WO2013178229A1

WO2013178229A1 - A biodegradable non-woven mesh with glue points

Info

Publication number: WO2013178229A1
Application number: PCT/DK2013/050162
Authority: WO
Inventors: Monica Ramos Gallego; Jakob Vange VANGE
Original assignee: Coloplast A/S
Priority date: 2012-05-30
Filing date: 2013-05-29
Publication date: 2013-12-05

Abstract

A biocompatible non-woven mesh comprising biodegradable fibres and glue points in the form of domains of biodegradable polymer wherein the fibres are interconnected by the glue points. The mesh is suitable for use as scaffold inthe treatment of pelvic organ prolapse (POP), stress urinary incontinence (SUI), or hernia.

Description

Title

A biodegradable non-woven mesh with glue points

Field of the invention

The present invention relates to a mesh suitable for medical application. In particular the present invention relates to biocompatible composite non-woven mesh useful for medical application.

Background

Scaffolds are structures, such as synthetic polymer structures used to guide the organization, growth and differentiation of cells in the process of forming new functional tissue at the site of a tissue defect or wound, typically used in conjunction with surgical intervention.

To achieve the goal of tissue reconstruction, scaffolds must meet some specific requirements. A high porosity and an adequate pore size are necessary to facilitate cell growth and diffusion throughout the whole structure of both cells and nutrients.

Biodegradability is essential since scaffolds need to be absorbed by the surrounding tissues without the necessity of a surgical removal.

Many different materials (natural and synthetic, biodegradable and permanent) have been investigated for use as scaffolds. Some of these materials have been known in the medical field before the advent of tissue engineering as a research topic, being already employed as bioresorbable sutures. Examples of these materials may be some linear aliphatic polyesters.

Conditions like stress urinary incontinence and Pelvic Organ Prolapse (POP) are indications for women seen as a result of multiparity, muscle weakness due to ageing and hormonal insufficiency. However, the same indications are also seen in younger inactive patients who have never given birth. Since the 1980's the use of synthetic meshes made from polypropylene has been in the preferred treatment. Examples of these meshes are: Prolene (Ethicon), Polyform (Boston Scientific) and Pelvitex (Bard). Over the last years, an increased number of side effects have been reported in up to 10% of the cases. Vaginal erosion and vaginal shortening are some of the more severe (Mistrangelo et.al, (2007).

To overcome these side effects, a lighter version (less material) of the traditional mesh has been developed and some, which have been made partly degradable by combining polypropylene with a degradable synthetic polymer-like Poliglecaprone (a glycolide- caprolactone copolymer)(Ultrapro, Ethicon). Cook Inc. has a xenografic approach which is completely degradable and based on decellurised extracellular matrix from porcine small intestines (Mantovani et al., 2003).

Summary

Electrospun meshes may be suitable for use as scaffolds. Electrospinning is a technique for making structures of non-woven nano- or microfibre. Because of the fibrous structure and the large surface area these structures can be useful as scaffolds for tissue regeneration. When made from a biodegradable polymer, scaffolds can first support formation of new tissue in-vivo and then disappear by being resorbed. Because electrospun mesh is non-woven, the strength is mostly achieved by friction between the fibres. When implanted into living tissue, cells will migrate into the structure and reduce this cohesion by lifting the fibres apart thereby weakening the scaffold.

This weakening of the mesh is undesirable. In a study where the mesh is implanted on the abdomen of rats according to Duprest, the mesh stretched during the study resulting in a hernia-like defect. Examples of this stretching are shown in Figure 2.

Thus, there is still a need for a mesh being more suitable for scaffolds.

One object of the present invention is to provide a mesh having improved strength and sustainability that contribute to the ease of the handling of an implant comprising said mesh under surgical implantation such as general prolapse surgery, pelvic organ prolapse, incontinence (sling) and hernia.

A further object of the present invention is to provide a mesh having improved strength that contribute to the support of tissue in applications such as general prolapse surgery, pelvic organ prolapse, incontinence (sling), hernia and enabling efficient cells migration into and within the mesh and adherence of the cells to the mesh.

In some aspects is provided a biocompatible non-woven mesh comprising

a) fibres of a biodegradable fibre material and

b) glue points in the form of domains of biodegradable polymer

wherein the fibres are interconnected by the glue points.

In some aspects the fibres have an in vivo degradation time that is higher than an in vivo degradation time of the glue points.

In some aspects the fibres have an in vivo degradation time of 2-48 months. In some aspects the glue points have an in vivo degradation time of 1 -52 weeks.

In some aspects the biodegradable fibre material comprises homo- or co-polymers of glycolide, L-lactide, DL-lactide, D-lactide,meso-lactide, ε-caprolactone, δ- valerolactone, 1 ,4-dioxan-2-one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3- hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane.

In some aspects the biodegradable polymer of the glue points comprises homo- or copolymers of glycolide, L-lactide, DL-lactide, D-lactide,meso-lactide, ε-caprolactone, δ- valerolactone, 1 ,4-dioxan-2-one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3- hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane.

In some aspects the fibres are electrospun fibres.

In some aspects the amount of glue points in the mesh is 1-70% (w/w). In some aspects the mesh has an area density in the range 2-20 mg/cm².

In some aspects the fibres have an average diameter size of 0.1 μηι-10μΓΤΐ. In some aspects said mesh further comprises a component that facilitates cell adhesion and/or migration into the mesh.

In some aspects said mesh further comprises a component selected from the group consisting of estrogen, estrogen derivatives, thrombin, ECM (Extra Cellular Matrix) powder, chondroitin sulfate, hyaluronan, Hyaluronic Acid (HA), heparin sulfate, heparan sulfate, dermatan sulfate, growth factors, such as Insulin-like growth factors (IGFs), such as IGF-1 or IGF-2, or Transforming Growth Factors (TGFs), such as TGF-alpha or TGF- beta, or Fibroblast Growth Factors (FGFs), such as FGF-1 or FGF-2, or 20 Platelet- Derived Growth Factors (PDGFs), such as PDGF-AA, PDGF-BB or PDGF-AB, or Mechano Growth Factor (MGF), or Nerve Growth Factor (NGF), or Human Growth

Hormone (HGH); fibrin, fibronectin, elastin, collagen, such as collagen type I and/or type II, type III, type IV, type V and/or type VII, gelatin, and aggrecan, or any other suitable extracellular matrix component.

In some aspects said non-woven mesh is suitable for supporting, augmenting and regenerating soft tissue.

In some aspects the mesh is for use in the treatment of pelvic organ prolapse, stress urinary incontinence or hernia.

In some aspects the glue points are in the form of substantially non-fibrous domains. In this context, non-fibrous means that the biodegradable polymer making up the glue point is not in fibre form. Substantially non-fibrous means that less than 5% (v/v) of the glue point is in fibrous form. The glue point may in some embodiments be entirely non-fibrous. In some embodiments, the glue points may comprise less than 25% (v/v) fibrous material, such as less than 20% (v/v) fibrous material, such as less than 15% (v/v) fibrous material, such as less than 10% (v/v) fibrous material, such as less than 5% (v/v) fibrous material, such as less than 2% (v/v) fibrous material, such as less than 1 % (v/v) fibrous material.

In some aspects the glue points are distributed homogeneously in the mesh.

Also provided is a biodegradable surgical implant for supporting, augmenting and regenerating soft tissue, where said implant comprises

a) fibres of a biodegradable fibre material, and

b) glue points in the form of domains of biodegradable polymer wherein the fibres are interconnected by the glue points.

In some aspects said implant is suitable for the treatment of pelvic organ prolapse, stress urinary incontinence or hernia.

Also provided is a method of preparing a non-woven mesh by electro spinning, said method comprising the steps of

a) dispensing a biodegradable fibre-forming solution on a collector (rotating cylinder), and

b) simultaneously or subsequently dispensing drops of a biodegradable

polymer solution by electrospraying. In some aspects the solution in step (b) comprises a solvent selected from the group consisting of ethyl acetate (EtOAc); isopropyl acetate (iPrOAc); a mixture of EtOAc and methanol (MeOH); and a mixture of iPrOAc and MeOH.

In some aspects said solvent is EtOAc.

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Biocompatible material: In the context of the present invention, a biocompatible material refers to a material that interacts with the surrounding tissue without eliciting any undesirable local or adverse systemic effects in the recipient or beneficiary of that therapy. Thus, in the context of the present invention biocompatible mesh or surgical implant refers to a mesh or surgical implant, which does not have undesirable local or adverse systemic effects in the recipient to which it is applied.

It is of importance that the metabolites from the biological degradation of the material are cleared from the body. Thus, a material falls under the present definitions of biocompatible materials only if the in vivo degradations products are cleared of the body of the subject.

Glue points: By glue points are herein meant domains of a non-fibre forming polymer distributed in the mesh thereby gluing the fibres together sporadically. The glue points may be in the form of droplets and/or irregular domains connecting adjacent fibres. The glue points are substantially non-fibrous, i.e. less than 5% v/v of the glue points are in the form of fibres.

Biodegradable: The term "biodegradable" refers to the capacity of a material to decompose over time as a result of biological activity, such as by enzymatic degradation or through simple hydrolysis. The material disappears over time; is biodegraded and vanish from the implantation site and the body within a given time. This is a huge clinical advantage as there is no remaining material to remove or to make complication afterwards. An example of a polymer, which is not biodegradable, is polyethylene oxide (PEO).

However, low molecular weight PEO (<40 kDa) is excreted through the kidney and expelled through the urine unaltered. Higher (>40 kDa) such as 100 kDa PEO is not excreted and tends to accumulate in the kidney.

Degradation time: The degradation time of a material is the time it takes for that material to be degraded. The in vivo degradation time is the time it takes for a given material to be degraded when said material is placed in vivo. For instance, biodegradable implants will degrade over time when implanted into an animal. A short degradation time means that the material degrades quickly, whereas a long degradation time means that the material degrades slowly. Composite materials may have different degradation times for the different components. For instance, the fibre material of the instant biodegradable meshes may have a degradation timer that is different from the glue points of the same meshes.

Non-woven mesh: A non-woven mesh refers to a sheet made from a layer of fibres bonded by random entanglement and/or physical, mechanical or chemical means as opposed to weave or knitted fabrics where the entanglements are highly ordered. The orientation of the fibres in a non-woven can be either random or have some degree of order. The bonds in non-woven formed by electrospinning are entanglement and sometimes connecting points between fibres formed by incomplete evaporation of spinning solvents when fibres hit the collector, causing the fibres to fuse where they touch each other. Mesh: The term "mesh" refers to a material (such as a sheet) providing a semi-permeable barrier. The mesh of the present invention is biocompatible. The term "mesh" refers to a material being porous and fibrous, preferably in the form of a layer or sheet, but may also have a more three-dimensional structure suitable for implants.

Fibre material: Fibre material refers to a material in the form of fibre of a uniform fibre thickness, or a range of thicknesses. Electrospinning: Electrospinning (or E-spinning) is a technique where a solution of polymer is stretched into a thin fibre by applying a voltage between solution and collector, typically resulting in an electrical field of 0.5-2 kV/cm. As the solution is stretched, the solvent evaporates, resulting in a fibre. The fibre can be collected as a non-woven fabric, either with a random orientation of the fibres, or with special setups, some degree of order. The technique can be used to form fibres with diameter ranging from <100nm to >10μηι.

In a non-aligned e-spun structure, the fibres are deposited randomly on the collector. In an aligned e-spun structure the fibres are deposited on the collector with a preferred direction. This can be done with special techniques, e.g air-gap e-spinning, or with a rapidly spinning collector.

Youngs modulus: Youngs modulus is the slope of the stress-strain curve for the elastic deformation of a sample.

da

Youngs modulus =—

oe

Where ε=(Ι-Ι₀)/Ι. I is the initial length of the sample and (l-l₀) is the elongation of the sample, σ is the stress obtained by dividing force with cross section of sample. In the calculations, the thickness used is the reduced thickness. This is the thickness of the sheet if compressed to a non-porous film. Reduced thickness = ( Area density)/p, where p is the density of the polymer (1.15 g/cm³ for polycaprolactone).

Area density refers to the weight of a given area of material. In one embodiment of the present invention, the area density is in the range of 0.1-50 mg/cm². Surgical implant: A surgical implant refers to a medical device manufactured to replace a missing biological structure, support a damaged biological structure, or enhance an existing biological structure. In the present context, the surgical implant is a biocompatible material that interacts with the surrounding tissue to perform the desired function with respect to a medical therapy, after which it is degraded and the metabolites are cleared of the body.

Tissue: The term "tissue" as used herein refers to a solid living tissue which is part of a living mammalian individual, such as a human being. The tissue may be a hard tissue (e.g. bone, joints and cartilage) or soft tissue including tendons, ligaments, fascia, fibrous tissues, fat, synovial membranes, muscles, nerves and blood vessels.

Detailed Disclosure

Provided is a biocompatible non-woven mesh comprising fibres of a biodegradable fibre material and glue points in the form of domains of biodegradable polymer wherein the fibres are interconnected by the glue points. The glue points may be in the form of non- fibrous domains. The biodegradable polymer forming the glue points may be a non-fibre forming biodegradable polymer.

The mesh of the present invention allows migration of cells into the mesh and adherence of the mesh in concert with the regeneration and augmentation of the tissue surrounding the implanted mesh, while retaining the strength needed for supporting the tissue. The effect is accomplished by applying a non-woven biodegradable mesh with glue points.

The fibres and the glue points may have different in vivo degradation times, one fast degrading and one slow degrading. At implantation the glue points contribute to the strength of the mesh by gluing fibres together. After implantation, the fast degrading elements are degraded and disappear from the mesh leaving only the slow degrading elements in a more porous configuration allowing better cell in-growth and vascularisation. At this point in time the newly formed tissue is not fully matured, but the slow degrading elements are still there to contribute strength while the newly formed tissue matures. The biodegradable fibre material may be made of homo- or co-polymers of glycolide, L- lactide, DL-lactide, D-lactide, meso-lactide, ε-caprolactone, 5-valerolactone, 1 ,4-dioxan-2- one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3-hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane. The biodegradable glue points may be made of homo- or co-polymers of glycolide, L- lactide, DL-lactide, D-lactide,meso-lactide, ε-caprolactone, 5-valerolactone, 1 ,4-dioxan-2- one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3-hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane.

In some embodiments the fibres degrade faster than the glue points. When the fibres are degraded as cells populate the mesh, it is secured that the added strength of the glue points is maintained through the period of degradation. Because these glue points reduce the porosity of the scaffold and therefore has the potential to reduce cell in-growth, it can be advantageous to make the glue points of a polymer that degrades faster than the fibres. This will ensure that the porosity of the scaffold increases over time as the glue points are degraded.

The in vivo degradation time of the fibres may be 1-48 months. For instance, the in vivo degradation time of the fibres may be 5-10 months, 10-20 months, 20-30 months, 30-40 months, 40-48 months, 5-20 months, 5-30 months, 5-40 months, 10-30 months, 10-40 months, 10-48 months, 20-40 months, 20-48 months, or 30-48 months.

The in vivo degradation time of the glue points may be 1-52 weeks. For instance, the in vivo degradation time of the glue points may be 1-10 weeks, 10-20 weeks, 20-30 weeks, 30-40 weeks, 40-52 weeks, 1-20 weeks, 1-30 weeks, 1-40 weeks, 1-52 weeks, 10-30 weeks, 10-40 weeks, 10-52 weeks, 20-40 weeks, 20-52 weeks, or 30-52 weeks.

Any of the above in vivo degradation times for the fibres may be combined with any of the above in vivo degradation times for the glue points. For instance, the in vivo degradation time of the fibres may be selected from the group consisting of 1-48 months, 5-10 months, 10-20 months, 20-30 months, 30-40 months, 40-48 months, 5-20 months, 5-30 months, 5- 40 months, 10-30 months, 10-40 months, 10-48 months, 20-40 months, 20-48 months, and 30-48 months, while at the same time the in vivo degradation time of the glue points is selected from the group consisting of 1-52 weeks, 1-10 weeks, 10-20 weeks, 20-30 weeks, 30-40 weeks, 40-52 weeks, 1-20 weeks, 1-30 weeks, 1-40 weeks, 1-52 weeks, 10-30 weeks, 10-40 weeks, 10-52 weeks, 20-40 weeks, 20-52 weeks, and 30-52 weeks. In some embodiments the fibres degrade slower than the glue points. When the glue points are degraded first, the porosity of the mesh is increased. Generally, having the glue points will lead to a slower in-growth of cells as compared to meshes without glue points. In order to ensure stability of the mesh during the entire in-growth of cells, it may be advantageous to have a relatively slow initial in-growth. Such a slow initial in-growth will help prevent newly in-growing cells from compromising the structure of the mesh before the newly formed tissue is strong enough. For instance, the glue points may ensure that newly in-growing cells do not push apart the fibres of the mesh and thereby compromise the strength of the mesh before a strong new tissue is formed by the in-growing cells. By having the glue points that degrade faster than the fibres, the stability of the mesh is ensured by the glue points during the early stages of cell in-growth. The faster degrading glue points will gradually degrade, thus gradually allowing faster in-growth without compromising the integrity of the mesh at a too early stage. After the glue points have been degraded, at least part of the fibres will remain to ensure a structured formation of the new tissue that is to replace the mesh once also the fibres have degraded.

In some embodiment the glue points degrade at least twice as fast as the fibres, such as at least 3 times as fast, such as at least 4 times as fast, such as at least 5 times as fast, such as at least 10 times as fast, such as at least 15 times as fast.

In some embodiments the mesh degrades at the same pace as the glue points. This may be obtained by making the glue-points of the same polymer as the mesh. Meshes of this kind are advantageous if maximum strength is required for the full duration of the treatment.

In some embodiments the mesh is made of electrospun fibres. Electrospinning is a superior way to produce fibres with a diameter of <10μηι to nanoscale. The amount of glue points can vary from 1-70 %(w/w) of the mesh.

The glue points may comprise 1-70% (w/w), such as 2-50% (w/w), such as 5-50% (w/w), such as 10-30% (w/w) of the mesh. The glue points may comprise at least 1 % (w/w) of the mesh, such as at least 2% (w/w) of the mesh, such as at least 5%(w/w) of the mesh, such as at least 10%(w/w) of the mesh, such as at least 20%(w/w) of the mesh, such as at least 30%(w/w) of the mesh, such as at least 40%(w/w) of the mesh, such as at least 50%

(w/w) of the mesh, such as at least 60%(w/w) of the mesh. The glue points may comprise no more than 70% (w/w) of the mesh, such as no more than 60% (w/w) of the mesh, such as no more than 50% (w/w) of the mesh, such as no more than 40% (w/w) of the mesh, such as no more than 30% (w/w) of the mesh, such as no more than 20% (w/w) of the mesh, such as no more than 10% (w/w) of the mesh, such as no more than 5% (w/w) of the mesh, such as no more than 2% (w/w) of the mesh, such as no more than 1 % (w/w) of the mesh.

An advantage of few glue points, such as 2-10% w/w, is that you obtain stability of the mesh, while maintaining the porosity of the fibres.

Increasing the concentration of glue points leads to the strength of the mesh being increased.

The mesh may have an area density in the range 2-20 mg/cm², such as 5-20 mg/cm², such as 5-15 mg/cm², such as 5-10 mg/cm².

The fibre material may be composed of fibres having an average diameter in the range of 0.1-10μηι. In one embodiment, the fibre material is a fibre having an average diameter in the range of 2-20μηι.

The mesh may be a non-aligned non-woven mesh.

The mesh may further comprise a component that facilitates cell adhesion and/or migration into the mesh. Such cell migration into the mesh is sometimes referred to as ingrowth. The mesh may further comprise a component selected from the group consisting of estrogen, estrogen derivatives, thrombin, ECM (Extra Cellular Matrix) powder, chondroitin sulfate, hyaluronan, Hyaluronic Acid (HA), heparin sulfate, heparan sulfate, dermatan sulfate, growth factors, such as Insulin-like growth factors (IGFs), such as IGF-1 or IGF-2, or Transforming Growth Factors (TGFs), such as TGF-alpha or TGF-beta, or Fibroblast Growth Factors (FGFs), such as FGF-1 or FGF-2, or 20 Platelet-Derived Growth Factors (PDGFs), such as PDGF-AA, PDGF-BB or PDGF-AB, or Mechano Growth Factor (MGF), or Nerve Growth Factor (NGF), or Human Growth Hormone (HGH); fibrin, fibronectin, elastin, collagen, such as collagen type I and/or type II, type III, type IV, type V and/or type VII, gelatin, and aggrecan, or any other suitable extracellular matrix component. The mesh may be suitable for supporting, augmenting and regenerating soft tissue and for use in the treatment of pelvic organ prolapse, stress urinary incontinence or hernia.

The glue points may be formed by introducing droplets, for example by spraying into the fibre mesh. The resulting glue points may be in the form irregular domains connecting the adjacent fibres. The glue points may preferably have an average diameter of 1-200μηι, such as 10-150μηι, such as 50-100μηι.

The glue points can be applied to the fibre material in different ways, for example:

• Uniformly: both fibre- and glue point-flow are kept constant throughout

manufacture, providing a homogeneous mesh.

· As a layer on either side of the mesh:

o Glue point-flow is turned off sometime before the fibre-flow - this will

provide glue points in the bottom layer of the mesh,

o Glue point-flow is turned on delayed relative to the fibre-flow - this will provide glue points in the top layer of the mesh.

· As a gradient - glue point-flow is varied during the manufacture.

• In patterns.

The invention also relates to a method of preparing a non-woven mesh by electro spinning, said method comprising the steps of dispensing a biodegradable fibre-forming solution on a collector (rotating cylinder), and simultaneously or subsequently dispensing drops of a biodegradable polymer solution by electrospraying.

The method is illustrated in Figure 1 , showing a cross section of setup for electrospinning with glue points. Nozzle 1 dispenses the fibre-forming solution in a layer on a collector 2, and nozzle 3 dispenses small drops of a non-fibre forming polymer solution by

electrospraying. Both the fibre and the glue points are deposited on the collector (2, a rotating cylinder) resulting in a composite of fibres glued together by small domains of polymer.

The invention further relates to a biodegradable surgical implant for supporting, augmenting and regenerating soft tissue, where said implant comprises biodegradable fibre material and glue points in the form of domains of biodegradable polymer wherein the fibres are interconnected by the glue points. The surgical implant may be suitable for the treatment of pelvic organ prolapse, stress urinary incontinence or hernia. Examples

Example 1 : Preparation of an electrospun mesh with glue points.

Fibre-forming solution: 27 g PCL (polycaprolactone), 165 g acetone, 12.5 g 1.3-dioxolane, 4.5 g methanol, sealed in a closed flask, was heated to 60°C overnight and stirred until homogeneous.

Glue point forming solution: 2,5 g methoxypolyethylene glycol-co-poly(DL-lactide-co- glycolide) 2-40 kDa with 85%(mol) DL-lactide, 20 g acetone and 5 g butanone sealed in a closed flash, was heated to 60°C overnight and shaken until homogeneous.

Fibre-forming nozzle (-28 kV, 20 cm from collector) fed with 25 mL/h of fibre-forming solution.

Glue point nozzle (+8.77 kV, 3 cm from collector) fed with 5 mL/h of glue point forming solution.

The collector was a rotating cylinder 025cm (0 kV), 1.3 s^"1, covered with an embossed PE- film (poly ethylene). The nozzles were mounted on a linear bearing moving slowly back and forth along the axis of the rotating cylindrical collector, thereby depositing the composite on the PE-film.

Both nozzles were dual feed coaxial: A 19 gauge inner needle and a 17 gauge outer needle. The inner needle was fed with the polymer containing solution and the outer needle was fed with a slow stream of pure solvent (1 ml_/h of 10% 1.3-dioxolane in acetone) to avoid clogging.

Tensile testing

Samples were tested on a TA.XT Plus Texture Analyser (Stable Micro Systems) with a 5 kg load cell. The test specimens are 40x10mm and approximately 7 mg/cm². The samples are weighed and the exact area density and thickness is determined from the weight. The gauge length is 20mm. Samples are tested at 1 mm/s. The wet samples were wetted with 0.5% Sodium dodecyl sulfate in water. The table below summarizes the modulus for various test conditions. Table 1 : Modulus e-spun PCL and e-spun PCL modified with glue points

At all conditions tested the modulus was higher for PCL when reinforced with glue points. Creep/stretch testing

The resistance of the mesh with regard to being stretched during use is tested. Samples of mesh beingl 5x130mm, approximately 7 mg/cm² were provided. Weights (made to weigh 100, 200 and 300 g under water) were attached. The samples were fixed at the other end so that the distance between fixtures without load was 120 mm. The samples were then hung in a water bath (23°C) and the increase in length was noted at least once a week. As seen in Figure 5, the presence of glue points in the mesh reduced creep for all 3 weights tested.

Testing with cells and immersion in media:

Samples preparation as Example A and tensile testing as above.

Primary fibroblast passage were harvested and seeded onto the samples (1.9x10⁶ cells/sample). The seeded samples were immersed in media (Dulbeccos modified Eagle Medium (DMEM) containing 10% fetal calf serum and antibiotics). The flasks containing 10ml media and cell seeded sample, were kept in an incubator, at 5% C0₂/37°C. Media was changed once a week. Control samples were kept in media without cells. Samples were harvested at 3 and 8 weeks. In Figure 6 is shown the modulus of e-spun PCL after coculturing with fibroblasts for 3 and 8 weeks. Control samples kept in media without cells. In Figure 7 is shown the modulus of e-spun PCL reinforced with glue points after coculturing with fibroblasts for 3 and 8 weeks. Control samples kept in media without cells. From Figure 6 and 7 it is seen that the mesh reinforced with glue points have a higher modulus after 3 weeks both when grown with cells and when kept in media without cells. At 8 weeks the modulus is comparable.

Example 2: Generating glue points with different solvents

It was found that generating glue points with previously tested solvent systems

(Acetone/MEK/MeOH) resulted in glue points with a less than optimal size range and also tended to lead to some degree of fibre formation. We hypothesized that because of the relatively poor fibre-forming properties of ethyl acetate (EtOAc) and similar solvents, such solvents could advantageously be used to make glue points. The glue point formation of PLGA dissolved in EtOAc, iPrOAc, EtOAc/MeOH and iPrOAc/MeOH was tested at various flows and voltages. We found that using the said solvents it was possible to make glue points with an advantageous narrow size distribution and with no or very low tendency to form fibres.

Electrospraying

PLGA: 2-40 85DL. All solutions were dyed with 0.2 mg Dil (1 , 1 '-Dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate).

Table 2

PLGA (g) Solvents Cone PLGA (%w/v)

861 1 1 ,5 8 g EtOAc 14,9

861 J 1 ,5 8 g iPrOAc 14,5

862A 1 ,3 6 g EtOAc 12,7

2 g MeOH

862B 1 ,3 7 g EtOAc 12,9

1 g MeOH

863G 7,1 ml. 861 1 8,6

3,5 mL EtOAc

864B 1 ,3 21 g EtOAc 12,9

3 g MeOH

865A 1 ,3 7,4 g EtOAc 13,0

0,6 g MeOH

865B 1 ,3 7,7 g EtOAc 13,0

0,3 g MeOH

These solutions are sprayed onto PET-film (50μηι) mounted on the collector (0 kV). The Coaxial nozzle(17G/19G) is fed with polymer solution to the inner tube and a rinse flow of acetone (1 ml_/h) to the outer tube. Image analysis

The PET films with glue points are imaged with a Leica DMI 4000B CLSM and analyzed with ImageJ. Images are analyzed with ImageJ (lower cutoff limit 200 μηι²) and areas are transformed to spherical diameters.

Results & discussion

The results are presented in Table 3 below.

Table 3

Exp# Solution Nozzle Flow Dist Cone PLGA Diameter+stdev (μηι) + Notes

(kV) (mL/h) (cm) (%w/v)

863A 861 1 18 5 6 14,9 52,7+8,3

863B 861 1 24 5 6 14,9 44,0+7,4

863C 861 1 18 8 6 14,9 68,0+9,9

863D 861 1 24 8 6 14,9 53,5+17,6

863E 861 J 18 5 6 14,5 Big GP - not analyzed

863F 861 J 24 5 6 14,5 Big GP - not analyzed

863H 863G 16 5 4 8,6 58.9+1 1 ,3

863I 863G 20 5 4 8,6 49,1 +8,3 863J 863G 16 8 4 8,6 62,2+24,9

863K 863G 20 8 4 8,6 60+13,1

863L 862A 16 5 4 12,7 Broad distribution - trace of fibres

863M 862A 20 5 4 12,7 Broad distribution - lots of fibres

863N 862A 16 8 4 12,7 Broad distribution - some of fibres

8630 862A 20 8 4 12,7 Broad distribution - lots of fibres

863P 862B 16 5 4 12,9 29,8+5,2

863Q 862B 20 5 4 12,9 21 ,6+4,8

863R 862B 16 8 4 12,9 25,7+10

863S 862B 20 8 4 12,9 23,4+6,5

863T 862B 12 5 4 12,9 22,5+1 1 ,6

863U 862B 10 5 4 12,9 25,3+17,1

867A 865A 16 3 4 13,0 26,6+5

867B 865A 16 5 4 13,0 38,9+7

867C 865A 16 8 4 13,0 35,3+9,9

867D 865B 16 3 4 13,0 37+7,3

867E 865B 16 5 4 13,0 44,2+6,9

867F 865B 16 8 4 13,0 45,2+18,3

With the exception of experiment 863LMNO all solutions formed glue points without fibres. By tweaking the parameters, it is possible to get a range of glue point-sizes with a narrow distribution. Glue point formation of PLGA dissolved in EtOAc, iPrOAc, EtOAc/MeOH and iPrOAc/MeOH was tested at various flows and voltages. We found that using the said solvents it was possible to make glue points with an advantageous narrow size distribution and with no or very low tendency to form fibres.

Example 3: Generating glue points at different flows and different voltages

Glue points containing Dil fluorescent dye were sprayed onto PET-film at different flows (7-10 mL/h) and different voltages (10-24 kV). The samples were analyzed with fluorescence microscopy and size distributions were determined using image processing.

Experimental

- Solution: 14.9%(w/w) MPEG-PLGA (2-40 80DL) in EtOAc with 0.4 mg Dil/20 ml_

(JV937E)

make-up-flow: 1 mL/h EtOAc

- Needle: 17-22G

Distance to drum: 6 cm

- 21 ,8°C, 42% RH

- Film: 3M Product 5932 lot HD 0096, non-silicone side Olympus BX60 microscope at 4x, EvolutionMP camera (MediaCybernetics). 10 pictures were taken of each sample, Image analysis with ImageJ (circularity 0.8-1 , size cutoff 500 μηι²).

Results and discussion

The s-Nozzles are the nozzles normally used for spinning fibres. A voltage is applied to these inactive nozzles to mimic the electrical fields that would be present when spinning a mesh with glue points. Results are displayed in the table below. The size increases with flow and decreases with voltage. The higher voltages (>3 kV/cm) give a more unstable and narrrow jet making them less advantageous for our purposes.

Table 4

d Flow drum ES- Shape of jet ml_/h Kv kV/cm D^m) stdev(μm) (cm) (mL/h) (kV) nozzle

Example 4: Pilot in vivo testing of mesh with glue points

The purpose of this study was to do a preliminary test of the PCL and PCL-GP implants in two rats. The model is a full thickness defect model in rats done according to M.L.

Konstantinovic et al. (Neurourology and Urodynamics 29:488-493 (2010)). Materials and methods

Two Wistar retired female breeder rats weighing 250-300 grams (Taconic, Denmark) were included in this study. Animal housing and caretaking were provided by the Panum Institute (P 10-268) according to the national guidelines. All animals were kept under equal conditions and were housed individually the first week after the operation after which the 2 animals were housed together. The study was approved by the national animal ethical committee (2012-15-2934-00242 , AEM protocol P 12 322).

Products

The implants were cut into 2.5 cm x 4.0 cm pieces. All implants and mesh were tested as sterilized products. The following products were tested:

- The implants

- Electrospun PCL implants (JV948-004)

- Electrospun PCL implants with glue point (JV948-022)

Anesthesia, analgesia and antibiotics

The rats were anesthetized with Hypnorm/Dormicum, 0.3 ml/ 100g s.c. and supplemented with half the dose every 20-30 min thereafter. All rats received peri-operativt Rimadyl

(0.1 ml/100 g s.c), Norostrep (0.2ml/ 300g s.c.) and at the end of the operation two depots of 0.5ml saline given s.c. in order to avoid dehydration of the rats. The rats were given Rimadyl (0.1 ml/ 100g) s.c. the following 2 days after the operation and Buprenorphin (0.03 mg/kg) mixed into a chocolate paste (Nutella) every 12 hours for the first 3 days after operation. The rats were trained to eat the chocolate before the operation.

Surgery

The abdominal area was shaved and disinfected by ethanol and iodide. A midline incision was made and by blunt dissection the skin was loosened from the abdominal muscle in the right side of the rats. A 1.5 cm x 3.0 cm full thickness defect was made in the abdominal muscle layer. The defect was afterwards repaired by the respective implant or mesh with a 0.5 cm overlap in all directions. The corners of the implants or mesh were independently fixated with suture followed by a continuously suture all the way around the implant or mesh. The suture in the corners was permanent Prolene 5-0 suture (Ethicon) in order to be able to find the implant and the continuously suture was Vicryl 4-0 (Ethicon). The skin was afterwards closed by skin staples. The rats were inspected at least once a week in order to observe the rats for hernia or other complications.

Explantation

After 8 weeks the 2 rats were killed by cervical dislocation and a midline incision was made in each animal and the implant-area dissected free from the skin. The implantations areas were inspected for signs of herniation, fluid-collection, infection, erosion, rejection or other signs of discrepancies. Digital pictures of the implantation areas were taken and the area was measured. Explants were harvested compromising the implantation area with surrounding tissue. This area was divided into four sections each 1 cm x 2.5 cm. The two midsections were saved for mechanical testing by placing the tissue in phosphate buffered saline (PBS) until testing later the same day of explantation. The two outer sections were divided further into 3-4 pieces, fixated in formalin buffered saline pH 7.4, embedded in paraffin and cut into 4μηι sections using a Leica RM 2255 microtome. The sections were stained with Meyer's Haematoxylin and eosin (HE).

Digital images were obtained using an Olympus BX-60 light microscope fitted with a digital camera. Images were analyzed using Image Pro plus 5.1 software (Media Cybernetics, Silverspring, MD, USA).

Mechanical testing

The tensile test was performed using a TA-XT plus, Stable Micro Systems. The samples for mechanical testing were stored in PBS after explantation and until testing. The two samples from each rat were measured by respectively having the grips placed at the ends of the implants or by having the grips in the tissue surrounding the implants. The grips were modified with 1 mm rubber sheet and 3M Safety-Walk grip paper. The grip pressure was set to 3 bar, the gauge length was respectively 10 or 30 mm for the placement of the grips and the thickness and width of the samples were measured before the test. Results

Explantation

Both rats survived the operation and the following 8 weeks. The first days after operation the rats were followed very closely in order to inspect the wounds and avoid any complications. None of the rats showed any signs of hernia or other type of complications during the 8 week study period. At explantation both types of implants were a little elevated from the abdominal muscle as an indication of an increase in thickness of the implants. Both operation areas showed macroscopic good tissue integration with the surrounding tissue. There were some adherences of the oment to the side of the implants facing the intestine.

The implants were 2.5 x 4 cm (10 cm²) when they were implanted in the rats. At explantation the implants were 2.2 x 3.5 cm (7.7 cm²) and 2.5 x 3.7 cm (9.1 cm²), respectively. The reason for the smaller implants 8 weeks after implantation is due to folds in the implants as seen in the pictures in Figure 1 (se appendix). There were no indications of an increase or decrease in the size of the implants which could be related to failure of the implants.

Mechanical test

The explants were compared to healed partial defects of the muscle layer. The tensile test of the explanted PCL and PCL-GP implants showed a comparable strength to the partial healed muscle layer.

Histology

The histology shows a good tissue integration of both types of implants. Both implant types have areas in the middle of the implants where the in-growth of cells seems to be reduced. In the PCL-GP implants these low in-growth areas are more pronounced compared to the PCL implants. The PCL-GP implants have small areas in the full thickness of the implants where there are no in-growth. These areas correspond to the glue points.

Conclusion

A generally good biocompatibility was found for both implant types. A reduced in-growth at 8 weeks was found in the areas with the glue points and in middle of the PCL-GP implants compared to the PCL implants. Slower in-growth in the PCL-GP implants may contribute to higher stability of the implants by not allowing the in-growing cells to push apart the individual fibres of the implants. Thus, the glue points may help maintain stability of the implant during in-growth.

Figures

Figure 1 shows a typical example of an electrospun structure (polycaprolactone fibres).

Figure 2a and 2b show a scaffold during implantation (Figure 2a) and after 3 weeks of implantation (Figure 2b).

Figure 3 shows cross section of setup for an electrospinning process with addition of glue points. Figure 4 shows electrospun polycaprolactone fibres bonded with glue points of MPEG- PLGA.

Figure 5 shows the result of a creep test of mesh. Figure 6 and 7 show E-modulus of mesh.

Claims

1. A biocompatible non-woven mesh comprising

a) fibres of a biodegradable fibre material and

b) glue points in the form of domains of biodegradable polymer

wherein the fibres are interconnected by the glue points.

2. The mesh according to claim 1 , wherein the fibres have an in vivo degradation time that is higher than an in vivo degradation time of the glue points.

3. The mesh according to any of the preceding claims, wherein the fibres have an in vivo degradation time of 1-48 months.

4. The mesh according to any of the preceding claims, where the glue points have an in vivo degradation time of 1-52 weeks.

5. The mesh according to any of the preceding claims, wherein the biodegradable fibre material comprises homo- or co-polymers of glycolide, L-lactide, DL-lactide, D-lactide,meso-lactide, ε-caprolactone, 5-valerolactone, 1 ,4-dioxan-2-one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3-hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane.

6. The mesh according to any of the preceding claims, wherein the biodegradable polymer of the glue points comprises homo- or co-polymers of glycolide, L-lactide, DL-lactide, D-lactide,meso-lactide, ε-caprolactone, 5-valerolactone, 1 ,4-dioxan-2- one, trimethylene carbonate, block-copolymers of MPEG or PEG and one or more of the monomers mentioned above, degradable polyurethanes, degradable polyurethane-urea, polypeptides, degradable polyesters, poly(3-hydroxybutyrate), polyhydroxyalkanoate and polyesterdiol-based polyurethane.

7. The mesh according to any of the preceding claims, wherein the fibres are

electrospun fibres.

8. The mesh according to any of the preceding claims, wherein the amount of glue points in the mesh is 1-70% (w/w).

9. The mesh according to any of the preceding claims, wherein the mesh has an area density in the range 2-20 mg/cm².

10. The mesh according to any of the preceding claims, wherein the fibres have an average diameter size of 0.1 μηι-10μΓΤΐ.

1 1 . The mesh according to any of the preceding claims, where said mesh further comprises a component that facilitates cell adhesion and/or migration into the mesh.

12. The mesh according to any of the preceding claims, where said mesh further comprises a component selected from the group consisting of estrogen, estrogen derivatives, thrombin, ECM (Extra Cellular Matrix) powder, chondroitin sulfate, hyaluronan, Hyaluronic Acid (HA), heparin sulfate, heparan sulfate, dermatan sulfate, growth factors, such as Insulin-like Growth Factors (IGFs), such as IGF-1 or IGF-2, or Transforming Growth Factors (TGFs), such as TGF-alpha or TGF- beta, or Fibroblast Growth Factors (FGFs), such as FGF-1 or FGF-2, or 20

Platelet-derived Growth Factors (PDGFs), such as PDGF-AA, PDGF-BB or PDGF- AB, or Mechano Growth Factor (MGF), or Nerve Growth Factor (NGF), or Human Growth Hormone (HGH); fibrin, fibronectin, elastin, collagen, such as collagen type I and/or type II, type III, type IV, type V and/or type VII, gelatin, and aggrecan, or any other suitable extracellular matrix component.

13. The mesh according to any of the preceding claims, where said non-woven mesh is suitable for supporting, augmenting and regenerating soft tissue.

14. The mesh according to any of the preceding claims for use in the treatment of pelvic organ prolapse, stress urinary incontinence or hernia.

15. The mesh according to any of the preceding claims, wherein the glue points are in the form of substantially non-fibrous domains.

16. The mesh according to any of the preceding claims, wherein the glue points are distributed homogeneously in the mesh.

17. A biodegradable surgical implant for supporting, augmenting and regenerating soft tissue, where said implant comprises

a) fibres of a biodegradable fibre material, and

b) glue points in the form of domains of biodegradable polymer

wherein the fibres are interconnected by the glue points.

18. The surgical implant according to claim 17, wherein said implant is suitable for the treatment of pelvic organ prolapse, stress urinary incontinence or hernia.

19. A method of preparing a non-woven mesh by electro spinning, said method

comprising the steps of

a) dispensing a biodegradable fibre-forming solution on a collector (rotating cylinder), and,

b) simultaneously or subsequently dispensing drops of a biodegradable

polymer solution by electrospraying.

20. The method according to claim 19, wherein the solution in step (b) comprises a solvent selected from the group consisting of ethyl acetate (EtOAc); isopropyl acetate (iPrOAc); a mixture of EtOAc and methanol (MeOH); and a mixture of iPrOAc and MeOH.

21. The method according to claim 20, wherein said solvent is EtOAc.