WO2005094911A2

WO2005094911A2 - Composite materials

Info

Publication number: WO2005094911A2
Application number: PCT/EP2005/002473
Authority: WO
Inventors: David Philip Knight
Original assignee: Oxford Biomaterials Limited
Priority date: 2004-03-05
Filing date: 2005-03-04
Publication date: 2005-10-13
Also published as: EP1729832A2; US8911761B2; US20120023677A9; US20070113355A1; GB0405045D0; WO2005094911A3

Abstract

The application discloses a composite material (30) comprising one or more silk elements (10) in an acrylic or cross-linked protein matrix (20). The silk elements (10) are made from the group of silk elements consisting of domestic silkworm silk, wild silkworm silk, spider dragline silk, and filaments spun from recombinant silk protein or protein analogues. The composite material is particularly useful for use in surgical implants.

Description

COMPOSITE MATERIALS Description

Field of the Invention

The invention relates to a composite material comprising one or more silk elements in an acrylic or cross-linked protein matrix, its method of manufacture and its use in surgical implants.

Prior Art

Biodegradable polymer materials made of silk fibroin elements are known, for example, from US-A-2004/0005363 (Tsukada et al.). This patent document teaches a composite material made from silk fibroin and another "secondary" substance, such as cellulose, chitin, chitosan (or derivatives), Keratin or polyvinyl alcohol. The composite material can be used as a sustained release substrate for medicines, a biological cell growth substrate, a metal ion-absorbing material and a biodegradable water-absorbing material.

Another example of a composite material in which proteins coat the surface of a surgical device is disclosed in WO-A-94/22584 entitled "Chronic Endothelial Cell Culture under flow". In this patent application, the inner lumen of a hollow polypropylene fibre was coated with a synthetic protein polymer Fibronectin F which contains multiple repeats of the RGD fibronectin binding site. This produced an inner lumen surface which was substantially uniformly coated with the synthetic protein polymer on which cells could be grown. The polypropylene fibres can be used in vascular grafts. The strength of the vascular graft is as a result dependent on the tensile strength of the polypropylene fibres.

The surgical device (stent grafts and vascular grafts) described in WO-A-01/38373 (Boston Scientific) exploits the strength of spider silk by providing an outer or interstitial sheath over the outside surface or the luminal surface of an inner stent. The inner stent is made from a multitude of materials such as synthetic textile materials, fluoropolymers and poly defines. Nylon, polyester and polyurethane are often used. These materials which can not be resorbed by the body. The material of the outer sheath is non-resorbable conventional man-made polymeric material or a combination of spider silk and man made polymeric material.

Summary of the Invention

These and other objects of the invention are solved by providing a composite material having one or more silk elements in an acrylic or cross-linked protein matrix. This material is highly biocompatible. Preferably the silk elements are made from wild silkworm silk, domestic silkworm silk, spider dragline silk, and filaments spun from recombinant silk protein or protein analogues or mixtures of these. A protein matrix is preferred because this means that both the protein matrix and the silk elements are resorbable.

In one embodiment of the invention, the silk elements are embedded in an acrylic matrix made of a cyanoacrylate, as this acrylate is known to be biocompatible. Alternatively, the silk elements can be incorporated into a matrix made of cross-linked fibroin or cross- linked casein.

In a particularly advantageous embodiment of the invention, the silk elements are made from the silk derived from wild silkworms (i.e. Tussah silk) as the resorbtion rate of the composite material formed from the cross-linked protein matrix and wild silkworm silk elements is slower than that of composite material with silk elements derived from spider silk.

In one embodiment of the invention, the silk elements formed from a plurality of short filaments with a staple length less than 120 mm are from carded filaments (i.e. filaments drawn to lie approximately parallel to one another by a combing process). In another embodiment of the invention, the silk elements are twisted into a thread which can also be further twisted into a cord or rope or woven, braided, embroidered, wound, stitched or knitted.

The composite material can be formed into a substantially cylindrical form with the silk elements wound at an angle in excess of 40 degrees to the long axis of the substantially cylindrical form or circumferentially to the long axis. On a luminal surface of the form, the silk elements can be wound parallel to the long axis of the substantially cylindrical form. This latter construction has the advantage that it should stimulate the longitudinal movement of nerve processes in a sleeve and therefore promote nerve regeneration.

Mineralisation of the composite material is advantageous as this allows the composite material to be used as a bone substitute and stimulates regrowth of the bone material.

The principal silk protein used in the silk elements contains at least eight repeats of the triplet RGD. The eight repeats of the triplet RGD are located immediately adjacent to turns or predicted turns of a structure of the principal silk protein. This is advantageous as this sequence when next to a turn specifically recognises and holds the fibronectin binding site of integrin molecules anchored to the surface of most metazoan cell types. In turn this leads to excellent cell adhesion and advantageous changes in cell physiology including polarisation of function, cell differentiation and changes in the cell cycle.

The composite material is in one use formed into surgically implantable devices, such as sutures, artificial ligaments and tendons, endoluminar devices, anastomosis devices, and sleeves to aid in the regeneration of nerve cells

The invention also comprises a method for the manufacture of a compound material with a first step of providing one or more silk elements and a second step of embedding the silk element in an acrylic or protein matrix. The silk elements can be obtained by degurnming and unwinding or combing silk from a cocoon.

Fibroin can be prepared for the protein matrix by dissolving domestic or wild silkworm silk or cocoons in one or more chaotropic agents, such as calcium nitrate solution and lithium thiocyanate solution.

The protein matrix is cross-linked using a cross-linking agent such as formaldehyde vapour, glutaraldehyde, polyglutaraldehyde, carbodiimide and genipin.

The acrylic or fibroin material is applied to the silk elements by dipping, painting, spraying or casting.

An apparatus for the manufacture of an object made from the composite material is also part of the invention. The apparatus has a storage region having silk elements, a substantially cylindrical former and finally a feeding means to place said silk elements about the former.

The feeding means comprises rollers and tension is maintained between the rollers and the former to pre-stress and straighten the silk elements so as to ensure a good final product.

In one embodiment of the invention, a take up drum is used to form an object which is then removed as a substantially cylindrical form from the take up drum. Advantageously, carded silk fibres are fed to a cylindrical former and continuously coated onto the substantially cylindrical drum and finally a composite sheet emerges continuously from the substantially cylindrical drum.

Description of the Drawings

Fig. 1 shows a composite material according to this invention. Fig.2 shows a first manufacturing method for the composite material. Fig.3 shows a second manufacturing method for the composite material. Fig.4 shows the distribution of the fibronectin binding triplet RGD in the repetitive part of the sequence of Antheraea pemyi (left column) and A. yamamai (right column) heavy chain fibroin. Fig 5 shows the consensus repeat sequence for the glycine-rich domains for the first 900 repetitive amino acids of the repetitive region of A. pernyii heavy chain fibroin. Fig.6 shows the position of a putative turn immediately adjacent to the RGD triplet in the consensus sequence from the glycine-rich domain of A. pernyi heavy chain fibroin.

Detailed Description of the Invention

Fig. 1 shows a composite material 30 comprising one or more silk elements 10 in a matrix material 20. The matrix material 20 can be an acrylic matrix, made for example from a cyanoacrylate, or a protein matrix. The protein matrix can, for example, be a fibroin matrix or a casein matrix. Other highly soluble proteins could also be used. The protein matrix is cross-linked using a cross-linking agent such as formaldehyde vapour glutaraldehyde, polyglutaraldehyde, formaldehyde carbodiimide and genipin. In one embodiment the protein matrix is cross-linked by heating substantially dry formaldehyde vapour generated by heating paraformaldehyde in a sealed container to 80- 100 degrees centigrade for 5 minutes to 3 hours.

The silk elements 10 are made from domestic silkworm soil, wild silkworm silk or spider dragline silk. The silk elements 10 could also be made from recombinant silk protein or protein analogues. The silk elements 10 produced from wild silkworm in general means those elements produced by Antheraea pernyi, Antheraea yamamai, Antheraea militta, Antheraea assama, Philosamia Cynthia ricini and Philosamia Cynthia pryeri. The silk of other Saturnid moths such as those of the genus Actias or Cecropia though not generally defined as wild silk worms yield a closely similar silk element and can be used in this invention as the silk elements 10.

In Fig. 1 a simple block of composite material 30 is known. However, other shapes are possible such as fibres, rods, sheets or tubes as will become clear from the discussion below.

The silk elements 10 can be made up of a plurality of silk elements which have been twisted together to form a thread. The silk elements 10 can be further made of a plurality of short filaments of silk, for example with a staple length as long as possible and at least 20 mm. The silk elements 10 can be twisted into pairs or multiples to form a thread which can also be further twisted into a cord or rope or woven, braided, embroidered, wound, stitched or knitted to form devices. In one advantageous embodiment of the invention, the silk elements 10 comprise carded filaments.

In one embodiment of the invention, the silk elements 10 are degummed from the silk cocoon. However, other methods of extracting the silk elements 10 can be used.

The fibroin for the protein matrix 20 is extracted by dissolving domestic or wild silkworm silk or cocoons in one or more chaotropic agents, such as calcium nitrate solution and lithium thiocyanate solution. In the case of wild silks, it is necessary to heat the chaotropic agent. The silk elements 10 are placed in the resultant solution which is then dialysed to remove the chaotropic agent or agents and concentrated or dried by evaporation or reverse dialysis. The concentrated fibroin solution can be dried into films or flakes for storage and redissolved in distilled water or other solvents when required.

US Patent US-A-5,245,012 (US Army) also teaches a method of dissolving silk protein extracted from different arachnid species using varying solvent systems. The teachings of this patent are incorporated herein by reference.

The composite material 30 of the invention can be used to make a variety of objects. One example of an apparatus for the manufacture of the objects 100 is shown in Fig.2 in which the silk elements 10 are initially held in a storage region 200 and are spooled (or otherwise fed) to a drum 210 over rollers 220 and wrapped circumferentially or helically about the drum 210. The function of the rollers 220 is to ensure that the silk elements 10 are pre-stressed and therefore firmly placed on the drum 210. The matrix material 20 is then coated by an application device 250 over the outer surface 240 of the drum 210 with matrix material 20 to affix and embed the silk elements 10. It will be understood that the drum 210 could also be a mandrel or more generally a former. It will be understood that the silk elements 10 can be laid down on the drum 210 or mandrel in many different patterns of orientation by rotating the drum 210 or mandrel and changing the spatial relationship between the drum 210 and the rollers 220. In a further embodiment a composite rope or cable structure is formed as follows. Two or more lengths of single, double or multiple ply silk threads are twisted into a thin rope. The acrylate or fibroin solutions for the matrix can be applied before or after twisting. After drying or partial drying, the matrix is then cross-linked by chemical treatment, preferably by hot formaldehyde vapour. It will be understood that by successive twisting varying hierarchical levels of construction to give cords, ropes or cables of thicknesses appropriate to end use. The resulting materials can be incorporated into a variety of prosthetic devices including tendon and ligament prostheses or embroidered or knitted devices.

The object 30 constructed in accordance with the teachings of Fig.2 can be advantageously made with silk elements 10 formed from carded silk filaments derived from silk brins. Carded silk filaments are very thin (30μm diameter) allowing the manufacture of thin- walled devices or composite sheets.

The drum 210 can be so arranged that a continuous sheet made of composite material 30 emerges from the drum 210 as shown in the Fig 2.

As the composite material 30 is biocompatible, it can be used to make an implant, such as an endolumnal device, a stent device, an anastomosis device, a suture, or a device to promote nerve regeneration. It is known that the silk elements 10 provide an excellent substrate for cell or tissue growth. Therefore some of the matrix material 20 of the object 100 can be abraded away to reveal some of the silk elements 10 on either an inner (or endolumnal) surface 230 of the object 30 or on an outer surface 240 of the object 30.

Another method for the manufacture of the object 30 in a tubular structure is shown in Fig.3 in which silk elements 10 are wrapped substantially longitudinally along the surface of a former 300 on both an inner surface 310 and an outer surface 320 of the former 300. The former has typically a diameter between 0.5 mm but can have a diameter up to 10 m and has a long axis 325. The former 300 could also be tapered. The matrix material 20 is applied over the silk elements 10 and allowed to dry and thus embed the silk elements 10 in the object. Application of the material is by dipping, painting, spraying or casting. The ends 330a and 330b of the object 30 can be cut away and the former 300 slipped out. A tubular object is thus created. Formers coated with a layer of wax or which can be caused to collapse into a smaller diameter can be used to facilitate removal of the object 30 from the former 300.

Examples

Example 1. Preparation of composite materials from carded Antheraea pernyii silk filaments.

Antheraea pernyii silk filaments prepared from degummed silk cocoons were obtained from a commercial supplier. The carded silk from the skein was smoothed into flat sheets and gently tensioned to pull the silk filaments parallel. Keeping the silk filaments tensioned they were wound on to cylindrical formers 025 mm to 30 mm in diameter. The largest cylinders were made of glass and had a substantially smooth surface. The smallest cylinders 025 mm were prepared by coating a thin stiff wire in low melting point wax. Cylindrical formers with intermediate diameters were made from low melting point wax. The silk filaments were generally laid circumferentially on the largest formers but were laid in a helical fashion on the smaller cylindrical formers.

Care was taken to ensure a uniform dense lay of filaments was achieved on the cylindrical formers. The silk filaments at the ends of the thin cylindrical formers were secured with cyanoacrylate adhesive from Loctite. For the larger cylindrical formers the silk filaments at the ends were secured by a thin line of superglue running parallel to the long axis of the cylindrical former. This allows the removal of the composite material from the cylindrical former making a cut along the thin line of adhesive.

The composite material was formed either by painting a thin coat of either cyanoacrylate adhesive or concentrated regenerated fibroin solution on to the silk filaments. The regenerated fibroin solution was prepared by dissolving commercial degummed Bombyx mori fibroin powder in aqueous 6.3 M lithium bromide with gentle stirring at room temperature. The silk solution was then dialysed in 18-20kDa MWCO Visking tubes with two changes for a total of 6 hours at 4 degrees centigrade against aqueous OϋM lithium bromide solution with the intention of allowing the protein to refold after dissolution in the concentrated lithium bromide. Thereafter water was removed from the fibroin solution by reverse dialysis against aqueous 40% aqueous - or dry powdered- polyethylene glycol (MW 15-20 kDa), pooling the partially concentrated fibroin after 12 hours-24 hours of dialysis to a fresh dialysis tube to obtain a final highly concentrated solution. Alternatively the dialysed silk fibroin solution was allowed to partially dry by exposing the sealed the dialysis bag to dry air. The matrix material was applied by painting the concentrated fibroin onto the silk filaments and allowed to dry. Thereafter the matrix was cross-linked by treatment with substantially dry formaldehyde gas by heating to 80- 100 degrees centigrade for 5 minutes to 3 hours in a sealed vessel containing substantially dry paraformaldehyde. Unreacted formaldehyde was removed by heating the material to 100 degrees centigrade in a stream of air or by exhaustive washing.

Testing of the composite materials for mechanical strength was carried out by cutting silk -acrylate strips made using the larger cylindrical formers as described below in Example 3. An Instron universal mechanical testing instrument fitted with pneumatic grips was used to test composite strips to failure in uniaxial tension parallel to the lay of the silk filaments.

Example 2. Demonstration of RGD putative integrin binding sites on Antheraea spp heavy chain fibroins and their location adjacent to turns.

The published sequences of silk proteins were searched on the TrEMBL and SwissPROT data bases for the well established cell binding sequence RGD and the putative cell binding sites PPSRN and KNEED. The sequence triplet RGD is of considerable interest as it forms part of the mechanism used in multicellular organisms to stick many types of cells to the connective tissue framework of the body. The RGD triplet on the silk-like connective tissue protein fibronectin binds specifically to integrins. Integrins are a class of cell adhesion molecules. They are found intercalated into the lipid bilayer of the cell membrane with the fibronectin binding (RGD recognition) site protruding into the extracellular space. Thus the RGD recognition site of integrins is available to bind cells to the connective tissue framework via RGD triplets on fibronectin molecules.

The putative cell binding sites PPSRN and KNEED were absent from all silks examined. However, multiple copies of the triplet RGD were found in the glycine rich domains of three heavy chain fibroins of Antheraea pemyi (TrEMBL 076786), A. yamamai (TrEMBL Q964F4) and A. militta (Q8ISB3). There were 12 and 14 repeats respectively in the complete sequence of A. pernyi and A. yamamai and an even higher density of repeats (7) in the partial sequence of A. militta. The remarkably repetitive location of the RGD repeats is shown for A. pernyii in Fig 4. The RGD motif recurs in a constant location, in the glycine-rich repeat immediately separated by one polyalanine repeat before the sequence SAARRAGHDRAAGS (sometimes truncated to RRAGHDRAAGS) or a closely similar sequence.

In all cases the RGD triplet immediately preceded the motif GGYG. A. militta differed from the two other Antheraea sp in having two RGD's instead of one in one of its glycine-rich domains.

The RGD triplet was absent from all other silk protein sequences examined with the exception of that for Samia ricini heavy chain fibroin which contained a single copy immediately preceding a YGSD motif in a glycine-rich domain close to the end of this very long sequence.

Thus the cell-binding sequence RGD is found in multiple copies in the heavy chain fibroins of three Antheraea species.

We then sought to discover if the RGD sequence is present in locations within the silk molecule likely to be accessible to cell surface integrins. To seek an answer to this we first used RADAR (Expasy tools) to determine the consensus sequence for the repetitive glycine rich domains of Antheraea pernyii heavy chain fibroin (see Fig.5).

This showed that the sequence GGYGXGDGGYGSDS (where X =W or R) was well conserved within the glycine-rich domains. We then used a sophisticated secondary structure prediction tool, SCRATCH (Expasy tools) to determine the location of a putative turn within the consensus sequence (see Fig.6), as previous research (Peng et al. 2004 in preparation) has shown that turns are present within silk molecules as part of a tertiary structure defined by the existence of antiparallel β-sheets. A turn in the A. pemyi consensus sequence was found to be centred on a glycine residue immediately adjacent to the RGD. As each RGD triplet in fibronectin are located immediately adjacent to a turn (Leahy, Hendrickson et al. 1992; Leahy, Aukhil et al. 1996). We conclude that the RGD in Antheraea silks is likely to be available for binding to integrins and is therefore likely to strongly promote cell adhesion to this silk.

Example 3. Tensile data on Antheraea pernyi cyanoacrylate composite

Table 1. Tensile test data (n=6) for wet strips of composite material containing a high density of well-oriented Antheraea pernyii filaments set in a cyanoacrylate matrix. The strips (50 mm gauge length, average width 10.5mm and thickness 0.4 mm) were strained parallel to the filament orientation

For comparison, the ultimate tensile stress of a typical low alloy steel is in the region of 830 Mpa and a modulus of 200 Mpa but steel has a density of approximately 6 times that of the silk composite thus weight for weight this steel is less than 4 times as strong as the silk composite. The tensile data for the silk composite are comparable with that of that of two synthetic implantable materials (table 2). Property Poly-L-Iactide Poly ε A. pernyi acrylate caprolactone composite

Ultimate tensile 45 MPa 22 MPa 31 MPa strength

Ultimate extension 3% 500% 16%

Initial modulus 2.7 GPa 0.4 GPa l GPa

Table 2. Tensile data for wild silk -cyanoacrylate composite compared with two synthetic implantable materials.

Claims

1. A composite material comprising one or more silk elements in an acrylic or cross- linked protein matrix.

2. The composite material of claim 1 , wherein the silk elements are made from the group of silk elements consisting of domestic silkworm silk, wild silkworm silk, spider dragline silk, and filaments spun from recombinant silk protein or protein analogues.

3. The composite material of claim 1 or 2, wherein the silk elements are made from the silk derived from wild silkworms.

4. The composite material of any of the above claims, wherein the acrylic matrix is a cyanoacrylate

5. The composite material of any of the above claims, wherein the cross-linked protein matrix is a cross-linked fibroin.

6. The composite material of any of claims 1 to 4, wherein the matrix is cross-linked casein

7. The composite material of any of the above claims, wherein the silk element has a shape selected from the group of shapes consisting of fibres, rods, sheets or tubes.

8. The composite material of any of the above claims, wherein the silk element comprises a plurality of silk elements twisted into a thread.

9. The composite material of any of claims 1 to 7, wherein the silk element comprises a plurality of short filaments with a staple length of at least 20 mm.

10. The composite material of any of the above claims, wherein the silk element is a woven, braided, embroidered or knitted silk element.

11. The composite material of any of claims 1 to 7, wherein the silk element comprises carded filaments.

12. The composite material of any of the above claims and having a substantially cylindrical form with a long axis, an outer surface and a luminal surface.

13. The composite material of claim 12 , wherein the silk elements are wound at an angle in excess of 40 degrees to the long axis of the substantially cylindrical form.

14. The composite material of claim 12, wherein the silk elements are wound substantially circumferentially on an outer surface of the cylindrical former.

15. The composite material of any one of claims 12 to 14, wherein the silk elements are wound substantially parallel to the long axis on the luminal surface of the substantially cylindrical form.

16. The composite material of any of the above claims wherein silk element has a principal silk protein containing at least eight repeats of the triplet RGD.

17. The composite material of claim 16, wherein at least some of the at least eight repeats of the triplet RGD are located immediately adjacent to turns or predicted turns of a structure of the principal silk protein.

18. The composite material of any of the above claims, wherein fibroin in the fibroin matrix is prepared by dissolving domestic or wild silkworm silk or cocoons in one or more chaotropic agents.

19. The composite material of any one of the above claims, wherein the cross-linked protein matrix is cross linked using a cross-linking agent selected from the group of cross-linking agents consisting of glutaraldehyde. polyglutaraldehyde, formaldehyde carbodiimide and genipin.

20. The composite material of any one of the above claims being mineralised

21. A surgically implantable device made at least partially from the composite material of any of the above claims.

22. The surgically implantable device of claim 21 being an endoluminar device.

23. The surgical implant able device of claim 21 being a suture.

24. The surgically implantable device of claim 21 being a stent device.

25. The surgically implantable device of claim 21 being an anastomosis device.

26. The surgically implantable device of claim 21 being a sleeve to guide regenerating nerve cell processes

27. A method for the manufacture of a compound material comprising: - a first step of providing one or more silk elements; and - a second step of embedding the silk element in an acrylic or a protein matrix.

28. The method of claim 27, wherein the first step of providing the one or more silk elements comprises a step of degumming a silk element from a cocoon.

29. The method of claim 27 or 28, further comprising a step of preparing fibroin for the protein matrix by dissolving domestic or wild silkworm silk or cocoons in one or more chaotropic agents.

30. The method of claim 29, wherein the one or more chaotropic agents are selected from the group of chaotropic agents consisting of calcium nitrate solution and lithium thiocyanate solution.

31. The method of one of claims 27 to 30, further comprising a step of cross-linking the protein matrix using a cross-linking agent selected from the group of cross- linking agents consisting of glutaraldehyde, polyglutaraldehyde, formaldehyde, carbodiimide and genipin.

32. The method of any one of claims 27 to 31 , wherein the first step comprises twisting one or more of the one or more silk elements into a thread.

33. The method of any one of claims 27 to 32, wherein the first step comprises a step of weaving , braiding , embroidering or knitting one or more of the silk elements .

34. The method according to any one of claims 27 to 33, wherein the silk elements are wound around a former or implanted prosthesis prior to embedding in the acrylic or protein matrix.

35. The method according to claim 34, wherein the former is a flat plate or a cylindrical former with a long axis.

36. The method according to claim 34, wherein the former is a cylindrical former with a diameter from 0.5 mm to 10m.

37. The method according to any one of claims 27 to 36, wherein the second step comprises a step of coating the one or more silk elements with acrylic or protein material to form the acrylic or fibroin matrix.

38. The method according to any of claims 27 to 37, wherein the second step comprises a step in which the acrylic or fibroin material is applied to the silk elements by dipping, painting, spraying or casting.

39. The method according to any one of claims 35 to 38, wherein the silk elements are wound substantially circumferentially around the cylindrical former.

40. The method according to any one of claim 35 to 38, wherein the silk elements are wound helically around the cylindrical former.

41. The method according to claim 40, wherein the silk elements are wound at an angle in excess of 40 degrees to the long axis.

42. The method according to any one of claims 35 to 41 , wherein the silk elements are wound substantially parallel to the long axis of the cylindrical former.

43. The method according to any one of the claims 27 to 42, wherein the compound material has an outer surface and an inner surface and further comprising a step of abrading the outer or the inner surface so as to expose the silk elements.

44. The method of any one of claims 27 to 43, further comprising a step of mineralising the composite material.

45. The method of claim 44, wherein the step of mineralising the composite material comprises a step of immersing the object in a bath of an apatite mineral.

46. An apparatus for the manufacture of an object comprising - a storage region having silk elements; - a substantially cylindrical former; and - feeding means to place said silk elements about said substantially cylindrical drum.

47. The apparatus of claim 46, wherein the feeding means comprises rollers.

48. The apparatus of claim 47, wherein tension is maintained between the rollers and the cylindrical former to pre-stress and straighten the silk elements

49. The apparatus of any one of claims 46 to 48, wherein the storage region contains carded silk fibres.

50. The apparatus of any one of claims 46 to 49, further comprising a take up device to take up the object emerging from the substantially cylindrical drum.

51. The apparatus according to claim 49, where the carded silk fibres are fed to a cylindrical former and continuously coated onto the substantially cylindrical drum and wherein a composite sheet emerges continuously from the substantially cylindrical drum.

52. Biocompatible material made from proteins selected from the group of proteins consisting of wild silkworm proteins, recombinant silkworm proteins or analogues thereof.

53. The biocompatible material according to claim 52, wherein the proteins are heavy chain fibroin proteins.

54. The biocompatible material according to claim 52 or 53, wherein the silkworm proteins contain at least eight repeats of the triplet RGD.

55. The biocompatible material of any one of the claims 52 to 54, wherein at least some of the at least eight repeats of the triplet RGD are located immediately adjacent to turns or predicted turns of a structure of the silkworm protein.

56. Use of the biocompatible material of claims 52 to 55 as a substrate for cell adhesion.

57. Implant made at least partially of the biocompatible material of any one of claims 52 to 55.

58. The implant of claim 57 being an endoluminar device.

59. The implant of claim 57 being a suture

60. The implant of claim 57 being a stent device.

61. The implant of claim 57 being an anastomosis device.

62. The implant of claim 57 being a sleeve to promote nerve regeneration.

63. The implant of claim 57, wherein the implant is made of a plurality of elements of the biocompatible material.

64. The implant of any one of claims 57 to 62, wherein the implant is substantially cylindrical and has a surface.

65. The implant of claim 64, wherein the elements of the biocompatible material are arranged longitudinally on the surface of the implant.

66. The implant of claim 64, wherein the elements of the biocompatible material are arranged in a helical manner on the surface of the implant.

67. The implant of claim 64, wherein the elements of the biocompatible material are arranged circumferentially on the surface of the implant.

68. The implant of any one of claims 57 and 63 to 67, wherein the biocompatible material has been mineralised

69. Method for the manufacture of an implant comprising: - a first step of applying elements of silk proteins to a former; - a second step of affixing the elements; and - a third step of removing the former.

70. The method of claim 69, wherein the second step of affixing the elements comprising applying acrylate or protein material to the elements of silk proteins.

71. The method of claim 69 or 70 , wherein the former has a substantially cylindrical shape with an outer surface and an inner surface, and the first step comprises applying longitudinal elements of silk proteins substantially lengthways along the outer surface of the former.

72. The method of claim 71 , wherein the first step also comprises applying the longitudinal elements substantially lengthways along the inner surface of the former.

73. The method of any one of claims 67 to 72, further comprising a step of removing the former.

74. The method of any one of claims 69 to 73, further comprising a step of mineralisation

75. The method of the claim 74, wherein the step of mineralisation is carried out by immersing the object in a bath of apatite mineral.