GB2425538A

GB2425538A - Substrate and method for modulating tissue formation or deposition

Info

Publication number: GB2425538A
Application number: GB0608477A
Authority: GB
Inventors: Kevin Quinlan; Richard Morgan; Matteo Santin; Gary Phillips
Original assignee: Porvair Filtration Group Ltd
Current assignee: Porvair Filtration Group Ltd
Priority date: 2005-04-29
Filing date: 2006-04-28
Publication date: 2006-11-01
Also published as: GB0608477D0; GB0508815D0

Description

SUBSTRATE AND METHOD FOR MODULATING

TISSUE FORMATION OR DEPOSITION

The present invention relates to a range of novel substrates used as such or in combination with methods for controlling, modulating or regulating tissue formation or deposition and related metabolic products as well as for regenerating different types of tissues in vitro and in vivo.

Cells are cultured in laboratories using tissue culture plates for in vitro studies and for tissue engineering applications.

The tissue culture plates are generally supplemented with nutrients to enable the cells and tissues to grow. However, the surface area of a tissue culture plate provides only a limited amount of space for the biological material and it does not mimic adequately the three dimensional structure of the real biological environment. It is desirable to be able to increase the amount of biological material that can be grown without using an excessive number of tissue culture plates.

EP 1 245 670 discloses a method of growing cell cultures using a scaffold formed from an open cell polymer foam that has been surface treated by an oxidative plasma discharge.

However, it would be desirable to be able to provide for and control tissue formation or deposition.

According to a first aspect of the present invention there is provided a three dimensional substrate for controlling or modulating tissue formation or deposition, the substrate comprising: a porous material in which at least one of the surface physical and chemical properties and architecture are arranged to regulate or modulate cell proliferation andlor differentiation.

Cell differentiation is defined as the process during which young, immature (unspecialised) cells take on individual characteristics and reach their mature (specialised) form and function (www.seniormag.com).

It has not previously been possible to provide for and control tissue formation and deposition in a three dimensional substrate. The ability to control tissue formation and deposition enables increased yields to be obtained or for tissue formation to be discouraged for example for medical devices, non-fouling or incubation. Tissue growth is a controlled cell growth embedded in consolidated extra cellular matrix (ECM).

The use of a porous material provides a three dimensional structure in which the combined surface area of the pores substantially increases the available area in which biological material may be grown, significantly increasing yields and offers a biometric environment for the growing cells and tissues.

The porous material may, for example, be a porous thermoplastic material such as an organic polymer, examples of which include polyolefins such as polyethylene and propylene, or nylons, polycarbonates, poly(ether sulfores), polystyrene and mixtures thereof.

The surfaces of the pores may be configured to encourage tissue growth thereon such as by one or more of using a plasma or vapour to activate the pore surfaces, ionising the pore surfaces, making the pore surfaces hydrophyllic, providing free radicals in the pore surfaces or removing oil from the pore surfaces.

When the substrate is plasma treated, the plasma is preferably at a pressure greater than 0.05 Ton. The pressure may be greater than 0.1 Ton or between 0.05 and 0. 5 Ton or between 0.1 and 0.3 Ton.

The pore surfaces preferably have a defined roughness or rugosity. The pore surfaces preferably have an average rugosity of not lower than 45nm, more preferably not lower than 48nm to encourage cell adhesion or not higher than 45nm, more preferably not higher than 42nm to reduce cell adhesion encouraging inertness for a superhydrophobic substrate. The pore roughness may for example be caused by cracking of the surface of the pores.

The porous material preferably has pores with a diameter that is between 5 and 200 times the size of the biological material to be grown. For bacteria which are usually approximately 1 im in length, the substrate pores preferably have a mean size between 5 and 200gm in diameter, more preferably between 50 and 1 50pm and more preferably still between 75 and 125pm. For an eukaryotic cell which is typically 5-20.tm, the substrate pores preferably have a mean size greater than 20gm in diameter.

According to a second aspect of the present invention there is provided a method of controlling or modulating tissue formation or deposition, the method comprising providing a three dimensional substrate formed of porous material and arranging at least one of the surface physical and chemical properties and architecture of the porous material to regulate cell proliferation andlor differentiation.

The surfaces of the pores may for example be treated by one or more of being activated using a plasma or vapour, ionising the pore surfaces, making the pore surfaces hydrophyllic, providing free radicals in the pore surfaces or removing oil from the pore surfaces.

If the substrate is plasma treated, the plasma is preferably at a pressure greater than 0.05 Ton. The pressure may be greater than 0.1 Ton or between 0.05 and 0.5 Ton or between 0.1 and 0.3 Ton.

Once tissue has been grown on the substrate it may be used in any desired manner, such as being implanted into a body.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows a flow diagram illustrating the steps taken to prepare a porous thermoplastic substrate; Figure 2 shows a scanning electron micrograph of osteoblast cell growth in the porous thermoplastic substrate; Figure 3 compares cell growth on the porous substrate and in a tissue culture plate at different incubation times; Figure 4 compares collagen synthesis by osteoblast cells on tissue culture plates and in the porous substrate at different times after seeding; Figure 5 shows typical porosities obtained at different plasma conditions; Figure 6 shows typical surfaces with different rugosity and Figure 7 shows typical surface mineralization nuclei.

In this example the porous material is prepared by sintering thermoplastic granules, powder or pellets.

As those skilled in the art are well aware, the ability of a thermoplastic to be sintered can be determined from its melt flow index (MFI). Melt flow indices of individual thermoplastics are known or can be readily determined by methods well known to those skilled in the art. hi general, however, the MFI of a thermoplastic suitable for use in the materials and methods of the invention is from about 0 to about 15.

Suitable thermoplastics that can be used to provide the porous thermoplastic substrate include, but are not limited to, polyolefins, nylons, polycarbonates, poly(ether sulfones), polystyrene and mixtures thereof. A preferred thermoplastic is a polyolefin. Examples of suitable polyolefins include, but are not limited to: ethylene vinyl acetate; ethylene methyl acrylate; polyethylenes; polypropylenes; ethylene-propylene rubbers; ethylene- propylenediene rubbers and mixtures and derivatives thereof. A preferred polyolefin is polyethylene. Examples of suitable polyethylenes include, but are not limited to, low density polyethylene, linear low density polyethylene, high density polyethylene, ultra- high molecular weight polyethylene, and derivatives thereof.

PRE-PROCESSJNG

Thermoplastic particles are made using cryogenic or ambiently grinding and screened to ensure a proper particle size distribution. This step gives the optimum pore size for cell growth.

MOULD FILLING

The powder is placed in a mould, or multiple moulds, such that there is an adequate number of cavities to meet the volume demands of the commercial application. Moulds are produced of carbon steel, stainless steel, brass, or aluminium, and may have a single cavity, or up to several hundred. The number of cavities is also a function of the part geometry, and the heating method required to assure consistent parts. Mould filling is preferably assisted by using commercial powder handling and vibratory equipment.

SINTERING

Thermal processing is carried out by introducing heat to the mold, using any of the various controllable heating means. Electrical resistance heating, electrical induction heating, or steam heat may be used. The applied heat is controlled as appropriate to allow softening of the polymer particles and allow inter-particle binding to occur. Processing of parts with consistent porosity, strength, and flow characteristics is dependent on carefully considered application of commercial process control equipment. Control of the temperature cycle must allow consistent part manufacture such that there are no problems with under-processing which leads to weak, unsintered parts, or overprocessing, leading to glazed, non-porous parts.

Another method is available where material is laid down on a suitable belt and passed through a sintering oven to make continuous sheet which can be later fabricated into required shapes.

MOULD STRIPPING

Product is removed from the mould.

SURFACE TREATMENT

In this example the porous substrate is subject to plasma treatment using one or more suitable gases e.g. oxygen, air, nitrogen, ammonia, carbon dioxide which generates the presence of suitable radical ions on the surface of the sintered porous plastic. Suitable plasmas for use in the method of the invention include non-equilibrium plasmas such as those generated by radio frequencies (RF), microwaves or direct current (DC). They may operate at atmospheric or sub-atmospheric pressures as are known in the art.

The effect of treatment can be measured by applying droplets of liquids of different surface tensions on to the treated material and observing the absorption of the liquid over time or measuring the contact angle of differing surface tension liquids and therefore determining the surface energy.

Parameters used in determining the effect of the treatment include time, pressure, power/unit area, 02 concentration,

EXAMPLE 1

MATERIAL PREPARATION

DOW 53050E high density polyethylene pellets were ambiently ground and polished to give a particle distribution of: >600tm 2% 425-600jtm 39% 300-425gm 31% <300pm 28%

SINTERING

A im x im sheet 3.2mm thick was prepared by sintering the ground pellets using a continuous belt method at the temperature of 2 10 C and a belt speed of 1.25m/min. The sintered material had a mean flow pore size of 95pm and a porosity of5l%.

PLASMA TREATMENT

12mm diameter discs were cut from the sheet and placed randomly in the plasma chamber between the electrodes. The plasma chamber was sealed and evacuated to 0.O2torr to remove any possible volatile contaminants from the sintered plastic. A continuous flow of air was admitted into the chamber to a pressure of 0.2torr. The plasma was then ignited by applying a high frequency voltage at 13.56 M}Iz at a power of 35W, continuously for a period of 20 minutes. The chamber was returned to atmospheric pressure and the treated material removed.

De-ionised water droplets were then placed on the surface of the treated material and were absorbed into the structure immediately indicating the production of a satisfactory porous substrate.

MATERIAL ANII METHODS

Cell adhesion and bone noduli formation: Cell line human MG63 cells (10 x i05 cell/ml, ATCC catalogue No. CRL-1427) were incubated in tissue culture plates (TCP, Nunc) for 24 h in Minimum Essential Media Eagle (MEM, Sigma Cat No. M2279) enriched with 0.292g/L L-Glutamine, 1% (vlv) non essential amino acids (Sigma Cat. No. M7145) and 10% (v/v) foetal calf serum at 37 C under a 95% air, 5% CO2 flow. The medium was made osteogenic by the addition of glycerophosphate (10pm) and ascorbic acid (250pm).

Cells were allowed to proliferate for 48h, washed with calcium-free phosphate buffered saline pH 7.4 (Oxoid, UK). After incubation MG63 osteoblasts were fixed in formalin for 1 h at room temperature, washed 3 times with PBS, underwent a rapid wash in deionised water and stepwise prior to preparation for examination by scanning electron microscopy.

Electron Microscopy 12mm diameter discs incubated with osteoblast cells were fixed for 1 hour in 2.5% glutaraldehyde and then dehydrated using a series of ethanol in water solutions (25% V/s, 50% "/.,, 75% "/, and 2 changes of 100%). The dehydrated samples were freeze dried (12 hours) prior to mounting on aluminum sample stubs fitted with adhesive pads. The discs were then sputter coated with palladium using a voltage of 2.2 kv. The cell bearing supports were imaged using a JEOL 6310 scanning electron microscope at an accelerating voltage of 5kv.

Collagen and protein synthesis. The synthesis of collagen produced by the MG63 osteoblasts was evaluated at different incubation times (24, 48, and 72h). The cells adhering on the tested substrates were trypsinised by a standard procedure, lysated by the addition on 01% (w/v) Triton, and centrifuged for 5 mm at 500x g and the supernatants (50.tl) tested for their collagen content by quantative Sirius Red method. A standard curve was obtained using collagen type I (Sigma, UK, catalogue No. C9879) in the concentration range 5gg/50p.1 to 20gI50pJ. The total protein content of each sample was evaluated by a Bradford's microplate assay (Biorad, UK, catalogue No. 500-0006).

Collagen data were expressed as collagen tg/g of protein mean standard deviation from n = 6. Protein data were expressed as protein jig/mi mean / standard deviation from n =6.

Alkaline phosphate (ALP) activity. Cells (1 05/mI) were incubated for 24 h, detached from the substrate by trypsin and lysated. The samples were centrifuged and the supematants stored at -70 C until use. An ALP microplate assay was performed as described elsewhere. Briefly, S0jil of each sample were added to 5Opi of 100 mM p- nitrophenoiphosphate (Sigma UK) and incubated for 30 mm at 37 C. The reaction was stopped by adding 5OpJ of 1M NaOH. The absorbance was read by a Titertek Multiscan Pluse microplate reader (ICN Flow, UK) at 405nm. The total protein content was also estimated by a Biorad microplate protein assay based on the Bradford's method (catalogue n. 500-0201). Data were expressed either as ALP activity (Sigma mU/ml) or as ALP specific activity (Sigma mU/jig of protein) mean standard deviation from n=6.

RESULTS

Figure 2 shows osteoblast cells growing over the surface of the polymer matrix (1) and forming tissue (2). The osteoblasts were seen to adhere and proliferate on the surface of the candidate substrate as shown in Figure 2. In particular the cells formed typical bone noduli which were not observed on tissue culture plastic. Unlike the tissue culture plate, these noduli were encouraged to develop into a 3-D space by the porous architecture of the substrate.

CELL DIFFERENTIATION

Figure 3 shows ALP activity of the cells on the porous substrate and TCP at different incubation times. Osteoblast differentiation on the candidate discs was evaluated as a measure of the alkaline phosphatase activity produced by the cells during their contact with the materials for 24hours (h), 48hours (h) and 72hours (h) and compared with their differentiation on conventional tissue culture plates (TCP). Data were normalised as ALP mU/pg of proteins. Although, a similar ALP activity peak at 48h was observed for both the substrate types, osteoblasts have a significantly higher degree of differentiation on porous scaffolds. Figure 3 clearly shows that the ALP activity was higher when the cells were incubated on the porous substrates.

Collagen Synthesis Figure 4 shows collagen synthesis by osteoblast cells on tissue culture plastic and candidate discs 24, 48 and 72 hours following seeding. Collagen synthesis by the osteoblasts on the porous substrate was shown to be significantly higher at 24 and not significantly different at longer incubation times as shown in Figure 4. These data combined with the cell adhesion and tissue formation studies suggest that this early production of collagen together with the porous nature of the material encourage the formation of 3-D bone noduli.

EXAMPLE 2

MATERIAL PREPARATION

DOW 53050E density polyethylene pellets were anibiently ground and polished to give a particle distribution of: >600jtm 2% 425-600jim 95% <425gm 3%

SINTERING

A im x im sheet 3.2mm thick was prepared by sintering the ground pellets using a continuous belt method at the temperature of 210 C and a belt speed of 1.25mlmin. The sintered material had a mean flow pore size of 1 50j.tm and a porosity of 51%.

PLASMA TREATMENT

12mm diameter discs were cut from the sheet and placed randomly in the plasma chamber between the electrodes. The plasma chamber was sealed and evacuated to 0.O2torr to remove any possible volatile contaminated from the sintered plastic. A continuous flow of air was admitted into the chamber to a pressure of 0.2torr. The plasma was then ignited by applying a high frequency voltage 13.56 MHz at a power of 35W, continuously for a period of 20 minutes. The chamber was returned to atmospheric pressure and the treated material removed.

De-ionised water droplets when placed on the surface of the treated material were absorbed into the structure immediately indicating the provision of a satisfactory porous substrate.

EXAMPLE 3

MATERIAL PREPARATION

DOW 53050E high density polyethylene pellets were ambiently ground and polished to give a particle distribution of: >425 4% 300-425 93% <300 3%

SINTERING

A im x im sheet 3.2nmi thick was prepared by sintering the ground pellets using a continuous belt method at the temperature of 210 C and a belt speed of 1.25mlmin. The sintered material had a mean flow pore size of 60 jtm and a porosity of 46%.

PLASMA TREATMENT

12mm diameter discs were cut from the sheet and placed randomly in the plasma chamber between the electrodes. The plasma chamber was sealed and evacuated to 0.O2torr to remove any possible volatile contaminated from the sintered plastic. A continuous flow of air was admitted into the chamber to a pressure of 0.2torr. The plasma was then ignited by applying a high frequency voltage at 13.56 MHz at a power of 35W, continuously for a period of 20 minutes. The chamber was returned to atmospheric pressure and the treated material removed.

The attached Table - Appendix 1 - indicates the results achieved for porous three dimensional substrates prepared with a variety of properties indicated in the table, for example pore size, various pressures and times of plasma treatment and mgosity of the pore surfaces.

The different plasma treatment conditions offer a series of materials which support cell adhesion and differentiation at different degrees or are able to prevent cell adhesion (SuperHydrophobic), where cell differentiation is defined as: "the process during which young, immature (unspecialised) cells take on individual characteristics and reach their mature (specialised) form and function (www.seniormag.com). According to this definition, plasma- treated three dimensional (3D) scaffolds supporting cell adhesion and differentiation offer the additional benefit of inducing the formation of tissue noduli and the deposition of a more consolidated, mineralized extracellular matrix. Indeed, the almost inverse relationship between concentrations of soluble collagen and levels of deposited ECM indicates that a tissue more stable than traditional 2D substrates is formed on the 3D scaffolds. Tissue noduli were obtained in traditional 2D cell culture conditions in 72 hours (h). SuperHydrophobic scaffolds are relatively inert towards cell adhesion (limited to the first 24 h only) and they are particularly suitable for testing cell adhesion properties of deposited coatings (e.g. biomaterials for biomedical applications) or grafted molecules (e.g. structural proteins) in a 3D environment. They can also be used as biomaterials for biomedical implants capable of supporting biomaterials or bioactive molecules with cell adhesion properties. The deposition of a stable coating of these specific cell substrates is ensured by the relatively high hydrophobicity of the treated 3D scaffold. The different degrees of cell adhesion which can be achieved on these scaffolds also widen the range of substrates available to test drugs in clinically-reflective conditions in vitro.

As can be seen from the table of Appendix 1, different conditions encourage or reduce cell adhesion. For example, if the plasma treatment occurs at a pressure greater than 0.05 Torr or greater than 0.1 Torr cell adhesion is increased. By way of a further example, if the rugosity is not lower than 45nm cell adhesion occurs whereas if the rugosity is not higher than 45nm cell adhesion is reduced.

Figures 5, 6 and 7 show graphically some of the results of Appendix 1. Figure 5 shows typical porosities obtained at different plasma conditions. Macroporosity was always greater than 300 jim as shown in Appendix 1. Some samples of Appendix 1 exhibited the formation of nanoporosity as shown in Figure 5C. These samples are denoted by a double asterix next to the porosity values. Figure 6 shows typical surfaces with different rugosity. Figure 6A shows an untreated surface, Figure 6B shows a superhydrophobic surface and Figure 6C shows a hydrophilic surface. The formation of nanopores is also shown in Figure 6C as indicated in Appendix 1. Figure 7 shows typical surface mineralization nuclei (brownlred precipitates). Figure 7A shows an example of a tissue culture plate (TCP) and Figure 7B shows an example of a 3D porous scaffold.

Many variations may be made to the examples, described above whilst still falling within the scope of the invention. For example instead of or as well as being subjected to plasma treatment, the pore surfaces may be treated in any suitable manner to encourage the growth of organic materials, such as one or more of ionising the pore surfaces, making the pore surfaces hydrophyllic, providing free radicals in the pore surfaces, removing oil from the pore surfaces or roughening the pore surfaces for example.

Appendix 1 Cell support properties of plasma-treated scaffolds ________________ ________________ ________________ ________________ Cell Features 2D substrate Untreated Super Hydrophilic Hydrophilic Hydrophobic treatment treatment ID RDM/1 1/175/2 RDM/1 1/175/3 RDM/1 1/175/4 RDM/1 1/175/5 Porvair Grade ____________ ____________ Vyon HP Vyon HP Vyon HP Vyon HP Material ___________ ___________ HDPE HDPE HDPE HDPE Pore Size' Max ___________ >300 >300 >300** >300** ____________________ Mean _____________ 90 90 90 90 ____________________ MIFP _____________ 25 25 25 25 Plasma Treatment Power / Watts - 40 40 40 ____________________ Time / mins _____________ - 20 20 20 __________________ Gas ____________ - PFEA Air Oxygen _____________________ Pressure / ton ______________ - 0.05 0.2 0.2 Rugosit(nm) _____________ none 63.23 8.69 36.39 9.22 51.56 5.41 48. 15 6.5 Protein synthesis - 120t 95* 92* lOlt (TCP%_at 24 h) ____________ ____________ _____________ _____________ _____________ _____________ Soluble Collagen - 120t 67* 73* 98* (TCP% at 24 h) ____________ ____________ _____________ _____________ _____________ _____________ ALP activity - 92* 91* 87* 96* (TCP% at 24 h) ____________ ____________ _____________ _____________ _____________ _____________ Cell-cell interactions Limited to cell tissue noduli apoptotic cell tissue noduli tissue noduli (SEM analysis at 72 h) ______________ lamellipodia _______________ aggregates _______________ _______________ Deposited ECM + ++ - ++++ ++++ (SEM analysis at 72 h) ______________ _____________ _______________ _______________ _______________ _______________ Tissue formation 2D 3D confluent none 2D confluent 3D confluent (SEM analysis at 72 h) _____________ Semi confluent ______________ ______________ ______________ ______________ Mineralization _____________ + -i--i-+ - + ++ Cell Features Hydrophilic Hydrophilic Hydrophilic treatment treatment treatment RDMI12/93/1 RDM/12/93/2 RDM112193/4 Porvair Grade ____________ Vyon HP Vyon HP Vyon HP Material __________ HDPE HDPE HDPE Pore Size' Max >300 >300** >300** ____________________ Mean 90 90 90 ___________________ MFP 25 25 25 Plasma Treatment Power / Watts 40 40 40 ____________________ Time / mins 20 20 60 ___________________ Gas Allylamine Air Air _________________ Pressure/ton 0.2 0.4 0.2 Rugosity ____________ 51.03 6.13 52.20 7.34 49.55 6.33 Protein synthesis 98.5t 106.6t 101.2t (TCP% at 24 h) ____________ _____________ _____________ _____________ Soluble Collagen 103t 104.4t 103.3t (TCP% at 24 h) ____________ _____________ _____________ _____________ ALP activity 98t 112t 129.6* (TCP% at 24 h) ____________ _____________ _____________ _____________ Cell-cell interactions Tissue noduli Tissue noduli Tissue noduli (SEM analysis at 72 h) _____________ ______________ ______________ ______________ Deposited ECM ++++ ++++ ++++ (SEM analysis at 72 h) _____________ ______________ ______________ ______________ Tissue formation 2D confluent 2D confluent 2D confluent (SEM analysis at 72 h) _____________ ______________ ______________ ______________ Mineralization _____________ +++ + + 1 Determined using Xonic Pormeter 3G, Xonics Corporation, Sunrise, Florida, USA 2 PFEA - 2-(Perfluorodecyl)ethyl acrylate 3. Rugosity definition: the quality or state of being rough, wrinkled, creased (www.thefreedictionary.com, http://dict.die.net). Typical graphical examples of surfaces with different rugosity are reported in Figure 5.

= value not significantly different from control tissue plate = value significantly different from 2D substrate at p<O.O5 **rrindicate the formation of nanoporosity as shown in Figure 5 C 2D= Two dimensional arrangement 2D substrate= commercial tissue culture plate 3D= Tn-dimensional arrangement Tissue noduli= cell clustering into a early tissue architecture Cell lamellipodia= cell membrane extensions driving cell/substrate and cell/cell adhesion ECM= extracellular matrix Apoptotic cell aggregates= cells which are not able to adhere onto the substrate and try to form aggregates among themselves, but soon entering a programmed cell death cycle.

Claims

1. A three dimensional substrate for controlling or modulating tissue formation or deposition, the substrate comprising: a porous material in which at least one of the surface physical and chemical properties and architecture are arranged to regulate or modulate cell proliferation andlor differentiation.

2. A substrate according to claim 1, wherein the porous material is a porous thermoplastic.

3. A substrate according to claim 2, wherein the porous material is an organic polymer.

4. A substrate according to any one of the preceding claims, wherein the surfaces of the pores are activated by a plasma.

5. A substrate according to any one of the preceding claims, wherein the surfaces of the pores are activated by a vapour.

6. A substrate according to claim 4 or claim 5, wherein the substrate is plasma or vapour treated at a pressure greater than 0.05 Ton.

7. A substrate according to claim 4 or claim 5, wherein the substrate is plasma or vapour treated at a pressure greater than 0.1 Ton.

8. A substrate according to claim 4 or claim 5, wherein the substrate is plasma or vapour treated at a pressure between 0.05 Ton and 0.5 Ton.

9. A substrate according to claim 4 or claim 5, wherein the substrate is plasma or vapour treated at a pressure between 0.1 and 0.3 Ton.

10. A substrate according to any one of the preceding claims, wherein the surfaces of the pores are ionised.

11. A substrate according to any one of the preceding claims, wherein the surfaces of the pores are hydrophyllic.

12. A substrate according to any one of the preceding claims, wherein the surfaces of the pores contain free radicals.

13. A substrate according to any one of the preceding claims, wherein the surfaces of the pores are substantially oil free.

14. A substrate according to any one of the preceding claims, wherein the surfaces of the pores have a defined roughness.

15. A substrate according to any one of the preceding claims, wherein the surfaces of the pores have cracks therein.

16. A substrate according to claim 14 or claim 15, wherein the surfaces of the pores have a minimum average rugosity of 45nm.

17. A substrate according to claim 14 or claim 15, wherein the surfaces of the pores have a maximum average rugosity of 45nm.

18. A substrate according to any one of the preceding claims, wherein the pores have an average diameter that is between 5 and 200 times the size of the organic elements to be grown.

19. A substrate according to any one of the preceding claims, wherein the pores have a mean diameter of between 5 and 200J.Lm.

20. A substrate according to any one of the preceding claims, wherein the pores have a mean diameter of between 50 and 150 jtm.

21. A substrate according to any one of the preceding claims, wherein the pores have a mean diameter of between 75 and l25m.

22. A method of controlling or modulating tissue formation or deposition, the method comprising providing a three-dimensional substrate formed of porous material and arranging at least one of the surface physical and chemical properties and architecture of the porous material to regulate cell proliferation andlor differentiation

23. A method according to claim 22, wherein the surfaces of the pores are treated by being activated by a plasma.

24. A method according to claim 22 or claim 23, wherein the surfaces of the pores are treated by being activated by a vapour.

25. A method according to claim 23 or claim 24, wherein the surfaces are plasma or vapour treated at a pressure greater than 0.05 Ton.

26. A method according to claim 23 or claim 24, wherein the surfaces are plasma or vapour treated at a pressure greater than 0.1 Ton.

27. A method according to claim 23 or claim 24, wherein the surfaces are plasma or vapour treated at a pressure between 0.05 Ton and 0.5 Ton.

28. A method according to claim 23 or claim 24, wherein the surfaces are plasma or vapour treated at a pressure between 0.1 and 0.3 Ton.

29. A method according to any one of claims 22 to 28, wherein the surfaces of the pores are treated by being ionised.

30. A method according to any one of claims 22 to 29, wherein the surfaces of the pores are treated to become hydrophyllic.

31. A method according to any one of claims 22 to 30, wherein the surfaces of the pores are treated to provide free radicals therein.

32. A method according to any one of claims 22 to 31, wherein the surfaces of the pores are treated to have oil removed therefrom.

33. A method according to any one of claims 22 to 32, wherein the surfaces of the pores are treated by being roughened.

34. A method according to any one of claims 22 to 32, wherein the surfaces of the pores are treated by having cracks introduced therein.

35. A method according to claim 33 or claim 34, wherein the surfaces of the pores are treated to produce a minimum average rugosity of 45nm.

36. A method according to claim 33 or claim 34, wherein the surfaces of the pores are treated to produce a maximum average rugosity of 45nm.

37. A substrate substantially as hereinbefore described with reference to the accompanying drawings.

38. A method of controlling or modulating tissue formation substantially as hereinbefore described with reference to the accompanying drawings.