• Ultimate stress

o Maximum stress sustained by the specimen before failure of the material upon stretching the material;

· Ultimate strain

o maximum strain sustained by the specimen before failure of the material upon bending the material;

• The linearity range of a fiber is the portion of the stress-strain curve which is linear. Fibers can be made of any material, given that the material allows formation of fibers. A general requirement of such fibers is that they are capable of deforming upon application of mechanical stress. Furthermore, it is important that the fibers display at least some adherence to the used shrinkable material during the buckling process, and the fibers can be fixated after the buckling process, so that their buckled shape is maintained. Further preferably, the fibers can be removed from the shrinkable material after the buckling process.

A suitable fiber material for use in the present method comprises a synthetic or natural polymeric material, a ceramic material, a metallic material, and any combination of these materials. A ceramic material for forming at least one fiber is an inorganic non-metallic material, which is generally solid and comprises at least one type of metallic element and at least one type of non-metallic element. A ceramic material may be a crystalline ceramic material or a non-crystalline ceramic material (glass). Examples of a ceramic fiber which can be used in the present method include zirconia, aluminum hydroxide, calcium phosphates, calcium carbonates, and bioglasses. Bioglass is a group of bioactive glasses which bind to various tissue types depending on composition. Bioglass generally comprises Si0₂, Na₂0, CaO and P₂0₅ in specific proportions. Examples of a suitable bioglass are bioglass 45S5, bioglass 8625 and ceravital, the composition of which is known in the art.

Generally, if a ceramic fiber is used in the present invention, this fiber must be able to deform upon shrinking the shrinkable material. This can for instance be achieved by selecting a ceramic fiber which either has not hardened yet after formation of the fiber, or which softens under the applied conditions. Conditions which may allow such softening include the used temperature, light and irradiation conditions, as well as solvent presence and polymer precursors. Also, powder-based processes, electrospinning ("ESP"), as well as casting and molding procedures may allow for formation of a fiber which allows deformation upon shrinking of the shrinkable material.

Preferably, the conditions which achieve softening of the ceramic material also are capable of inducing shrink of the shrinkable material. Thus, thermoshrinkable, electroshrinkable, solvent-shrinkable and irradiation-shrinkable materials are preferred for buckling fibers of ceramic materials, preferably thermoshrinkable materials.

Fibers of ceramic materials may have a diameter of for instance 10 nm to 100 urn, preferably 50 nm to 20 urn, more preferably 100 nm to 10 urn.

A metallic material for forming at least one fiber is a powdered or whole metal, comprising essentially metallic elements or a mixture of metallic elements, and possibly comprising other elements to tune the properties of the material. Metallic materials for forming a fiber may be conductive or non-conductive. If powdered, a means of retaining fiber shape of the fiber material is required, although deformation of the fiber material while retaining fiber shape, at least during the shrinking of the shrinkable material, should be possible, also.

A metallic fiber may preferably be preformed or formed in situ during the deposition. Suitable metallic materials include tungsten, titanium, copper, silver, nickel- palladium alloys, nickel-cobalt alloys, and magnesium-zinc alloys. Preferably, a metallic material is relatively flexible to allow deformation of the fiber upon shrinking the shrinkable material, but alternatively, the conditions employed during shrinking may induce the metallic fiber to become flexible. Preferably, a metallic fiber can attain the required flexibility by adjusting the temperature to a temperature just below the melting

temperature. This temperature is metal-dependent, and the skilled person knows where to find suitable temperatures under which conditions the metal fiber allows deformation upon shrinking the shrinkable material.

Preferably, the conditions under which the metallic fiber attains the required flexibility also are capable of inducing shrink of the shrinkable material. Thus, a thermoshrinkable is preferred for buckling at least one fiber made of a metallic material. In case of a thermoshrinkable material used in combination with a metallic fiber, it is preferred that the shrinking temperature T_s of the shrinkable material is below the melting point T_M of the metallic material.

Fibers of metallic materials may have a diameter of for instance 10 nm to 100 urn, preferably 50 nm to 20 urn, more preferably 100 nm to 10 urn.

A natural polymer for forming at least one fiber must also be able to deform upon shrinking the shrinkable material. Natural polymers, usually, have at least some flexibility which allows for deformation during the shrinking of the shrinkable material.

However, it is preferred that natural polymers do not loose their natural functionality during the process of buckling, or thereafter. This may mean that the temperature at which the shrinking is performed should not be too high. Too high temperatures may result in denaturation or degradation of the natural fibers.

Similarly, other means of inducing shrink in the shrinkable material may result in denaturation or degradation of the natural polymeric materials. Therefore, it is preferred that the shrinking of the shrinkable material is induced by a parameter of such type and magnitude that the natural polymeric material retains its natural composition and function after having been buckled.

Thermoshrinkable materials, irradiation shrinkable materials and solvent- (in particular water-) shrinkable materials are preferred to buckle natural polymeric fibers in the method of the invention, preferably thermoshrinkable materials. In case of a thermoshrinkable material, it is preferred if the shrinking temperature T_s of the shrinkable material is below the denaturation or degradation temperature T_D of the natural fiber material.

The denaturation or degradation temperature is the temperature at which the natural polymeric fiber material degrades or denatures, whatever happens first upon increasing the temperature. Degradation in this respect is the loss of covalent binding between a significant portion of the molecules comprised in the fiber. Denaturation in this respect is the loss of a significant portion of non-covalent binding between the molecules comprised in the fiber. Loss of a "significant portion" of bonding may be material dependent, but means in essence that natural functionality of the fiber in terms of for instance cell sustenance, strength, shape and/or polarity, among other parameters, is lost to a large extent.

A natural polymer for forming at least one fiber to be buckled in the present invention include for instance polypeptides, elastin, collagens, silk and polysaccharides, such as for instance, amylose, pectin, (non-chemically modified) cellulose and chitosan. Preferably, such polymers include elastin and collagen, most preferably collagen.

Fibers of natural polymers may have a diameter of for instance 10 nm to 100 urn, preferably 50 nm to 20 urn, more preferably 100 nm to 10 urn.

A synthetic polymer for forming at least one fiber can be any synthetic polymer, but preferably is a thermoplastic synthetic polymer. A thermoplastic polymer is a polymer which softens substantially at a polymer-dependent characteristic temperature, the glass transition temperature T_G, without becoming liquid. The glass transition temperature of a thermoplastic polymer is substantially below the melting temperature T_M of the thermoplastic polymer, or it is substantially below the degradation temperature T_D if the polymer degrades before melting upon increasing the temperature. The glass transition temperature of a polymer is a well-known parameter in the art, and can be retrieved for instance from Physical Properties of Polymers Handbook edited by James E. Mark, or similar handbooks.

A thermoplastic polymer can be a simple polymer or a co-polymer, and if it is a co-polymer, it may be of any type, such as for instance an alternating, random, or block copolymer, as long as the polymer can be formed into a fiber. A thermoplastic polymer which is easily shaped into a fiber is preferred. Suitable polymer types are for example polyolefins, polydienes, polystyrenes, polyesters, poly(alkylene oxides), polyoxyalkylenes, polyhalogenoalkylenes, polyalkylenephthalat.es or terephthalates, polyphenyl or phenylenes, poly(phenylene oxide or sulphide), polyvinyl acetates), polyvinyl alcohols), polyvinyl halides), poly(vinylidene halides), polyvinyl nitrites), polyamides, polyimides, polycarbonates, polysiloxanes, acrylic or methacrylic acid polymers, polyacrylates or polymethacrylates, polyethers, polylactams, chemically modified cellulosics, poly(ethylene glycols terephthalates) and poly(butylene terephthalates).

Examples of polyolefins are the homopolymers, copolymers and terpolymers of ethylene, propylene, butene-1 ,4-methyl pentene-1 , isobutylene and co and terpolymers thereof, including co and terpolymers with dienes and with polar monomers. Examples of fluorocarbon polymers are polytetrafluoroethylene and polytrifluorochloroethylene.

Examples of vinyl polymers are polyvinyl chloride, polyvinyl acetate), polyvinyl alcohol), poly(acrylonitrile )and the co- and terpolymers thereof, including co- and terpolymers with other monomers such as maleic anhydride and maleic acid.

Examples of styrenic polymers are polystyrene, poly omethyl styrene, the co- and terpolymers thereof and with other monomers such as acrylonitrile, methyl methacrylate and the like. Examples of acrylic and methacrylic polymers are polyacryclic acid and polymethacrylic acid, their copolymers, esters and salts. Examples of polydienes are polybutadiene, polyisoprene, polychloroprene, polycyanoprene and copolymers thereof. Examples of polyacetals are polymethylene oxide, polytrioxane and copolymers thereof.

Examples of polyesters are poly(ethylene terephthalate), poly(propylene terephthalate) (PPT), poly(ethylene glycol), poly(butylene terephthalate), poly(ethylene isophthalate), poly(lactic acid), poly(glycolic acid), poly(e-caprolactone), poly(trimethyl carbonate) and their copolymers.

Examples of polyamides are poly(hexamethylene adipamide) and poly(hexamethylene sebacamide).

An example of a polycarbonate is the reaction product of a bisphenol A with diphenyl carbonate and an example of a polysulfone is the reaction product of an alkaline salt of bisphenol A with ρ,ρ'-dichlorophenyl sulfone.

Examples of polyurethanes are the fiber-forming polymer made from hexamethylene diisocyanate and tetramethylene glycol and the elastomeric polymer made from diphenylmethane ρ,ρ'-diisocyanate, adipic acid and butanediol 1 ,4.

Examples of thermoplastic cellulosics are ethyl cellulose, cellulose acetate, cellulose butyrate and hydroxy propyl cellulose.

A preferred type of polymer is a copolymer of poly(ethylene oxide terephthalate) and poly(butylene terephthalate), such as for example Polyactive®.

Polymers, such as thermoplastic polymers, may be used with any type of shrinkable material in order to buckle the at least one fiber, as long as the fiber at least partially adheres to the shrinkable material. As such, electric shrinkable, solvent- shrinkable, irradiation shrinkable, thermoshrinkable and elastic materials can be used. Preferred however is the use of thermoshrinkable materials, because it is easy to combine a suitable shrinkable material with a suitable polymeric fiber such that the required flexibility during the buckling process is attained.

Preferably, in case the shrinkable material is a thermoshrinkable material, the shrinking temperature T_s of the thermoshrinkable material is below the melting point (or degradation point) of the thermoplastic polymer. Further preferably, the shrinking temperature T_s of the thermoshrinkable material is above the glass transition temperature T_G of the thermoplastic polymer.

Fibers of synthetic polymers may have a diameter of for instance 10 nm to 100 urn, preferably 50 nm to 20 urn, more preferably 100 nm to 10 urn.

Combinations of two or more different types of fiber material are also contemplated in the present invention. A fiber material comprising two or more different thermoplastic synthetic polymers, two or more different ceramic material, two or more different metallic materials or two or more different natural polymers can be used, as well as combinations of one or more thermoplastic synthetic polymers, one or more ceramic materials, one or more metallic materials and/or one or more natural polymers with one or more of thermoplastic materials, ceramic materials, metallic materials and/or natural polymers.

A single fiber comprising multiple different materials is called a composite fiber, and the different materials in a composite fiber may have any relative orientation, such as side-by-side, sheath-core, "islands in the sea", citrus fibers, segmented pie types, etc. For example, a composite material may have the advantage of combining the strength, conductivity, and resistance of one or more metals with the flexibility of one or more polymers (e.g. a carbon fiber).

Generally, the fiber has a diameter of between 10 nm and 100 urn, preferably between 50 nm and 20 urn, more preferably between 100 nm and 10 urn.

Shrinkable material

A shrinkable material for use in the present method is any material that is capable of shrinking in at least one direction. A direction of shrinkage is understood as a direction in which the material decreases in length upon shrinking. A shrinkable material may have more than one direction of shrinkage, but it must have at least one direction of shrinkage. Shrinkable materials are among others characterized by a shrink ratio. The shrink ratio is the ratio of the length of the shrinkable material in one shrink direction after shrinking to the length of the shrinkable material in the same shrink direction before shrinking; if a shrink material has multiple directions of shrink, the material may have different shrink ratio's along different lengths. For some embodiments, a shrinkable material with one direction of shrinkage is preferred. In other embodiments, it is preferred to use a shrinkable materials with 2 directions of shrink, In this case, the two directions of shrink are preferably aligned approximately perpendicular.

The shrink ratio is highly dependent on the shrinkable material, and may be optimized for a certain buckling effect by suitable selection of an appropriate shrinkable material, and/or by altering the conditions of shrink. The shrink ratio may be for example between 10 and 90 %, preferably between 25 and 90 %, more preferably between 40 and 90 %, such as optimally between 70 and 80 %.

Shrinking of a shrinkable material may be activated by various means.

Suitable shrinkable materials include:

An elastic shrinkable material is a material which shrinks upon loosening of the stretched material. Examples of such materials are rubbers and elastomers, and may also be found among semi-crystalline polymers.

An electric shrinkable material is a material which shrinks upon application of an electric current. Examples of such materials are piezoelectric materials.

A solvent-shrinkable material is a material which shrinks upon contact with a solvent. Such solvent may be any solvent, such as water, an alcohol such as methanol or ethanol, an ether such as diethyl ether, or any other suitable solvent.

An irradiation-shrinkable material is a material which shrinks upon irradiation, such as irradiation with (visible, UV or IR) light, with an electron beam, or with other forms of irradiation. Examples of such materials can be found among thermosetting polymers.

A pH-shrinkable material is a material which shrinks upon changing the pH, such as by submersion in acid or base. The pH may usually be changed by acids or bases known in the art, as long as the fiber that is being buckled can withstand all pH- values that it is subjected to. Alternatively, in the case of using sacrificial fibers (see below), it may be advantageous if the sacrificial fiber dissolves slowly after changing the pH.

A thermoshrinkable material is a material which shrinks upon increasing the temperature, or alternatively, when decreasing the temperature. Preferably, a thermoshrinkable material is a material which shrinks upon increasing the temperature. Thermoshrinkable materials are well-known in the art, and are usually polymeric materials which have been subjected to a stretching force during formation, such that their molecules are aligned in a thermodynamically unfavorable state. Increasing the temperature for such materials allows reorganization of the polymeric molecules by an increase in entropy, thereby at least partially removing the molecular alignment which results in a shrinking of the material. The shrinking temperature T_s at which a

thermoshrinkable material displays significant shrink upon increasing the temperature may be any temperature, but is generally 10 to 200 degrees Celsius, preferably 40 to 150 degrees Celsius, more preferably 65 to 120 degrees Celsius.

In the context of the present method, it is highly preferred if the shrinkable material comprises a thermoshrinkable material. More preferably, the shrinkable material is a thermoshrinkable material. In that case, shrinking the shrinkable material can be achieved by raising the temperature or decreasing the temperature, such as in using an oven or other means known in the art to adjust temperature, to at least the shrinking temperature.

Adjusting the temperature can for example be to a temperature of -20-200 °C, but may be much lower or higher where required to shrink the shrinkable material.

Preferably, adjusting the temperature is done to a temperature of 10-150 °C, more preferably 30 - 90 °C.

Adjusting the temperature should be of sufficient duration to allow at least some shrinking of the thermoshrinkable material, preferably essentially full shrinking of the thermoshrinkable material, to the maximum shrink ratio; optionally, adjusting the temperature can be performed for a duration which allows only partial shrinking, so that the effective shrink ratio experienced by the at least one fiber can be reduced, to tune the degree of buckling. The duration of subjection of the thermoshrinkable material to the adjusted temperature to attain essentially full shrinkage of the shrinkable material can be for example 5 s - 3 days, such as 10 s - 3 days for poly(lactic acid) (PLA) when heating from 65 to 80 °C and 10 min - 24h for polystyrene (PS) with heating from 100 to 140 °C .

Suitable thermoshrinkable materials are for example thermoshrinkable materials made from polyethylene, polypropylene, polystyrene , poly(lactic acid), poly(glycolic acid), poly(e-caprolactone), poly(lactic-co-glycolic acid), polyvinyl chloride), poly(acrylonitrile, butadiene, styrene), copolymer of ethylene vinyl acetate and

poly(ethylene terephthalate. Fibers are deposited on a surface of the shrinkable material. It is preferred if a direction of shrinkage is oriented such that the surface decreases in area upon shrinking the shrinkable material.

The shrinkable material can be of any shape. For instance the shrinkable material is sheet- or tube shaped. In case of tube shapes, the at least one fiber may be deposited on the inner as well as on the outer surface, or both; in the case of a sheet- shape, the fiber may be deposited on either side, or on both sides; preferably, the fiber is deposited on one side, such as for example the upper side if the sheet-shaped shrinkable material is positioned horizontally.

The deposited fiber at least partially adheres to the shrinkable material.

Adherence in this context means that the fiber at least partially sticks to the shrinkable material, such as for example by non-covalent interaction, by ionic interaction, by hydrophobic or hydrophilic interaction, or by a combination of these mechanisms.

Adherence also means that upon application of a mechanical force with a component directed parallel to the surface of the shrinkable material on a deposited fiber, the fiber does not slide over the surface without resistance, but instead a certain degree of non-slip exists between fiber and surface, which results in at least some measurable resistance against motion over the surface of the shrinkable material. This resistance to slip, or adherence, provides the mechanical force through which a fiber can be buckled upon shrinking the shrinkable material.

Adherence is preferably present at least partially along the length of the fiber, such that upon shrinking the shrinkable material, the fiber buckles by the mechanical force provided by the shrinking of the shrinkable material and by the non-slip conditions provided by the adherence of the fiber to the surface of the shrinkable material.

Preferably, the fiber is deposited such that the fiber adheres to the surface essentially along its full length, although there may be portions of the fiber which do not touch the surface, or adhere to the surface. At least two points of adherence of the fiber to the shrinkable material are preferred. Adherence may be optimized by selection of an appropriate combination of a shrinkable material and a material for forming fibers.

However, in case of the buckling of a layer of fibers thicker than about 1 fiber diameter, such as multiple layers of fibers, it is understood that fibers in a layer distant from the surface of the shrinkable material by one or more other fiber layers do not themselves adhere to the surface of the shrinkable material. In such cases, at least partial adherence of a fiber layer to a fiber layer below the fiber layer of interest functions similar to adherence of a fiber layer to the shrinkable material, and similarly results in the buckling of fibers in that layer.

Fiber deposition

The at least one fiber is deposited on the surface of the shrinkable material in any way which results in at least partial adherence of the fiber to the shrinkable material. A fiber may be deposited by dropping a pre-formed fiber on a surface of the shrinkable material, such as by hand or by automated positioning. In this embodiment, fibers can be made from any material as defined above, and bought or formed as fibers even outside the context of buckling according to the present method.

Alternatively, it is possible to immediately buckle formed fibers. Preferably, this is the case when fibers are formed just before deposition on the surface of the shrinkable material, or while the fiber material already resides on the surface of the shrinkable material.

For example, it can be possible to deposit material of any shape on the surface, and form the fibers in situ prior to shrinking the shrinkable material with the aim of buckling the in-situ formed fibers. Formation of fibers in situ can be done by any type of molding, but also by use of increasing or decreasing the temperature, by various electromagnetic fields such as light, or, in case of metallic materials, or by the use of magnetic fields. A combination of these methods to form fibers in situ is also within the reach of the skilled person.

Fibers can also be formed just before deposition on the surface. For example, fibers can be molded during the deposition process, by for instance stretching a fiber- forming material into a fiber shape while depositing it on the surface of the shrinkable material.

It is preferred if fibers are deposited on the surface by electrospinning (ESP). It is well-known to form fibers from natural and synthetic polymers by ESP. Electrospinning is a technique in which one or more fiber material solutions in a suitable solvent are combined and ejected from a hollow exit point under the influence of an electric field. The formed electrically charged solvent/polymer jet is aimed at the deposition site. During deposition, the solvent largely evaporates, thereby forming fibers which can be collected from the deposition site.

Preferably, ESP is performed such that the formed fibers are deposited immediately on the surface of the shrinkable material. Thus, deposition of the fibers on the shrinkable material by ESP is preferred.

Fiber deposition, such as by ESP, results in at least one fiber, which may be aligned in any orientation relative to the direction of shrinkage; control over the alignment of the fiber(s) relative to the direction of shrinkage of the shrinkable material is readily achievable by proper positioning of the shrinkable material, for instance in the ESP equipment. Also, it is readily feasible to collect a layer of fibers, either randomly distributed or aligned, or a combination thereof. Multiple layers of fibers can also be easily deposited, such as through ESP, whereby it is possible to have multiple different fiber alignments throughout the fiber layers, but a fully random fiber layer, or any combination of such layers can also be obtained.

It is an advantage of ESP that it is possible to create fibers which comprise a single polymer type, or composite fibers, in any spatial arrangement as described above. Also, it is possible to create fiber collections in which fibers of different type or composition are arranged in the fiber layer(s), in any way, relative orientation or distribution.

The concentration of the solution used for electrospinning may be any suitable concentration for fiber forming in question, as long as it allows spinning. Electrospinning is preferably done with a polymer concentration of 0.05% to 60% weight by volume., preferably 1 % to 40%, more preferably 5% to 25%.

The voltage applied for ESP may be any suitable voltage for the fiber forming in question, as long as it is spinnable. A suitable voltage range is 1 KV to 30 KV, preferably 4 KV to 25 KV , more preferably 8KV to 20KV.

The feeding rate of polymer solution during ESP maybe any suitable rate for the fiber forming in question. A suitable feeding rate range is 0.01 ml/h to 30ml/h, preferably 0.1 ml/h to 20ml/h, more preferably 1 ml/h to 15ml/h.

The temperature used for ESP maybe any suitable temperature for the fiber forming as long as it not over the shrinking temperature of the shrinkable material. A suitable temperature range is 10 °C to 60 °C, preferably 15 °C to 50 °C, more preferably

20 °C to 40 °C. Most preferably, the temperature is around room temperature, i.e. about

21 degrees Celsius, such as between 20 and 25 degrees Celsius.

The humidity used for ESP may be any suitable humidity for the fiber forming in question. But it generally is between 5% to 80%. preferably between 20% and 70 %, more preferably from about 30% to 40 %.

The solvent used for ESP may be any suitable solvent for the fiber material in question, as long as it is capable of at least some evaporation during the process of ESP. Examples of suitable solvents are chloroform, water, ethanol, dichloromethane, dichloroethane, methanol, acetone, acetic acid, tri-fluoro acetic acid, dioxane, n-methyl pyrrolidone, xylene, 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP), dimethylformamide (DMF), tetrahydrofuran and any combination of these solvents. A preferred solvent is a mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP), such as for example in a 80:20 mixture.

The distance from the fiber solution exit point to the deposition site, preferably the shrinkable material, may be any distance which allows for fiber formation. Preferably this distance is between 5 and 30 cm, preferably between 10 and 25 cm, more preferably from about 15 to 20 cm.

The diameter of the fiber can be adjusted by suitable adaptation of the ESP parameters, as is well-known to the skilled person.

It is preferred if the ESP of at least one fiber, in particular a natural or synthetic polymer fiber, for buckling with the present method is achieved with a straight jet. That means that ESP preferably does not include deformation of the jet by the electrical bending instability. Furthermore, it is preferred if no secondary branching occurs during or after deposition by the jet. The skilled person knows how to adjust the voltage, concentration, feeding rate, solvent, temperature, humidity, and distance so that these problems do not occur.

Preferably, ESP using natural or synthetic polymer fibers is done using any combination of the above options for voltage, concentration, solvent, feeding rate, temperature, humidity and distance mentioned above.

Further preferably, deposition of fibers by ESP results in a mat of fibers, i.e. a collection of multiple fiber layers, which fiber layers may be random fiber layers or aligned, single fiber layers, or anything in between, and which orientation may be the same or different than fiber orientation in other layers of the same mat. Preferably, for some embodiments, a mat of fibers thus obtained is non-woven. However, in other

embodiments, a woven mat of fibers may be preferred.

Besides ESP, other techniques of fiber deposition also are contemplated. Examples of these techniques are melt electrospining, melt and wet spinning, rotary jet- spinning (Nano lett. 2010, volume 10, issue 6, pages 2257-2261 ), molecular self- assembly and additive manufacturing.

At least one fiber deposited on a shrinkable material can be deposited in any form or alignment. For instance, it can be a single fiber which can be of random shape and/or aligned in any relative orientation to a direction of shrinkage.

Also, at least one fiber can be a layer of fibers either randomly distributed, or aligned in any relative orientation to a direction of shrinkage, or a combination thereof. At least one fiber can also be multiple layers of fibers which have multiple different fiber alignments throughout the fiber layers, including one or more fully random fiber layers, or any combination of such layers.

The at least one fiber is preferably at least partially aligned non-perpendicular relative to a direction of shrinkage of the shrinkable material.

In a further preferred embodiment, at least one fiber is oriented randomly on the surface of the shrinkable material.

In a further preferred embodiment, the fiber is aligned more or less in a straight line on the surface of the shrinkable material. The orientation relative to the direction of shrinkage may be any orientation lower than 90 degrees relative to a direction of shrinkage, and up to and including 0 degrees relative to a direction of shrinkage.

It is highly preferred in this case if the at least one fiber is oriented substantially non-perpendicular to a direction of shrinkage, such as for example 0 - 70 degrees relative to a direction of shrinkage, or 0 - 45 degrees relative to a direction of shrinkage. In a much preferred embodiment, the at least one fiber is oriented at approximately 45 degrees of a direction of shrinkage. In a further, much preferred embodiment the at least one fiber is aligned approximately parallel with a direction of shrinkage, i.e. at approximately

0 degrees relative to a direction of shrinkage. Optimally, the at least one fiber is oriented substantially parallel to a direction of shrinkage of the shrinkable material.

Optionally, different deposited fibers may comprise different materials, such that one of the materials may be removed after buckling. This results in a buckled fiber with different regularity as observed for a fiber buckled without removal of one of the fiber types after buckling. Removal of one or more fiber types after buckling generally has the result that the resulting buckled fiber has higher amplitude.

Fibers which are included with the aim of removing them after the buckling process to alter the shape of the buckled fiber are called sacrificial fibers in the present context, and it is a distinct advantage that inclusion of such fibers allows for further control over the shape of the buckled fiber or the buckled fiber mesh. Removal of a fiber may be achieved for instance by dissolution in a suitable solvent, by irradiation or by mechanical means, such a pulling out. The fiber mesh

The term "fiber mesh" is used herein to indicate a three dimensional structure or fabric, comprising holes that are bordered by fibers. The fiber mesh may be made of fibers loosely twisted, knotted or woven together at regular or irregular intervals. The terms "matrix", "fiber mat", "mat" or "scaffold" are used interchangeably in the art and mean the same as "fiber mesh". These terms are also often used to indicate structures comprising or consisting of a network of spaces surrounded by fibers. It is commonly understood by the person skilled in the art that these terms all indicate the same structure of a coherent, interwoven or intertwined structure or network. A "buckled fiber mesh" is a fiber mesh comprising buckled fibers, and has the same meaning as a scaffold comprising a buckled fiber.

Buckling

In the method of the invention, fibers are buckled. This means that fibers subjected to the present method, which initially may be straight or essentially straight, attain a crimped, curved and/or more or less sinusoidal shape by the action of shrinking of the shrinkable material. Usually, the buckled fibers assume a two-dimensional buckled shape, which is the same as a more or less sinusoidal shape, and is also called crimp. It is also possible, however, to obtain three-dimensionally buckled fibers, such as fibers with a curl pattern.

The buckled fibers generally have a more or less regular pattern, for which it is possible to determine an average wavelength and an average amplitude, which both depend on a variety of factors, including the shrink ratio of the shrinkable material, the diameter of the fiber, the fiber material, the adherence of the fiber to the shrinkable material, the alignment of the fiber with respect to the direction of shrinkage, and the molecular composition of the fiber. An advantage of the method of the invention is that it is highly reliable and predictable, resulting in reproducibly buckled fibers and fiber meshes with predictable wavelength and amplitude.

Generally, the average wavelength may be between 1 .5 and 30 times the fiber diameter. Preferably the average wavelength is between 2 and 20 times the fiber diameter, more preferably the average wavelength is between 4 and 13 times the fiber diameter.

Further generally, the average amplitude is between 0.1 and 20 urn, preferably between 0.2 to 10.0 um, more preferably the average amplitude is between 0.5 to 5.0 um, more preferably the average amplitude is between 1.3 to 3.0 um.

After deposition of the fibers, the fibers are buckled by shrinking the shrinkable material, resulting in separately buckled fibers or in a buckled fiber mesh. The adherence of the fibers to the shrinkable material makes that the act of shrinking provides a mechanical force from the shrinkable material to the fiber by the non-slipping of the fiber over the surface of the shrinkable material, thereby buckling the fiber. A high shrink ratio of the shrinkable material with a high adherence results in strongly buckled fibers, whereas a lower shrink ratio in combination with a high adherence generally results in less, but efficient buckling. Low adherence in combination with a high shrink ratio generally results in relatively more slip, thereby obtaining fibers which have been buckled to a lesser degree.

The shrink ratio of the shrinkable material determines the local shrink force experienced by a fiber. Because the orientation of the fiber relative to the direction of shrinkage is proportional to the shrink ratio, a fiber aligned parallel along a direction of shrinkage experiences the maximum local shrink force. This is preferred if high buckling is required, in particular for combinations of fiber and shrinkable material which display strong adherence and/or little slip. By way of an example: A fiber aligned at about a 45 degree angle relative to a direction of shrinkage, experiences a proportional local shrink force in that location, i.e. approximately 0.71 times the shrink force for fibers aligned parallel. This has the advantage that single layers with various relative alignments, such as perpendicular alignments, may be buckled with a relatively high and equal shrinking force for the various layers, while still allowing for the interesting properties that arise from buckled fiber meshes comprising perpendicularly aligned single fiber layers.

Preferably, the buckled fiber or the buckled fiber mesh is fixated after the making of the buckled fiber or the buckled fiber mesh. Fixation means that after buckling, the buckled fiber(s) and/or buckled fiber mesh substantially retain their buckled shape. This can be achieved by many means imaginable to the skilled person, among which irradiation, decreasing temperature, application of a fixation layer, e.g. a polymeric, ionic or a salt layer, appropriate reactive gases or liquids, or their vapors.

Preferably, fixation is achieved through lowering the temperature relative to the temperature at which the shrinkable material was shrunk. An advantage of using temperature as a fixation means is that it is easily applicable, and works well for many different fiber types. In particular thermoplastic polymeric fibers can be fixated by decreasing the temperature at which the shrinkable material was shrunk, in particular when the shrinkable material was shrunk at a temperature higher than the T_G, and the temperature is lowered to a temperature below the T_G.

In the most preferred embodiment, the shrinkable material is a

thermoshrinkable material which can be shrunk at a temperature above the T_G, and preferably below the T_M of fibers made of a polymeric material, preferably a thermoplastic polymeric material. Raising the temperature to shrink the shrinkable material, for instance in an oven, results in softened fibers which can easily be buckled under the mechanical action provided by the shrinking of the shrinkable material. Subsequent lowering the temperature, such as to room temperature, lowers the temperature to below the T_G of the thermoplastic polymer fibers, which results in hardening of the fiber(s) and thereby in fixation of the fiber(s) in their buckled shape.

Preferably, the T_M of the thermoplastic polymer is between 10 °C and 230 °C, preferably between 60 °C and 200 °C, more preferably between 80 °C and 180 °C.

Further preferably, the T_G of the of the thermoplastic polymer is between -20 °C and 120 °C, preferably between 0 °C and 100 °C, more preferably between 20 °C and 80 °C.

Further preferably, the buckled fiber or the buckled fiber mesh is removed from the shrinkable material after forming the buckled fiber or the buckled fiber mesh. Preferably, removal is done after fixating the buckled fiber or the buckled fiber mesh.

Removal can be done in any way. The buckled fiber or buckled fiber mesh may simply be removed by hand from the shrinkable material, but alternatively, the shrinkable material may be dissolved, mechanically removed, torn, cut, liquefied or evaporated to obtain the buckled fiber and/or the buckled fiber mesh separately from the (at least partially) shrunk shrinkable material.

Buckled Fiber Mesh

In case the at least one fiber comprises a multitude of fibers, various options can be distinguished. The multitude of fibers can be buckled such that separately buckled fibers result. Alternatively, the multitude of fibers can be buckled such that the fibers after buckling display some degree of entanglement and/or coherence, which is called a buckled fiber mesh in the context of the present invention.

A random fiber layer can comprise a multitude of fibers oriented in random directions relative to each other. A random fiber layer may comprise substantially straight fibers, but also substantially bent or otherwise non-straight fibers. A random fiber layer generally has a thickness of more than one fiber diameter, such as at least 2 fiber diameters, at least 3 fiber diameters, or at least 5 fiber diameters.

Alternatively, a multitude of fibers may form a single fiber layer. Fibers in a single fiber layer are generally oriented approximately parallel relative to each other. In this case, fibers are generally more or less straight. In this case, also, the fiber layer generally has a thickness of approximately one fiber diameter, but a single fiber layer may also comprise multiple layers of substantially parallel aligned fibers, attaining a thickness of multiple fiber diameters.

A (single or random) fiber layer may comprise fibers comprising one material or composite fibers as described above, and also the layer may comprise various different fibers. Preferably, a layer consists of fibers of a single fiber material.

The present invention also discloses making a buckled fiber mesh, wherein a layer comprising a multitude of fibers is buckled simultaneously by

a) depositing a fiber layer on a surface of a shrinkable material, which material is shrinkable in at least one direction, wherein the deposited fibers at least partially adhere to the shrinkable material;

b) shrinking the shrinkable material in at least one direction, thereby buckling the fibers. A fiber mat, in the context of the present invention, generally comprises multiple fiber layers, such as at least 5 layers, in which the fiber layers may be one or more single fiber layers and/or one or more random fiber layers. Also, a fiber mat may comprise multiple fiber layers with various alignments relative to each other. A fiber mat may have for example a thickness of at least 50 times the average diameter of a fiber, preferably at least 100 times the average diameter of a fiber, more preferably at least 250 times the average diameter of a fiber, and even more preferably at least 500 times the average diameter of a fiber. Thus, a fiber mat has a thickness of substantially more than one fiber diameter.

The average diameter of a fiber is defined as the average diameter over the length of a single fiber, or, if fibers of different chemical constitution are used, the average diameter of the different fiber types. The average diameter over the length of a single fiber can be measured by measuring the diameter from for example scanning electron microscopy analysis. For ease of reading, the average diameter of a fiber is also referred to as the "fiber diameter". It was noted during the experiments that buckling of a fiber mat deposited on a shrinkable material in a perpendicular orientation relative to a direction of shrinkage results in a buckled fiber mesh. In addition, any alignment relative to a direction of shrinkage in which at least one fiber can be buckled to obtain a buckled fiber according to the present invention, also results in a buckled fiber mesh if a layer comprising a multitude of fibers, or a mat comprising multiple layers of fibers are buckled simultaneously. Thus, to obtain a buckled fiber mesh, a fiber mat may have any orientation relative to a direction of shrinkage of the shrinkable material.

The buckled fibers present in a buckled fiber mesh according to the invention do not necessarily attain a similar configuration as a single fiber (of same chemical composition) buckled under the same conditions. In many cases though, the single fibers in a buckled fiber mesh display a similar buckling pattern as to the case had they been buckled under the same conditions as a single fiber.

However, a buckled fiber mesh is distinguished from a buckled multitude of fibers because the mesh displays entanglement of the constituent fibers, so that a mesh of at least some degree of coherence can be obtained. A buckled fiber mesh comprises fibers or combinations of fibers as defined elsewhere, and may therefore comprise many different materials, or combinations of materials.

A buckled fiber mesh obtained from at least a multitude of fibers, such as a fiber mat, has an improved Young s modulus compared to fiber mesh before buckling. For tensile tests, the buckled fiber mesh was fixed in a standard clamps and aligned to the 500N load cell of a tensile tester. Generally, the average Young s modulus of buckled fiber mesh may be between 1 times and 6 times of fiber mesh before buckling. Preferably, the average Young s modulus of buckled fiber mesh may be between 2.4 times and 4.7 times of fiber mesh before buckling.

In a preferred embodiment, the buckled fiber mesh has a wave pattern. This can be obtained by the method of the invention wherein the layer has a thickness of at least 50 times the average diameter of a fiber, to obtain a buckled fiber mesh which has a wave pattern.

Preferably, the wave pattern is obtained by buckling a fiber mat of at least 50 fiber diameters, more preferably at least 100 fiber diameters, even more preferably at least 200 fiber diameters, more preferably of at least 300 fiber diameters.

Alternatively, a buckled fiber mesh with a wave pattern is obtained from buckling a fiber mat with a thickness of at least 100 urn, preferably at least 150 urn, more preferably at least 200 um, even more preferably at least 250 urn, and more preferably at least 300 um.

The wave pattern of a buckled fiber mesh is characterized by the wave periodicity and amplitude. It is a distinct advantage of a buckled fiber mesh with a wave pattern that the wave pattern is highly predictable and reproducible, and can be optimized to obtain wave patterns of any periodicity. This is true for any fiber material, in particular for polymeric fiber materials, such as natural or synthetic polymeric materials, in particular synthetic polymeric materials.

Periodicity, in the context of this invention, is a word which describes the wavelength of a wave pattern of a buckled fiber mesh. The periodicity is the wavelength of the wave pattern of a buckled fiber mesh, which can be for example 20 to 500 um, preferably 50 to 300 um, more preferably 70 to 200 um. The periodicity of the wave pattern may be different from the wavelength of the single fibers comprised in the buckled fiber mesh.

The wave pattern in a buckled fiber mesh also has an amplitude, which amplitude may be different from the amplitude of the single fibers comprised in the fiber mesh. The amplitude of the wave pattern in a buckled fiber mesh can be 0.1 to 10 um, preferably 0.1 to 6.0 um , more preferably 0.5 to 3.0 um, and even more preferably 0.5 to 2.0 um.

A buckled fiber mesh with a wave pattern has particular advantages in various applications, such as increased porosity, decreased density, increased flow-through at similar fiber density, increased surface area and increased resemblance with naturally occurring buckled fibers, relative to fiber meshes of the same composition but without having been buckled by the method of the invention.

Accordingly, the invention also discloses a buckled fiber, which is obtainable by the method of the invention. Furthermore, the invention discloses a buckled fiber mesh, obtainable by the method of the invention.

Changing simple parameters, like the quantity and alignment of fibers, composition of the layer, selection of fiber and shrinkable material and layer thickness provides a high degree of control over the shape of a buckled fiber or a buckled fiber mesh. Optionally, inclusion of sacrificial fibers allows further control over the shape of the buckled fiber or buckled fiber material. A few simple experiments lead to optimization of the wanted buckle pattern, where after buckled fibers or buckled fiber material with that pattern can easily and reproducibly be obtained, at high scale if necessary. It is a further advantage of the present invention that a buckled fiber mesh can be obtained by buckling a multitude of fibers simultaneously.

The wave pattern of a buckled fiber mesh can have a more or less regular periodicity, or a gradient periodicity. To obtain a gradient periodicity, it is preferred if the fibers are deposited not only on the shrinkable material, but fully cover the shrinkable material and stretch beyond it in at least one direction; this makes that only those fibers deposited on the shrinkable material are buckled, while the fibers stretching beyond the shrinkable material are not buckled, thereby constraining the buckling of the fibers on the shrinkable material at least on one side, which leads to a gradient in the wave pattern, generally on that side.

A more or less regular periodicity is obtained by depositing the multitude of fibers only on shrinkable material, without stretching beyond the shrinkable material. Preferably, a regular wave pattern is obtained by depositing the multitude of fibers such on the shrinkable material, that it covers no larger area than the area of the shrinkable material after shrink. More preferably, the area covered in this case is located in the middle region of the shrinkable material.

The buckled fiber and buckled fiber mesh display buckling on a first level, which is reflected in the sinusoidal wave pattern of the separate fibers. On a next level, the wave pattern in a buckled fiber mesh may have different periodicity than the separate fibers. The wave pattern obtained in certain buckled fiber meshes is not the same as the sinusoidal pattern obtained for single fibers; it is an additional effect at a higher, macroscopic level of spatial organization, in addition to the microscopic organization of the sinusoidal buckled fibers.

As such, in a buckled fiber mesh which does not display a wave pattern, the separate fibers comprised in the material are buckled in much the same way as for a single fiber, but entangled at the same time to display at least some coherence. In a buckled fiber mesh, also, the single fibers are buckled, but in addition, the higher-level organization of the wave pattern emerges. The increased three-dimensionality is a factor which allows for various uses of buckled fibers and/or a buckled fiber mesh, in particular a buckled fiber mesh with a wave pattern.

For a buckled fiber mesh with a wave pattern, it is preferred to deposit a random or aligned fiber layer on the shrinkable material, comprising one or more types of fiber material, preferably synthetic or natural polymeric materials. In an alternative embodiment, it is preferred to deposit multiple single fiber layers, preferably a synthetic or natural polymeric material or a combination thereof, on the shrinkable material, to obtain a buckled fiber mesh with a wave pattern.

Applications of buckled fibers and buckled fiber meshes

A buckled fiber mesh according to the invention can be used in various applications, among which for example in tissue engineering applications in vivo or in vitro, as a filter, as a catalytic material, or as a textile.

In one embodiment, a buckled fiber or a buckled fiber mesh can be used as a textile, or in textile. Preferably, a buckled fiber mesh is used. In this embodiment, it is preferred if the fibers comprised in the buckled fiber mesh display at least some flexibility, and that the buckled fiber mesh itself also displays at least some flexibility. Thus, for use as textile, natural and synthetic polymer fibers are preferred materials for making a buckled fiber mesh according to the invention. Preferably in this embodiment, the buckled fiber mesh has a sheet-like shape. Further preferably, the buckled fiber mesh has a wave pattern. Further preferably, the buckled fiber mesh has suitable softness and density.

Examples of suitable materials for creating buckled fibers and/or a buckled fiber mesh according to the present invention for use as a textile are polyolefins, polydienes, polystyrenes, polyesters, poly(alkylene oxides), polyoxyalkylenes, polyhalogenoalkylenes, polyalkylenephthalat.es or terephthalates, polyphenyl or phenylenes, poly(phenylene oxide or sulphide), polyvinyl acetates), polyvinyl halides), poly(vinylidene halides), polyvinyl nitrites), polyamides, polyimides, polycarbonates, polysiloxanes, acrylic or methacrylic acid polymers, polyacrylates or polymethacrylates, polyethers, polylactams, chemically modified cellulosics, poly(ethylene glycols

terephthalates) and poly(butylene terephthalates).

Accordingly, the invention further provides a textile comprising a buckled fiber or a buckled fiber mesh obtainable by the method of the invention.

In another embodiment, the buckled fiber, or the buckled fiber mesh, can be used as a catalytic material, or as a support in a catalytic material. In this embodiment, the buckled fiber, or the buckled fiber mesh may have any shape and comprise any type of fibers, but preferably, a fiber is a metallic or a ceramic material, or a combination thereof, if the mesh is to be used as a catalytic material itself; if the mesh is to be used as a support for a catalytic material, any type of fiber is suitable which can support the catalytic material and which can withstand the conditions under which the catalytic material is to catalyze a reaction, including metallic and ceramic materials as well as natural and synthetic polymers. As such, the invention also provides a catalytic material comprising a buckled fiber or a buckled fiber mesh obtainable by the present method.

Preferably, a buckled fiber mesh is used in this embodiment, preferably in a three-dimensional structure which displays at least some resistance to deformation.

Alternatively, a buckled fiber mesh in this embodiment has a sheet-like shape. Further preferably, the buckled fiber mesh has large surface area per unit mass and supports high catalyst loading.

Examples of suitable materials for creating a buckled fiber and a buckled fiber mesh according to the present invention for use as a catalytic material are

polystyrene(PS), titanium tetraisopropoxide (Ti0₂), copolymers of acrylonitrile and acrylic acid (PAN-AA), polyolefins, polydienes, polystyrenes, polyesters, poly(alkylene oxides), polyoxyalkylenes, polyhalogenoalkylenes, polyalkylenephthalat.es or terephthalates, polyphenyl or phenylenes, poly(phenylene oxide or sulphide), polyvinyl acetates), polyvinyl alcohols), polyvinyl halides), poly(vinylidene halides), polyvinyl nitrites), polyamides, polyimides, polycarbonates, polysiloxanes, acrylic or methacrylic acid polymers, polyacrylates or polymethacrylates, polyethers, polylactams, chemically modified cellulosics, poly(ethylene glycols terephthalates) and poly(butylene

terephthalates), zirconia, aluminum hydroxide, tungsten, titanium, copper, silver, nickel- palladium alloys, nickel-cobalt alloys, and magnesium-zinc alloys.

In another embodiment, the buckled fiber or buckled fiber mesh can be used as a filter. Preferably, a buckled fiber mesh is used as a filter. In this embodiment, the buckled fiber mesh preferably has a wave pattern because higher surface area to volume ratio and resulting higher surface cohesion than a comparable non-buckled fiber mesh, or buckled fiber mesh without wave pattern. Therefore, the filtration efficiency can be improved.

Suitable materials for creating a buckled fiber and a buckled fiber mesh according to the present invention for use as a filter maybe synthetic or natural polymeric materials, ceramic materials, metallic materials, and combinations of these materials.

Ceramic fibres can be created from slurries including ceramic micro- or nanopowder and thermoplastic binders, but of course also binders based on duromer

(e.g., thermoset) or elastomeric precursors. Buckling would then preferably happen before the binder in the 'green fibre' is burned out pyrolized and the powder particles are sintered. The fibres can of course also stay composite fibres. Collagen can also be processed in a mixture with a (thermoplastic) polymer. Due to potential denaturation of collagen, unlocking of the pre-stretched/oriented shrinkable material should then be not by heat/temperature but, e.g., by a solvent (mixture) in a liquid or vapour phase.

Examples of natural materials are polypeptides, elastin, collagens, silk and polysaccharides, such as for instance, amylose, pectin, (non-chemically modified) cellulose and chitosan. Examples of_synthetic materials are polyolefins, polydienes, polystyrenes, polyesters, poly(alkylene oxides), polyoxyalkylenes, polyhalogenoalkylenes, polyalkylenephthalates or terephthalates, polyphenyl or phenylenes, poly(phenylene oxide or sulphide), polyvinyl acetates), polyvinyl alcohols), polyvinyl halides), poly(vinylidene halides), polyvinyl nitrites), polyamides, polyimides, polycarbonates, polysiloxanes, acrylic or methacrylic acid polymers, polyacrylates or polymethacrylates, polyethers, polylactams, chemically modified cellulosics, poly(ethylene glycols terephthalates) and poly(butylene terephthalates); Examples of ceramic materials are zirconia, aluminum hydroxide; Examples of metallic materials are tungsten, titanium, copper, silver, nickel- palladium alloys, nickel-cobalt alloys, and magnesium-zinc alloys.

Thus, the invention also provides a filter comprising a buckled fiber or a buckled fiber mesh obtainable by the present method.

In another embodiment, the buckled fiber or buckled fiber mesh can be used in tissue engineering applications in vivo or in vitro. Preferably, a buckled fiber mesh is used, more preferably a buckled fiber mesh which has a wave pattern. An advantage of a buckled fiber mesh, preferably with a wave pattern, in these applications is the high surface area and/or the high porosity, and/or the easier diffusion of cells and solutions through the buckled fiber mesh, and/or the special surface topography of the buckled fiber mesh. Accordingly, the invention further provides a tissue engineering scaffold comprising a buckled fiber or a buckled fiber mesh obtainable by the method of the invention.

Preferably, the buckled fiber mesh comprises fibers which are biocompatible, such as fibers of natural polymeric materials and fibers of synthetic polymeric materials, preferably thermoplastic synthetic polymeric materials. The skilled person knows which synthetic polymers are biocompatible. Examples of natural polymers which may be used to form buckled fibers in a buckled fiber mesh for tissue engineering applications are: polypeptides, elastin, collagens, silk and polysaccharides.

Examples of synthetic thermoplastic polymers which may be used to form buckled fibers and/or a buckled fiber mesh for tissue engineering applications are:

polyolefins, polydienes, polystyrenes, polyesters, poly(alkylene oxides), polyoxyalkylenes, polyhalogenoalkylenes, polyalkylenephthalates or terephthalates, polyphenyl or phenylenes, poly(phenylene oxide or sulphide), polyvinyl acetates), polyvinyl alcohols), polyvinyl halides), poly(vinylidene halides), polyvinyl nitrites), polyamides, polyimides, polycarbonates, polysiloxanes, acrylic or methacrylic acid polymers, polyacrylates or polymethacrylates, polyethers, polylactams, chemically modified cellulosics, poly(ethylene glycols terephthalates) and poly(butylene terephthalates).

Tissue engineering applications suitable for use with the present invention include but are not limited to use of a buckled fiber mesh as a tissue engineering scaffold. A tissue-engineering scaffold preferably has a wave pattern, and can be used as a more or less solid support on or in which cells of any type may be seeded and cultured. It will be understood that the tissue-engineering scaffold can also be implanted without cells having been seeded onto it. In a preferred embodiment, the invention also provides a method of creating an artificial tissue, comprising seeding a buckled fiber mesh with at least one target cell in a culture medium and culturing said at least one cell to create an artificial tissue. An artificial tissue in terms of the present invention is a three-dimensional cell- colony which has been formed at least partially within a tissue engineering scaffold comprising a buckled fiber material, preferably with a wave pattern. Artificial tissue may be formed in vitro or in vivo, and formed artificial tissue may subsequently be implanted into living tissue.

In one embodiment, the tissue engineering scaffold comprises a

biodegradable polymer. This has the advantage that engineered tissue may be preformed before use. Suitable biodegradable polymers are poly(ethylene glycol), poly(butylene terephthalate), poly(ethylene isophthalate), poly(lactic acid), poly(glycolic acid), poly(e- caprolactone), poly(trimethyl carbonate) and their copolymers.

In another embodiment, the tissue engineering scaffold comprises a natural polymer, such as a polymer occurring in the extracellular matrix of living tissues. Suitable polymers in this embodiment are proteins and polysaccharides, such as elastin, collagens, amylose, pectin, (preferably non-chemically modified) cellulose and chitosan.

In a tissue engineering scaffold, a buckled fiber mesh with a wave pattern has as a distinct advantage that the wave pattern closely mimics the natural wave pattern. In addition, the structure of the wave pattern may be optimized by appropriate modification of parameters such as the shrink ratio, fiber thickness and alignment, layer structure and adherence, as is understood by the skilled person, so that wave patterns similar to the natural wave pattern of any tissue can be substantially recreated.

As such, an artificial tissue can be formed by electrospinning or otherwise depositing a suitable polymer on a shrinkable material and subsequent buckling, to obtain tissue of the same molecular composition and a very similar wave pattern as natural tissue.

The advantage of this way of forming an artificial tissue is that a wave pattern similar to the natural wave pattern can be obtained much more reliably and predictably, and therefore easier, cheaper and faster. Thus-formed tissue may for instance be implanted to replace natural tissue, for instance by allowing cell diffusion from the surrounding tissue. Alternatively, it may be used for research purposes.

An artificial tissue can also be formed by seeding a buckled fiber mesh comprising synthetic and/or natural fibers, with cells which subsequently form the natural tissue inside the buckled fiber mesh. This can be done in vivo or in vitro. Preferably, the fiber is biocompatible. Further preferably, the buckled fiber mesh has a wave pattern.

In this case, the buckled fiber mesh is used as a scaffold, in which the seeded cells form the tissue. The advantage of this method lies in the increased porosity and diffusion through a buckled fiber mesh, in particularly those with a wave pattern. This sustains more cell-growth than in known materials, and allows formation of a tissue more resembling natural tissue. In addition, certain cell types only form natural-like tissue in an environment which sufficiently resembles the natural three-dimensional tissue. It has been found that a buckled fiber mesh with a wave pattern resembles natural tissue sufficiently to allow tissue formation, whereas known materials which lack the wave pattern do not.

Fibers for use in the latter method may be biodegradable, which can have benefits for certain tissue engineering applications, in particular in applications where tissue growth is quick and/or where dangerous surgery is required. Alternatively, nonbiodegradable fibers can also have benefits, for instance in applications where continued tissue support by a scaffold is preferred, such as in engineering of slowly growing or heavily stressed tissue, or in research. Cells which can be used for seeding include for example

- a pluripotent or multipotent stem cell if the artificial tissue is any tissue;

- an osteoblast if the artificial tissue comprises bone tissue;

- a chondrocyte if the artificial tissue comprises cartilage;

- a tenocyte if the artificial tissue comprises tendons;

- a ligament cell if the artificial tissue comprises ligaments;

- an adipocyte if the artificial tissue comprises fat tissue;

- a smooth muscle cell if the artificial tissue comprises smooth muscle; - a cardiomyocyte cell if the artificial tissue comprises the heart muscle;

- an endothelial cell if the artificial tissue comprises a vessel;

- an eptithelial cell if the artificial tissue comprises an epithelial tissue;

- a neuronal cell if the artificial tissue comprises nerve or brain tissue; - a fibroblast if the artificial tissue comprises connective tissue.

It is preferred if the buckled fiber mesh comprises a fiber type which is naturally present in the tissue of interest. It is further preferred that a naturally present fiber type is combined with a cell type that is naturally present in the tissue type of interest.

It has been shown that a buckled fiber mesh, in particular one with a wave pattern, displays high potential for cell growth, higher than for other scaffold materials. The shape (topography) of the buckled fiber mesh directly influences cell growth, not only by the altered properties as discussed above, but also by the mere chemical-mechanical surrounding the buckled fiber mesh provides.

In order to survive, cells rely on interactions with the surroundings and with other cells. When a buckled fiber mesh is seeded with cells, cells expand under the influence of culture medium, and take advantage of the scaffold to sense their

surroundings (mechanotransduction). This allows for the cell to behave like a transducer, converting mechanical information of the surroundings into biochemical responses, resulting in gene- and protein-level modulation. In addition, the topography of the material has the normal mechanical effects on cells.

For example, cells spread on a non-buckled fiber mesh display a higher spread, such as a monolayer, due to the flatness of the surface. Cells seeded on a buckled fiber mesh with a wave pattern assumed a more tight initial packing, but also displayed much higher penetration into the material. In addition, it was shown that the shape as well as the biochemical response of the cells to their surroundings was different, with the cells on the buckled fiber mesh with a wave pattern behaving more similar to cells in a natural environment.

Also, the wave pattern influences growth of the cells. Larger grooves prevents cells from spreading over different grooves, whereas smaller grooves allows cells to bridge the peaks between the grooves by elongation of the cell itself, thereby resulting in a different distribution and different cell shape. This is true both on the surface of the buckled fiber mesh, as within the buckled fiber mesh, leading to different three- dimensional distributions of cells, and therefore different differentiation and different tissue formation. Cells cultured in a buckled mesh with a wave pattern behaved more similar to cells in their natural surroundings.

Also, the higher porosity and higher "randomness" of porosity in a buckled fiber mesh allows faster ingrowth of cells than for other electrospun scaffolds. This is true in particular for a buckled fiber mesh with a wave pattern.

In a different embodiment, it was shown that a buckled fiber mesh with a wave pattern had the same mechanical effect on the cells responsible for formation of tendons and ligaments, modeled by TGFD cells. A buckled fiber mesh with a wave pattern comprising a synthetic thermoplastic polymer (in this case: a poly(ethylene glycol/oxide terephthalate) - co - poly(butylene phthalate) copolymer) was shown to have an increased activation effect on TGF-D relative to the same non-waved material. It could further be shown that this effect was due to the wave pattern in the buckled fiber material, which had similar periodicity as natural collagen tissue. These results are expected to be applicable to other tissue types, given the similarity in obtainable wave pattern for various polymers with natural wave patterns of various tissue.

In an alternative embodiment, an artificial tissue may be formed by creating a buckled fiber mesh, preferably with a wave pattern, from a suitable polymer. Examples of suitable polymers in this regard are collagen and elastin. Such tissue may for instance be implanted into natural tissue of the same type in order to repair tissue defects.

The invention will further be illustrated by the following, non-restricting examples.

Examples:

Example 1 : Buckled fiber

METHOD

Three different methods were performed to change the shape of fibers through buckling.

(1 ) Polyactive^® 300/55/45(PA) (a block copolymer composed of poly(ethylene oxide terephthalate) (PEOT) and poly(butylenes terephthalate) (PBT) with a weight ratio of 55 o 45 for the two segments, respectively, and a molecular weight of the starting poly(ethylene glycol)(PEG) segments of 300Da used in the co-polymerization process) from PolyVation® (Groningen, the Netherlands), was dissolved in a 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature.

The feed rate of the polymer solution during ESP was controlled by a pump at a rate of 2 ml/h. The distance between the needle tip and the collector was set to 15 cm and the applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. Aligned fibers were collected on a poly (lactic acid)

(PLA) monoaxially oriented thermoshrinkable material ("film") with a shrink ratio around 60 % which was put on the top of a pair of electrodes. Fiber collection time was 2 seconds. Changing the position of the PLA film during the electrospinning allows the deposition of fibers according to specific orientations. Parallel, diagonal and perpendicular orientations relative to the direction of shrinkage were used in this experiment. PA fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber.

(2) For tailoring of the buckled pattern of fibers, PA fibers were deposited on a PLA frame, instead of a PLA film, which was put on the top of a pair of electrodes. The frame, having a void area in its middle, allows the fibers to buckle also along the fiber thickness (transversal direction with respect of the PLA frame). Fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA frame and formation of a buckled fiber mesh.

(3) For tailoring of the buckled pattern of fibers, a second layer of polyvinyl alcohol) (PVA) fiber was deposited on top of the PA fiber layer. PVA and PA fibers were interspersed by spinning layer-by-layer in an alternating manner. Briefly, 8% (w/v) of PVA solution was prepared by dissolving PVA powders in a mixture solvent of ethanol/water (V/V = 1/4). The ESP parameters for PVA were the same as that for PA fiber described in (1 ). Fiber-film constructs were then transferred to an oven pre-heated to 75 °C ± 1 °C for 1 min to allow shrinking of the PLA film and formation of a buckled fiber mesh. After that, the fiber-film constructs were immersed in water for 30 min to remove PVA fibers.

To check the morphology of fibers, samples were sputter-coated with gold and then observed under scanning electron microscopy (SEM) (Philips XL-30) at an accelerating voltage of 10kV. The measurements were made using the software for the microscope control.

RESULTS

Figure 1 shows a schematic of fabricating buckling fibers using

thermoshrinkable PLA material. The effect of buckling random fibers with a shrinkable material is clear from a comparison of the fiber shape before and after buckling (from linear to a buckled configuration, sinusoidal shapes can be observed) (Figure 2). Figure 3 depicts a comparison between fibers deposited in different directions before and after shrinkage. The average wavelength and amplitude of the buckled fibers was 3 urn and 1.3 urn for parallel oriented fibers, and 4.3 urn and 1 .7 urn for diagonally oriented fibers

(Figure 4). Perpendicularly oriented fibers did not result in a measurable buckled pattern.

The buckle pattern can be changed by using a modified PLA film, a PLA frame, from which a geometry part (it can be any shape) was removed. Figure 5 depicts fibers depositing on a PLA frame before and after shrinking. After shrinking, a buckled pattern was observed at the fiber level.

The buckled pattern can also be tailored by sacrificing a second layer of fibers. After removing the PVA fiber layer by dissolution in water, the buckled pattern of the fiber mesh is similar in shape to the signal of a cardio electrogram (Figure 6B). The highlighted regularity in peaks that occurred (Figure 6C) was the result from scarifying of PVA fiber. The effect of changing buckling patterns on fibers by using this methods is clear from comparing the fiber shape (Figure 6D) without using a sacrificed fiber.

Example 2: Buckled fiber mesh METHOD

Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands), was dissolved in an 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between needle tip and collector was set to 15 cm and applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. Aligned fibers with their orientation parallel or perpendicular relative to the direction of shrinkage were deposited on a poly(lactic acid) (PLA) monoaxially oriented

thermoshrinkable material ("film") with a shrinking ratio around 60 % which was put on the top of a pair of electrodes. The fibers were collected from 10 seconds to 45 min. Fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh. The morphology of the fibers was analyzed by scanning electron microscopy as described above. Fiber layers aligned parallel and diagonally relatively to the direction of shrinkage were tested. RESULTS

Buckled fiber meshes were successfully prepared using electrospinning and subsequent buckling with a shrinkable material. The buckling pattern was dependent on the thickness of the fiber layer.

For fiber layers with a thickness less than around 0.28 ± 0.10 mm (for perpendicular orientations relative to the direction of film shrinking) or 0.25 ± 0.08 mm (for parallel orientations relative to the direction of film shrinking), buckling occurred as described for single fibers in Example 1. Above this thickness threshold, buckling transferred to the whole electrospun fiber mat, resulting in a higher layer organization visible by a wave pattern (Figure 7).

Example 3: Tailoring the buckled pattern on fiber meshes by sacrificing fibers METHOD

Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands) into chloroform/HFIP (V:V=4:1 ) at the concentration of 20% (w/v) and stirred overnight at room temperature until complete dissolution. This solution was then loaded into a 5 ml syringe mounted with a needle with diameter of 0.8 mm. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between the needle tip and the mandrel was 12 cm and the applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. The polyvinyl alcohol) (PVA) solution was prepared by dissolving of PVA into ethanol/water (1 :4) at a

concentration of 8% (w/v) and stirred overnight before electrospinning. The feeding rate of PVA solution was controlled by a syringe pump at a rate of 1 ml/h. The applied voltage was16 kV and the distance between needle tip to mandrel was 12 cm. Poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable film with a shrinking ratio around 60 %were covered on the surface of mandrel to collect fibers. Fibers were collected for 45 min for each sample with a speed of mandrel at 300rpm/min.

Fiber-film constructs were then transferred to an oven pre-heated to 75 ±1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh. After that, the fiber-film constructs were immersed in water for 30min to sacrifice PVA fibers by dissolution. RESULTS

The buckle pattern of a fiber mesh can be changed by using sacrificial fibers (Figure 9). Figure 9A shows a brief concept of blending ESP techniques used in spinning PA PVA fiber materials. After sacrificing the PVA layer, the structure of PA/PVA collapsed in some content thus changing the pattern of the fiber mesh (Figure 9D).

Example 4: Buckled tube mimicking the structure of trachea

METHOD

Buckled fiber material mimicking the structure of trachea was fabricated by using a mandrel wrapped with a cylinder-shaped Poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable film with a shrinking ratio around 60%. Briefly, Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands) was dissolved in a 80/20( V/V) mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature before

electrospinning. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between the needle tip and the mandrel was set to 12 cm and the applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. The fibers were collected for 30min with a rotating speed of mandrel at150rpm/min. After removing the fiber-film tube from mandrel, it was transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh.

RESULTS

A buckled fiber mesh which structure mimics trachea was successfully prepared using electrospinning and subsequent buckling with a shrinkable tube material. Figure 10 depicts the morphology of fibers tube before and after shrinkage.

Example 5: a buckled fiber mesh in tissue engineering using bone marrow derived human mesenchymal stromal cells (hMSCs)

METHOD

The buckled pattern created in a fiber mesh increases space between fibers. Human mesenchymal stromal cells (hMSCs) were cultured on the buckled fiber meshes from Example 2 to instigate the improvement of cell infiltration into the scaffold. hMSCs were cultured in basic medium which contains minimal essential medium (alpha-MEM, Life Technologies, Gaithersburg, MD), 10% fetal bovine serum (FBS, Lonza), 0.2 mM L- glutamine(lnvitrogen), 0.2 mM ascorbic acid (Sigma, Aldrich), 100 units/ml penicillin (Life Technologies) and 100 mg/ml streptomycin (Life Technologies). Buckled fiber meshes from example 2 were sterilized with 70% ethanol two times and then incubated in basic medium for 3 hours before cell seeding. Cells were seeded with a cell density of 5000 cells/cm² and cultured up to 5 days. The medium was refreshed every two days.

After 5 days of culture, cell infiltration was evaluated by staining the cross- section of scaffold with DAPI. In order to get cross-sections, the circular samples were cut in half and embedded with Cryomatrix™ mounting media on mounting blocks with the cross-section facing upwards. Using the Shandon Cryotome®, evenly-spaced (80 urn) sections were cut with a thickness of 8 urn. Samples were checked under a fluorescent microscope (Nikon Eclipse E600).

RESULTS

Cell infiltration was investigated by staining the cross-section of fiber meshes with DAPI . Cell nuclei will be stained in blue. The fiber meshes before buckling was set as a control. At day 2, higher cells numbers were present on the cross-section of buckled fiber meshes compared to non-buckled fiber meshes, which means an improvement of hMSCs infiltration after buckling fiber meshes (Figure 1 1 ). This trend can also been seen at day 5. So the buckled fiber meshes show an improvement of cell infiltration when compared to fiber meshes before buckling. Example 6: Buckled fiber materials in tendon and ligaments tissue engineering

METHOD

Given the resemblance of the obtained fibers with the buckled patterns typical of tendons and ligaments, we investigated if the wave pattern of a buckled fiber mesh could regulate a key signaling pathway involved in tendon and ligament maintenance, the TGF-β pathway.

Mink lung epithelial cells (MLEC), which are stably transfected with an expression construct containing a truncated plasminogen activator inhibitor-1 (PAI-1 ) promoter fused to the firefly luciferase reporter gene as reported by Abe (Anal. Biochem. 1994, volume 216, issue 2, Pages 276-284).

These cells are highly sensitive and specific to TGF-β expression, based on its ability to induce PAI-1 expression. MLEC were cultured in Dulbecco's Modified Eagle's Medium (Life Technologies, Gaithersburg, MD) supplemented with 0.2 mM L-glutamine (Invitrogen), 0.2 mM ascorbic acid (Sigma, Aldrich), 100 units/ml penicillin (Life Technologies) and 100 mg/ml streptomycin (Life Technologies). The medium of control samples was added with 2ng/ml fetal bovine serum (FBS, Lonza) to work as TGF-βΙ . The buckled fiber meshes from example 2 were sterilized with 70% ethanol for 3 times, each time stay 15min, and incubated in the described medium (without TGF-βΙ ) overnight before cell seeding. Cells were seeded at a cell density of 50000 cells/cm2 on the scaffolds and tissue culture plate (TCP) as control, the cell culture were carried on up to 5 days.

The activation of the reporter was analyzed by Luciferase assay. Samples were washed thoroughly with PBS, lysis buffer (5x) diluted (5:1 ) in Milli-Q water was added and stored at -80°C for at least 1 h. Then, samples were defrosted on a plate shaker with luciferase assay substrate (Promega) and read the light produced on Victor 3 ™ plate reader (PerkinElmer®) column by column to ensure that the reaction is still stable (1 min). The data presented was normalized with the DNA amount. The amount of DNA on scaffold were investigated by using CyQUANT® cell proliferation assay kit (Invitrogen) according to its instruction. Samples were lysed and incubated for 1 h at room

temperature. Meanwhile, a standard curve was prepared using the DNA standard provided. After the incubation period, the CyQUANT® GR dye was mixed with lysis buffer and added to the samples. Fluorescence was measured on Victor 3™ plate reader (PerkinElmer®).

RESULTS

Cells seeded on a buckled fiber mesh with a wave pattern ("wavy scaffold") emitted more light than those on a buckled fiber material without a wave pattern "flat scaffold"). (Figure 12A). At day 2, cells on wavy scaffolds show significant higher light single than that on flat scaffold with a p-value < 0.05 (flat: 7654 ± 1988 vs wavy: 13248 ± 3349). The fetal bovine serum (FBS) control worked just as the TGF-βΙ and showed that cells were ok. Even normalized with the DNA amount, the trend presented (Figure 12B) shows that flat scaffolds induced less luciferase activity than wavy scaffolds at day 2 and day 5. Based on the above results, the wavy scaffold displays high potential in tendon tissue engineering.

Example 7: buckled fibers using other shrinkable materials

METHOD

Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands), was dissolved in a 80/20 v/v mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final polymer concentration of 20%(w/v), and stirred overnight at room temperature. The flow of the polymer solution during ESP was controlled by a pump at a feeding rate of 5 ml/h. The distance between the needle tip and the collector was set to 15 cm and the applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. For random fibers, polystyrene (PS), biaxially oriented thermoshrinkable material ("film") with a shrinking ratio around 50 % was put on the top of a static plate collector. For aligned fibers, a polystyrene (PS) film was covered on the surface of a mandrel with a high speed of 5000 rpm/min. The thickness of fibers layers was controlled by the deposition time.

Fiber-film constructs were then transferred to an oven pre-heated to 120 °C for 15 min to allow shrinkage of the PS film and formation of buckled fiber materials. The morphology of the fibers was analyzed by scanning electron microscopy.

RESULTS

Figure 13 depicts buckled fibers were successfully fabricated using biaxial oriented polystyrene film. The buckling pattern on both random fiber and aligned fiber is clear after shrinking compared to the shape of fiber before shrinking.

Example 8: buckled fibers using other polymers

METHOD

Polyvinyl alcohol) (PVA) solution was prepared by dissolving PVA into ethanol/water (1 :4) at a concentration of 8% (w/v) and stirred overnight before

electrospinning. The feeding rate of PVA solution was controlled by a syringe pump at a rate of 1 ml/h. The applied voltage was 16 kV and the distance between needle tip to collector was 15 cm. Poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable film with a shrinking ratio around 60 %were covered on the top of electrodes collector to collect fibers. The collecting time is range from 5s to 30min.

Fiber-film constructs were then transferred to an oven pre-heated to 75 ±1 for 1 min to allow shrinkage of the PLA film and formation of buckled fiber or materials. The morphology of the fibers was analyzed by scanning electron microscopy.

RESULTS

Figure 14 depicts buckling fiber from PVA were successfully fabricated using monoaxial oriented PLA film. The buckling pattern on aligned fiber and aligned fiber mesh are clear after shrinking compared to the shape of fiber before shrinking.

Example 9: Similarities between native tissues and the buckling fiber materials METHOD

The follow set of images shows a few examples that with some adjustments could be recreated with the buckled fiber material here proposed.

Figure 15 shows an artery wall composed of curled collagen fibers interconnected by a matrix. This pattern could be recreated by using the following method.

Polyactive^® 300/55/45(PA) (a block copolymer composed of poly(ethylene oxide terephthalate) (PEOT) and poly(butylenes terephthalate) (PBT) with a weight ratio of 55 o 45 for the two segments, respectively, and a molecular weight of the starting poly(ethylene glycol) (PEG) segments of 300Da used in the co-polymerization process) from PolyVation® (Groningen, the Netherlands), was dissolved in a 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature.

The feed rate of the polymer solution during ESP was controlled by a pump at a rate of 2 ml/h. The distance between the needle tip and the collector was set to 15 cm and the applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. Aligned fibers were collected on a poly (lactic acid) (PLA) monoaxially oriented thermoshrinkable material ("film") with a shrink ratio around 60 % which was put on the top of a pair of electrodes. Fiber collection time was 2 seconds and its orientations is parallel to the direction of shrinkage were used in this experiment. PA fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber. Collagen from the dermis of the skin (Figure 16) also displays a specific crimp pattern. This pattern could be mimicked by the following way. Briefly, Polyactive^®

300/55/45 (PolyVation®, Groningen, the Netherlands), was dissolved in an 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between needle tip and collector was set to 15 cm and applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. Aligned fibers with their orientation perpendicular relative to the direction of shrinkage were deposited on a poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable material

("film") with a shrinking ratio around 60 % which was put on the top of a pair of electrodes. The fibers were collected 45 min. Fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh.

The figure above shows two SEM pictures of the ciliary body (Figure 17 a) and the iris (figure 17 c), both part of the human eye. The ciliary body pattern could be recreated by using our methods. Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands), was dissolved in an 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3- hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between needle tip and collector was set to 15 cm and applied voltage was 16 kV. The temperature was approximately 25 °C and the humidity was approximately 30%. For recreating structure mimicking ciliary body, aligned fibers with their orientation perpendicular relative to the direction of shrinkage were deposited on a poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable material

("film") with a shrinking ratio around 60 % which was put on the top of a pair of electrodes. The fibers were collected 30 min. For mimicking the structure of iris, random fibers were deposited on PLA monaxially oriented film by using a static ground collector and fiber collecting time is 30min.

Then fiber-film constructs were transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh.

Accuracy of recreation can be observed by SEM pictures of the buckled fiber meshes (Figures 17 b and d). Example 10: The potential application of buckled fiber mesh in textile field METHOD

Polyactive^® 300/55/45 (PolyVation®, Groningen, the Netherlands), was dissolved in an 80/20 V/V mixture of chloroform and 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) with a final concentration of 20%(w/v), and stirred overnight at room temperature. The flow of the polymer solution during ESP was controlled by a pump at a rate of 5 ml/h. The distance between needle tip and collector was set to 15 cm and applied voltage was 16 kV.

The temperature was approximately 25 °C and the humidity was

approximately 30%. Poly(lactic acid) (PLA) monoaxially oriented thermoshrinkable film with a shrinking ratio around 60 %were covered on the surface of mandrel to collect fibers. Fibers were collected for 30 min for each sample with a speed of mandrel at 150rpm/min.

Fiber-film constructs were then transferred to an oven pre-heated to 75 ± 1 °C for 1 min to allow shrinkage of the PLA film and formation of a buckled fiber mesh. The wettability of the fibers was analyzed by contact angle measurement.

RESULTS

Figure 18 depicts the wettability of fibers mesh before (black line) and after (red line) buckling were analyzed by contact angle measurement. The contact angle of non-buckled fiber mesh decrease from 129.34° to 1 1 .0 ° in 100651 ms while the contact angle of buckled fiber mesh decrease from 162.9 ° to 13.3 ° in 226543 ms. This significant difference can also be seen in Figure 19. The liquid drop on non-buckled fiber mesh disappeared around 1 17 s. However, the time span of the liquid drop completely disappearing on buckled fiber mesh was around 227 s which is almost two times compared to fiber mesh before buckling.

Claims

1 . A method of making a scaffold comprising a buckled fiber, the method comprising: a. depositing a polymer fiber onto a thermoshrinkable material by

electrospinning, wherein the depositing results in a construct of a fiber mesh of multiple layers of fibers, adhered to the thermoshrinkable material, wherein the fiber mesh has a thickness of at least 50 times the average diameter of the fiber,

b. heating the construct in order to shrink the thermoshrinkable material and c. cooling down the construct in order to obtain a scaffold comprising a buckled fiber, adhered to the thermoshrinkable material, and

d. optionally, removing the scaffold comprising a buckled fiber from the

thermoshrinkable material.

2. A method according to claim 1 wherein the polymer fiber is a thermoplastic,

synthetic fiber.

3. A method according to claim 1 or 2 wherein the polymer fiber is a biocompatible polymer fiber.

4. A method according to any one of claims 1 - 3 wherein the thermoshrinkable

material is shrinkable in at least one direction, preferably in two directions.

5. A method according to any one of claims 1 - 4, wherein at least one fiber is at least partially aligned non-perpendicular relative to a direction of shrinkage of the shrinkable material.

6. A method according to any one of claims 1 - 5, wherein the scaffold comprising a buckled fiber is fixated to retain shape after step b) or c).

7. A method according to any one of claims 1 - 6, wherein the thermoshrinkable material has a shrinking temperature TS which is below the melting point TM of the electrospun polymer.

8. A method according to any one of claims 1 - 7, wherein the thermoshrinkable material has a shrinking temperature TS which is below the denaturation or degradation temperature TD of the electrospun polymer.

9. A method according to any one of claims 1 - 8 wherein the thermoshrinkable material has a shrinking temperature TS which is above the glass transition temperature TG of the electrospun polymer.

10. A scaffold comprising a buckled fiber obtainable by a process according to any one of claims 1 - 9.

1 1 . A scaffold comprising a buckled fiber according to claim 10, wherein the buckled fiber comprises a wave pattern with an average wavelength between 1 .5 and 30 times the fiber diameter and an average amplitude of between 0.2 to 5.0 urn.

12. A scaffold comprising a buckled fiber according to claim 10 or 1 1 , wherein the buckled fiber comprises a wave pattern with a wavelength between 20 to 500 urn and an amplitude between 0.1 to 6.0 urn.

13. Use of a scaffold comprising a buckled fiber according to any one of claims 10 - 12 in in vitro tissue engineering, as a filter, as a catalytic material or as a textile.

14. In vitro method for creating an artificial tissue, comprising seeding a scaffold

according to any one of claims 10 - 12 with at least one cell in a cell culture medium, and culturing said cell to create a three-dimensional cell colony at least partially within the scaffold, thereby creating an artificial tissue.

15. Method according to claim 14 wherein the cell is selected from the group

consisting of a pluripotent or multipotent stem cell, an osteoblast, a chondrocyte, a tenocyte, a ligament cell, an adipocytes, a smooth muscle cell, a cardiomyocyte cell, an endothelial cell, an epithelial cell, a neuronal cell and a fibroblast.

16. An artificial tissue obtainable by a method according to claims 14 or 15.