WO2006059090A1

WO2006059090A1 - Polymer scaffold

Info

Publication number: WO2006059090A1
Application number: PCT/GB2005/004579
Authority: WO
Inventors: Phylip Rhodri Williams; Mohan Srinivasarao
Original assignee: Uws Ventures Limited
Priority date: 2004-11-30
Filing date: 2005-11-30
Publication date: 2006-06-08
Also published as: GB0426282D0

Abstract

This invention is in the field of tissue engineering and relates to methods of preparation of three-dimensional biodegradable microporous, mesoporous or nanoporous polymer scaffolds. Preferably said scaffold is seeded with cells, antibiotics, proteins, nutrients, media, stem cells or other drugs or genetic material during the manufacturing process to aid the tissue regeneration process. Said scaffold may preferably incorporate microcapillary tubes, microscopic wires, microfibres or optical fibres at the time of scaffold preparation to aid the tissue regeneration process and modify the mechanical, chemical, optical and electrical properties of the scaffold.

Description

Polymer Scaffold

TECHNICAL FIELD

This invention is in the field of tissue engineering and more specifically relates to methods of preparation of three-dimensional biodegradable polymer scaffold structures incorporating microporous, mesoporous or nanoporous void structures and which may incorporate embedded microcapillary, microfibre, microwire or optical fibre structures.

BACKGROUND ART

AU documents cited herein are incorporated by reference in their entirety.

Many surgical procedures involving the replacement or repair of tissues or organs require tissue or organ substitutes. Biomimetic materials and devices have been created which can be used in such surgical procedures. However these materials, for example heart valves, artificial hearts and breast implants, are subject to wear, fatigue and fracture and may induce unwanted immune responses. They also do not behave physiologically like true organs or tissues and remodel with time (i.e. they cannot grow or change shape in response to loads put on them).

Tissue engineering methods aim to replace organs and tissue using biodegradable, synthetic polymer scaffolds around which new tissue can be grown in the body. Such scaffolds are typically made from poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their co-polymers. However, in theory, any biodegradable, non-toxic material may be used. Solvent casting is the most widely used method for making scaffolds (Mikos, et ah, Polymer, 1994, 35:1068-77; de Groot, et al., Colloid Polym. ScL, 1991, 268:1073-81; Laurencin, et al, J. Biomed. Mater. Res., 1996, 30:133-8). In porous scaffolds created by crystal templating methods, the dominant length scale of the resulting solid is fixed by the template, making dynamic control of the length scale virtually impossible.

Polymer scaffolds provide the preliminary structural, vascular or cellular support for the development of new extracellular matrix (ECM). The scaffold is ideally completely absorbed into the body, leaving just the new ECM. However, inducing ordered tissue growth is not that simple, as cells require external signals for growth. These signals may be mechanical, structural, electrical or chemical. If these signals are lacking, then cells do not differentiate properly or dedifferentiate (become non-specific cell types), become disorganised and/or die. Physical interaction between cells affects the shape and function of those cells. Various growth factors are involved in cell differentiation and development. In bone, they control the migration of cells from one site to another, morphogenesis from one cell type to another and mitogenesis. These growth factors may act in an autocrine manner, act on neighbouring cells, act on cells far away by travelling through the bloodstream or induce a single cell to pass a signal onto a neighbouring cell by direct cell-cell interaction. These growth factors are not only used during growth and development, but also during times of remodelling and after injury.

Ideally a polymeric scaffold would be seeded with cells and/or growth factor(s) before being placed in the body in an attempt to organise cell growth into tissues or perhaps whole organs. This method has been used in laboratories to create various tissue analogues including skin, cartilage, bone, liver, nerve and blood vessels. However, only tissue engineered skin has been commercialised.

The major problem with such scaffolds is how to ensure that cells, growth factors, scaffold materials and microstructures are distributed in a regular, controlled manner within the scaffold. This problem exists due to the methods used for the manufacture and seeding of the scaffolds. Typically, the scaffolds are prefabricated and then seeded with cells by placing the scaffolds in cell suspensions so that the cells diffuse into and attach to the scaffolds. While cells can easily migrate into the outer part of the scaffold, it is more difficult to ensure an even distribution throughout the scaffold due to various factors including random motility and the limited nutrients that diffuse through the scaffold.

One solution would be to add cells to the scaffold during the scaffold fabrication process to ensure a better control over cell distribution. This is, however, precluded from most scaffold manufacturing processes as they involve heat or chemicals that would damage or destroy living cells and/or nutrient functionality. Even if cells did survive this process, they would lack a vascular supply to provide them with nutrients and so the cells would die shortly afterwards from a lack of nourishment. If the scaffold were implanted straight after manufacture, there would still not be enough time for a vascular system to develop to supply the required nutrients to the cells.

There is therefore a need for a method of producing tissue scaffolds of controlled morphology and furthermore, of appropriately seeding the interior structure of said scaffolds with cells, preferably during the process of scaffold formation. DISCLOSURE OF THE INVENTION

The present invention provides a method for making biodegradable polymeric scaffold(s) structures of controlled morphology incorporating regular arrays of void structures with microporous, mesoporous or nanoporous dimensions.

Specifically, the present invention provides a method for making a polymeric scaffold comprising:

providing a mobile film layer comprising a polymer dissolved in a solvent;

embedding an array of non-coalescing liquid droplets in said film layer; evaporating said solvent from said polymer; and evaporating said liquid from said polymer, wherein

said liquid is immiscible with said solvent and has a higher boiling point than that of said solvent.

The liquid film layer of polymer dissolved in a solvent is mobile due to the low shear viscosity of the solution. A layer of non-coalescing liquid droplets is then applied to the film layer.

In one embodiment, a stream of humidified air is passed across a layer of polymer solution resulting in the formation of liquid droplets on the surface of the polymer by condensation. The diameter of these droplets can be controlled by altering the humidity, temperature and speed of the stream of humidified air. In an alternative embodiment, the liquid droplets are produced by a vibrating orifice aerosol generator, which can produce droplets from 20 to 400μm diameter. These liquid droplets then sink into the mobile film layer. As the solvent evaporates, the liquid droplets become embedded in the polymer layer. The liquid droplets then evaporate, leaving empty voids (pore chambers).

Preferably the polymer used in the invention is biodegradable. The polymer used in the invention is more preferably selected from the group consisting of polyglycolic acid, polylactic acid, polylactic co-glycolic acid, polycalprolactone, collagen or hyaluronic acid.

The polymer is preferably dissolved in a solvent selected from the group consisting of chloroform, methyl chloride, p-dioxane, toluene, carbon disulphide, acetone, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethylacetate, methylethylketone, and acetonitrile. The liquid used to humidify the air and thus provide the liquid droplets that form voids in the polymer may be aqueous or non-aqueous. Preferably the liquid is aqueous. The liquid may also contain cells which may include hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smooth muscle cells, skeletal muscle cells, ocular cells, integumentary cells, keratinocytes. Preferably the liquid contains stem cells.

The liquid may also contain one or more of: growth factors, such as epidermal growth factor, retinoids, TGF-β growth factor, estrogen, tripeptide-copper complex (GHK-Cu) and hyaluronic acid (HA); nutrients and cell attachment mediators including the peptide containing variations of the "RGD" integrin binding sequence known to affect cellular attachment, biologically active ligands, and substances that enhance or exclude particular varieties of cellular or tissue ingrowth.

Such substances include osteoinductive substances, such as bone morphogenic proteins (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor

(PDGF), insulin-like growth factor (IGF-I and II), TGF and antibiotics, such as penicillin or kanamycin. Most preferably the liquid is a growth medium.

The porous polymeric scaffold structures may be formed by the solidification of a continuous polymer phase containing entrained, immobilised liquid droplets (water or other solutions, usually aqueous and optionally containing microbial cells, antibiotics, growth factors, proteins, stem cells or other biological or genetic materials). The distribution of liquid droplets within the resulting solid polymer scaffold is controlled so that polymer scaffolds of varying internal morphologies may be produced to order. The droplets are applied while the polymer is in its pre- gel point state. As evaporation of the solvent proceeds, the shear viscosity progressively increases. The polymer matrix undergoes a phase transition caused by the controlled evaporation of solvent, thus immobilising the arrays of ordered, non-coalesced liquid droplets in a high viscosity polymer phase. During the final stage of formation, the liquid droplet array acts as an embedded template for the porous network structure which is formed within the polymer matrix. The liquid droplets, which may become temporarily frozen at low temperature within the polymer solution film as a result of evaporative (solvent) cooling, are themselves then allowed to evaporate, leaving 2-dimensional monolayer arrays or 3 -dimensional multilayer arrays of mutually interconnected voids created by the exclusion of polymer and eventual evaporation of the liquid. The resulting solid polymeric scaffold has a highly monodisperse, regular and highly porous microstructure, possessing an exceptionally high degree of interconnectivity and displaying exceptional structural characteristics. The same process may be adapted to produce arrays of discrete (non-interconnected) pores by increasing the inter-pore spacing during the liquid droplet nucleation stage. The discrete pores may function as microscopic (picolitre or sub-picolitre capacity) containment vessels for microscopic assays, or as receptacles for the deposition and development of cell cultures in liquid media.

In a further embodiment, the polymer scaffold may contain one or more additional structural components. Such structural components are placed on the mobile liquid film layer prior to the addition of the array of non-coalescing liquid droplets. The scaffold may include any one or any combination of a microcapillary tube, preferably made from glass, nylon, carbon or plastic; electrically conducting wire; an optical fibre; or microfibres. Preferably, microfibres are added to the mobile liquid film layer for incorporation into the scaffold. These microfibres are preferably made from carbon or nylon. The microfibres may be added singly, in pairs or in bundles.

Nucleation

The initial formation of the liquid droplets may occur in one of two regions. Humidified air may be passed over a layer of polymer solution such that nucleation may be initiated at or immediately above the polymer solution-air interface. The polymer is at a lower temperature than the humidified air, resulting in the condensation of the humidified air, forming water droplets on the layer of polymer solution. The polymer solution may be cooled in order to promote or enhance this process. Alternatively, standard, commercial technology may be used in which liquid droplets are produced away from the solution-air interface i. e. in the airstream prior to introduction of said airstream above the polymer solution.

The liquid used to form the droplets may be aqueous or non-aqueous. Rather than just using water, it is possible to form the liquid droplet arrays using solutions or media incorporating growth factors, cells such as stem cells or microbial cells, antibiotics or other drugs, proteins, nutrients or nucleic acid in the liquid. This material, being incorporated within the liquid droplets, is subsequently transferred into, and incorporated within, the porous scaffold, being distributed within and throughout the entire porous void microstructure upon formation of the scaffold and, in residing at the surfaces of the pore chambers (which may subsequently be flooded with other liquids) can help to provide a preferential degree of biocompatability, or stimulate and direct the growth of desirable cells and cell structures, or suppress or discourage the development of undesirable microscopic species.

Growth

The liquid droplets are ultimately required to form the template around which the polymer is cast. Therefore the morphology of the final polymeric scaffold depends on the size and concentration of the droplets. A controlled growth of the liquid droplets is therefore required. In the case of nucleation at the interface, the size of the droplets is primarily controlled by regulating the humidity, air stream velocity and temperature difference at the solution-air interface.

It is possible to produce a distributed liquid phase within the film of polymer solution comprising uniform, monodisperse, hexagonally close-packed non-coalescing liquid droplets of equal diameter. The non-coalescing droplets assemble into an ordered, hexagonally-packed array by a process of self-organisation, the benefits of which are that it can provide the requisite highly regular pore size and ordered spatial geometry. Two-dimensional water droplet arrays have been produced, as colloidal crystal monolayers, in which the nominal droplet diameter lies between 0.1 μm and 100 μm.

Distribution

The distribution of the liquid droplets in the polymer solution is regulated by several factors. These include the temperature difference across the liquid film to control the competitive effects of thermocapillary and buoyancy forces acting on the water droplets, i.e. controlling the depth of penetration into the polymer. It is also possible to control the shear forces imposed at the upper interface by the action of the air cross-flow stream, thus regulating the assembly of the network structure of the water droplets and therefore controlling the porosity of the construct. It is also possible to control the onset and development of Benard-Marangoni type convection flow cells within the polymer solution by controlling the temperature gradients developed through the film of polymer solution. This may be achieved using thermoelectric (Peltier) assemblies below the surface on which the polymer solution is deposited. Polymers

The polymers used in this invention are those commonly used in tissue engineering applications and preferably include polylactic acid (PLA) and poly lactic co-gly colic acid (PLGA). Alternative bio-polymers include polycaprolactone, collagen and hyaluronic acid.

Solvents

The preferred solvents currently include chloroform, methyl chloride and p-dioxane. Further possible solvents are toluene and carbon disulfide.

Method

The method may be employed to produce either 2-dimensional monolayer arrays or 3- dimensional multilayer structures within the bulk polymer scaffold, depending on the extent of the induced thermocapillary convection, Benard-Marangoni convection, immersion capillary forces and buoyancy forces. The buoyancy force is readily manipulated through an appropriate choice of solvent. Those solvents with sufficiently reduced specific gravity more readily permit the downward penetration of water droplets away from the solvent-air interface. Sufficient depletion of water droplets at the interface permits the nucleation of a new generation of droplets which then penetrate the bulk polymer solution. The Benard-Marangoni convection flows, which form hexagonal, pentagonal or square convection flow cells as a result of the vertical temperature gradient established by solvent evaporation from the film of polymer solution, assist the downward penetration and distribution of water droplets away from the solvent-air interface within the polymer solution and may be controlled through the establishment of appropriate temperature gradients within the thin film of polymer solution using thermoelectric temperature control systems as mentioned above.

The invention may be further improved by incorporating within the scaffold, at the time of its formation, additional structural components, which may be functional, active, inert or biocompatible. These structural components may include:

(i) microcapillary tubes or hollow pipes (formed from glass, nylon, carbon or plastic) having solid or porous outer walls which may be used to facilitate the distribution of liquid media into the scaffold pore structure, or to withdraw samples of liquid media from within the scaffold pore structure by suction, or both; (ii) electrically conducting wires which may be used to facilitate the control of intra-scaffold temperature or temperature gradients in order to stimulate, regulate, modify or preferentially suppress the development of cellular material, micro-organisms or new tissue within the porous scaffold interior by a thermoelectric (Peltier) effect; or may be used to function as embedded thermocouples in order to measure temperature or temperature gradients within the scaffold. The embedded wires may also be used to set up, regulate and modify magnetic fields within the scaffolds;

(iii) microfibres (carbon, nylon or other materials), either singly, in pairs, in bundles or arranged in 3 -dimensional structures, which may be used to stiffen the surrounding solid porous polymer scaffold in which it is embedded such as to modify the mechanical characteristics (such as shear rigidity or torsional rigidity) of the scaffold in a beneficial way, e.g. in order to improve its mechanical performance such as its resistance to in vivo shear stresses; or

(iv) optical fibres (fibre optic cables) which may be used to admit light energy to the interior pore structure of the scaffold and which may be used to stimulate, regulate, modify or preferentially suppress the development of cellular material, micro-organisms or new tissue within the porous scaffold interior.

The tubes, wires, microfibres or optical fibres may be incorporated either singly, or in bundles, within the polymer scaffold by causing them to be covered by the polymer solution prior to the introduction of the liquid droplets. The polymer solution flows over and about the structures, either completely or partially immersing them as desired. By choosing structures whose characteristic dimension (diameter) is similar to that of the pores formed within the scaffold, the presence of the structures does not cause more than a highly localised perturbation of the scaffold's pore morphology. This method of formation avoids the necessity of having to insert wires, tubes or fibres within a solid polymer scaffold and thereby avoids the damage or disruption to scaffold structure which results from such action. The embedded structures referred to in (i)-(iv) above provide a means of inducing ordered tissue or cellular growth using external signals for growth. These signals may be mechanical, structural, electrical, chemical, optical or magnetic.

BRIEF DESCRIPTION OF DRAWINGS The present invention is now illustrated by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows a scaffold with a 5μm diameter thermocouple wire that was incorporated during formation of the scaffold;

Figure 2 shows a scaffold that contains two fibre optic cables, (the scaffold has been cracked to reveal the embedded cables);

Figure 3 shows a scaffold incorporating a 5μm diameter carbon fibre, (the scaffold has been cracked to reveal the embedded fibre);

Figure 4 shows the discrete chambers that can be formed in the scaffold; and

Figure 5 shows a scaffold incorporating a 5μm diameter microcapillary pipe.

MODES FOR CARRYING OUT THE INVENTION A polymer solution is made by adding a polymer (e.g. PLA) to a solvent (e.g. chloroform) to give a solution with a composition in the range of 0.1-10 wt% of polymer. The polymer and solvent are stirred at room temperature (20° C) in a sealed container to reduce both the evaporation rate of the volatile solvent and the preparation time. The dimensions of the container are such that it promotes a laminar air flow across the surface of the polymer solution. The container is attached to the horizontal translation stage of an optical microscope which is attached to a high speed video camera in order to monitor the process of liquid droplet growth and the establishment of the 2-dimensional droplet template array at the surface of the polymer solution.

After the complete dissolution of the polymer, a measured quantity of solution is deposited using a metered syringe, or by means of a metering micro-pump, as a thin layer on a smooth horizontal glass surface. The underside of the glass slide is etched with a cross— hair mark and serves as the primary location reference in subsequent characterisation of the solid polymer scaffold.

Structures such as microcapillary tubes, microscopic wires, microfibres or microscopic optical fibres, either singly, or in bundles, may be placed as desired on the upper surface of the glass slide, prior to deposition of the polymer solution. After its deposition, the polymer solution flows over and about the structures, either completely or partially immersing them as required.

The glass surface rests upon a temperature-regulating piezo-electric (Peltier) element which provides heating or cooling of the liquid on the glass surface which is contained within a laminar cross-flow cell filled with dry filtered air. The cross-flow cell is connected to a temperature regulated air filter and humidifier system via a 3 -way valve such that the cross-flow cell is isolated while the humidity, temperature and flowrate of the air is adjusted. Typical conditions of operation include an air stream of temperature 2O⁰C, humidity at 50-55% RH with a laminar cross flow velocity in the range of 0.1-1 m/s. A source of compressed air is first passed at a set flowrate through a flowmeter calibrated for air at STP and then bubbled through a series of Dreschel bottles containing a mixture of glycerol and water, the proportions of which are varied to produce the desired humidity. The humidified air is passed through a buffer flask which serves to damp pulsation due to flow through the humidification bubblers. The buffer flask also contains glass wool to remove entrained water droplets from the air stream. After passing an inline thermocouple and hygrometer, the humidified air is switched via a 3 -way valve such that it may be vented directly to ambient or first caused to pass through the flow chamber.

Following the introduction of the polymer solution, a continuous stream of humidified air is introduced to the cross flow cell and directed over the polymer solution. Cooling of the polymer solution (either by evaporative cooling alone due to solvent evaporation or augmented and regulated by the Peltier system) to temperatures below that of the air stream (around 5⁰C) initiates nucleation of water droplets at the polymer-air interface. These water droplets, whose primary size (within the range 0.1 μm to 50μm) is dictated by their residence time in the airflow and hence whose size may be controlled by regulation of the airflow velocity in the range noted above, are formed by the condensation of the airborne water vapour, and continue to grow at the fluid interface in the presence of regulated amounts of excess water vapour. The subsequent growth and packing of the colloidal system is monitored in real time using a high speed video microphotographic system and hence the conditions of humidity, residence time and airflow (under which the droplets grow to the desired size and distribution) may be regulated. Immediately following the nucleated condensation of water vapour, the solution - air interface is located using the high speed microphotographic system's fine focus adjustment and the focus thereafter is maintained by manual focus tracking, this being necessary due to depletion of the solution by evaporation of the solvent. In this system, the non-coalescing water droplets remain partially submerged in the film layer while assembling into the requisite template monolayer. If desired, the temperature may be reduced further to initiate freezing of the entrained water. Under the action of an appropriate magnitude of shear force applied by regulating the cross flow velocity of the air, the continuous water droplet phase is driven into an ordered hexagonal- packed structure. Evaporation of the solvent increases the concentration of the polymer, thereby increasing the viscosity of the solution. The solvent continues to evaporate at the interface through the interstices in the overlying water droplet monolayer until the bulk rheological properties of the film are such that the mobility of the water droplets is progressively reduced and they are observed to become finally immobilised in the polymer gel matrix. The temperature of the polymer film is allowed to equilibrate such that the temperature in the polymer film approaches that of the air stream, following which the water droplets evaporate, leaving hollow spheroidal shells or cavities (voids). In the case of embedded multilayers (those in which the porous microstructure consists of close packed, 3-dimensional colloidal layers of voids or pores), the temperature may be raised further to promote a higher rate of evaporation. The film is then placed in a reduced pressure cell to expedite evaporation of any residual solvent, yielding the final continuous scaffold construct containing the requsite network of pores and embedded wires, microfibres or microcapillary pipes.

Larger diameter spherical networks can be produced by substituting the nucleation-growth phase by a technique incorporating pulsed disintegration of a liquid jet. This method incorporates an established commercial technology for the production of aerosol particles. A vibrating orifice aerosol generator (VOAG) is used to produce monodisperse liquid droplets by the periodic segmentation of a liquid jet. The water droplets produced by the controlled break up of the jet issuing from a micrometer scale orifice are uniform and can be varied from 20-400μm diameter using vibrational frequencies in the range IkHz to IMHz.

Following jet disintegration and subsequent dilution in the airborne phase, the water droplets are continuously introduced to the polymer solution by passing the carrier stream over the surface of a cooled polymer solution. It is necessary to maintain a sufficiently low concentration of the airborne droplets in the carrier stream to prevent coalescence immediately above the solution surface. Integration of the droplets into larger quantities of solution is aided by forced circulation of the polymer or simple mechanical mixing, thereby supplying the polymer-air interface with liquid previously located in the bulk solution. To produce structures comparable to those described previously, it is necessary to arrest the mixing when a sufficient droplet concentration is observed such that the thermocapillary-buoyancy forces may then dictate the ordering process.

As mentioned previously, substitution of water with media or solution containing a growth factor and/or other solutions/particulates provides a method of further improving the invention. The chilling and freezing of the droplets whilst in the organic solvent, until the template is formed and the solvent removed, protects any cells, nutrients, stem cells or other genetic material in the liquid in that droplet from degradation by the solvent. Example 1

To produce the scaffold shown in Figure 1, PLA was added to chloroform to give a composition of 10 wt% of polymer. The mixture was stirred at room temperature in a sealed container.

The container was attached to the horizontal translation stage of an Olympus optical microscope which is attached to a Kodak Ektapro HS4540mx high speed video camera fitted with a Hadland

ILS-3 low light level intensifying lens system in order to monitor the process of liquid droplet growth and the establishment of the 2-dimensional droplet array at the surface of the polymer solution. The microphotographic lens system attached to the camera consisted of a Mitutoyo M-

Plan Apo compound microscope lens with X50 magnification, a working distance of 13.2mm and focal distance of 4.1mm, aNikkor manual focus 105mm, f/1.8 telephoto lens at 10 mm from the telephoto objective lens (attached by a custom mount converter) and additional magnification was provided by a 330 mm extension tube attached to the HS 4540mx camera.

A thermocouple wire was placed on a glass slide and 2ml polymer solution was deposited on the slide using a metered syringe. The airstream across the top of the polymer was regulated to provide a cross flow velocity of 0.5 m/s at a temperature of 5°C and humidity of 55% RH. This produced water droplets of 5 micron diameter on the polymer.

Similar methods were used to produce the scaffolds shown in Figures 2-5.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims

1. A method for making a polymeric scaffold comprising:

providing a mobile film layer comprising a polymer dissolved in a solvent;

embedding an array of non-coalescing liquid droplets in said film layer;

evaporating said solvent from said polymer; and

evaporating said liquid from said polymer, wherein

2. A method according to claim 1 wherein said polymer is biodegradable.

3. A method according to claim 1 or claim 2 wherein said polymer is polyglycolic acid, polylactic acid, polylactic co-glycolic acid, polycalprolactone, collagen or hyaluronic acid.

4. A method according to any one of claims 1-3 wherein said solvent is chloroform, methyl chloride, p-dioxane, toluene, carbon disulfide, acetone, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethylacetate, methylethylketone or acetonitrile.

5. A method according to any one of claims 1-4 wherein said liquid is aqueous.

6. A method according to any one of claims 1-5 wherein said liquid contains cells.

7. A method according to claim 6 wherein said cells are hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smooth muscle cells, skeletal muscle cells, ocular cells, integumentary cells, keratinocytes or stem cells.

8. A method according to any one of claims 1-7 wherein said liquid contains one or more growth factors.

9. A method according to claim 8 wherein said growth factors are selected from the groupd consisting of epidermal growth factor, retinoids, TGF- β growth factor, estrogen, tripeptide-copper complex (GHK-Cu), hyaluronic acid, fibroblast growth factor, platelet- derived growth factor, insulin-like growth factor and TGF.

10. A method according to any one of claims 1-9 wherein said liquid contains nutrients.

11. A method according to any one of claims 1-10 wherein said liquid contains antibiotics.

12. A method according to claim 11 wherein said antibiotic is penicillin or kanamycin.

13. A method according to any one of claims 1-12 wherein said liquid contains proteins.

14. A method according to claim 13 wherein said proteins are bone morphogenic proteins.

15. A method according to any one of claims 1-14 wherein said liquid contains nucleic acid.

16. A method according to any one of claims 1-15 wherein said liquid is a growth medium.

17. A method according to any one of claims 1-16 further comprising adding a structural component to the mobile film layer prior to the embedding of the array of non-coalescing liquid droplets.

18. A tissue scaffold manufactured according to the method of any one of the previous claims.

19. A tissue scaffold according to claim 18 wherein said scaffold further comprises a microcapillary tube.

20. A tissue scaffold according to claim 19 wherein said microcapillary tube is made from glass, nylon, carbon or plastic.

21. A tissue scaffold according to claim 19 or claim 20 wherein said microcapillary tube is porous.

22. A tissue scaffold according to any one of claims 18-21 wherein said scaffold further comprises electrically conducting wires.

23. A tissue scaffold according to any one of claims 18-22 wherein said scaffold further comprises microfibres.

24. A tissue scaffold according to claim 23 wherein said microfibres are made from carbon or nylon.

25. A tissue scaffold according to any one of claims 18-24 wherein said scaffold further comprises optical fibres.

26. A tissue scaffold according to any one of claims 18-25 for use in the repair of tissue damage.