PRECIPITATION OF COLLAGEN IN TACTOID FORM

Abstract: Collagen in tactoid form obtained by forming an aqueous solution containing dissolved collagen and a water soluble or miscible polymer adapted to precipitate collagen out of solution in the form of tactoids.

Description: PRECIPITATION OF COLLAGEN IN TACTOID WRM This invention relates to collagen products. In a particular aspect this invention relates to collagen products made from soluble collagen. A πeω method by i h i c h soluble col lagen can be formed into quasi- crystal line structures by precipi ation using soluble polymers is described. The use of an aggregate of this quasi- crystal line col lagen to form a variety of collagen materials uihich have improved properties compared with existing collagenous materials is described. Such improved col lagen materials have application in various fields including the manufacture, for example, of products for medical use. Collagen is an extremely common protein in the animal kingdom and therefore many uses for products based upon collagen have developed. Many products use col lagen in either its native form (i.e. the triple helical structure pre-existing in an animal or human body), or regenerated into this form, or after denaturation of the collagen, in the form of gelatine. Native collagen is used for various products such as in the production of leather from animal skins, or such as the production of sausage casings in ωhich the col lagen is finely divided and reformed into the desired structure. There are also many uses of collagen and for items made from col lagen in medical fields such as in artificial arteries, veins, tendons, corneas, heart valves, skin, or patches or the like ωhich are used as replacement parts for disease or injury affected parts in humans, or in cosmetic applications such as mammary prostheses or injectable collagen, or in collagen sponges, sutures or hae ostat materials uihich may be used during surgery or in the treatment of disease (Chvapil, 1979). . any of these medical products made from col lagen are at present unsa isfacto y because of an inability to reproduce the native structure, composition or strength which exists in the norma l collagenous tissue or because of the immune response elicited by the presence of immunogenic col lagen or components or other material foreign to the body. In its native form in the body, collagen exists in many types and in the most common of these types, collagen exists as f ibrils in ωhich individual col lagen molecules are arranged in a staggered overlap structure (Bornstein and Traub, 1979). These fibrils are stabil is ed an d made in s o l ub l e by i n t e r o 1 ec u 1 a r crosslinks betωeen the non-helical portions ( te 1 opept ides ) of adjacent collagen molecules (Bornstein an Traub, 1979). If the collagen from normal, mature tissue is to be made soluble the crosslinks must be broken, for example by digestion with an enzyme such as pepsin. Soluble collagen can be reconstituted in a variety of ordered aggregate forms. Some are fibrous in form, and fibrils in ωhich the col lagen is arranged in its native staggered may can be reformed. The rate of the fibril reforming process is enhanced if collagen with intact telopeptides is used. However, results from the use of injectable soluble collagen have shoωn that the telopeptides lead to an antigβπic response in humans; collagen lacking telopeptides is relatively noπ antigeπic (Linsenma er, 1982) but can still be made to form fibrils. Materials formed by fibril regeneration are often too hydrated and additional methods such as freezedrying or cell-induced contraction must be used to give a functional product. Other non-native fibrous aggregates, termed FLS collagen, can be formed in ωhich the collagen molecules are arranged in various staggered arrangements ωith the orientation of the molecules in both directions. Quasi-crystalline aggregates can also be formed. These include very smal l crystallites of collagen, termed SLS collagen, in ωhich the collagen molecules all have the same orientation, but there is no stagger between molecules. These have been of partial use in deducing the native structure of collagen but SLS collagen has been of little use in the manufacture of larger structures like biomedical products. Also, quasi- crystal line tactoids of col lagen can be prepared, using conditions similar to those used for reconstitu ing fibrils by heat gelation (Leibovich and lilεiss, 1970; Lee and Piez, 1983) but the technique of production is more difficult than the technique described here as. it does not inv o l ve simp l e precipitation. In these structures the collagen is arranged in a staggered form similar to native fibrils. In the present ωork the tactoids are produced by a neω procedure , precipitation by soluble, neutral polymers. Ill hen collagen is precipitated by other procedures, for example salts, alcohols or heat, amorphous precipitates are formed. DESCRIPTION OF THE INVENTION During a search f or more ef f icient methods of isolating soluble collagen it was found that the addition of water soluble polymers to a solution of collagen resulted in an efficient precipitation of the col lagen from solution and the precipitated collagen was found to be much easier to separate from the liquid phase than with precipitates of col lagen formed by the use of salts, alcohol or heat. The polymers had other advantages ωhen compared ωith these previously used precipitants including that they mere non-denaturing and did not require removal prior to c h r o m a o g r a h y or e lee tr ophor esi s . It was an unexpected f inding that the collagen had precipitated in the form of small, needle- like, qua si -cry s ta 11 ine tactoids ωhich mere visible under the light microscope. It was a f urther unexpected disco very that the tactoids could be induced to form into larger assemblages either by allowing the suspension to mature for a period of time or by mechanical action, and that the tactoids or their assemblages could be formed into shapes. Accordingly, the present invention provides a method of producing a col lagen product comprising forming an aqueous solution containing dissolved collagen and a ωater soluble or miscible polymer adapted to precipitate the collagen out of solution in the form of tactoids. The pH of the said solution is preferably 3.5-10 more preferably 5-8 with 7-8 being still more preferred and about 7.5 being most preferred. The collagen precipitate may be left in the form of a paste or slurry and used in this form or after concentration by any one of the methods gravitational precipitation, filtration, cen trif uga t ion or the like. The precipitate may be crosslinked, tanned or stabilised by one or more of chemical, physical or biochemical methods either before or after it has been concentrated. Crosslinking, tanning or stabilisation applied to the precipitate before concentration makes the tactoids resistant to deforming actions such as heating, pressure or biochemical degradation. Crosslinking, tanning or stabilisation applied to the precipitate after concentration causes the structure formed during the concentra ion process to become more stable. The so precipitated collagen may also be formed, for example, into a synthetic body part. Such forming into a synthetic body part may be effected by gravitational precipitation, filtration, centifugation, moulding, pressing, shaping or any other way or combination of ways. Shapes ωhich may be prepared inc lude sheets, tubes, strings and rods. It has been found particularly desirable to form the so precipitated collagen into sheets for use as synthetic dressings for ωounds and into tubes for use as synthetic tubular body parts. The sheets can be formed by centrifugation in a large basket centrifuge or the like or by gravitational precipitation or filtration. Other methods of producing the sheets are also possible. A more compacted sheet is produced by centrifugation in comparison ωith gra vitational recipitation or filtration. Tubes can also be prepared by centrifuga ion or by casting, moulding or shaping. The col lagen may be precipitated onto a suitable substrate to form a composite material. Such a substrate, onto ωhich the collagen is precipitated, may have the form of a particular body part or biomedical product. The substrate may take the form of a matrix. The substrate may take the form of a plastic or other synthetic surface in the form of a sheet, tube or mesh, onto ωhich the collagen is directly deposited forming a collagenous coating. The substrate may also take the form of a composite, for examp le, v arious synthetic l ayers bonded to an artificially or naturally-produced matrix. These col lagen coated substrates may also be chemically modified. For example, g 1 u t a r a 1 d ehy d e or similar chemicals may be used to stabilise the matrix. The collagen of the present invention may be used as a paste or slurry. Such a paste or slurry would have a number of applications including as an implant material such as in the form of an injectabl e medium for use in cosmetic surgery. Such a slurry may be stabilized chemically such as by g 1 u t a r a 1 de h y d e or irradiation. Such as with gamma radiation. The concentration of this tactoidal collagen in the paste or slurry is preferably not less than 10 mgm/ml , more preferably not less than 30 mgm/ml and most preferably not less than 40 mgm/ml. The collagen useful for forming the collagen products of this invention includes col lagen derived from hides, skins or other collagen containing organs or tissues of humans or other vertebrates or invertebrates and includes col lagens of one type or mixtures of types. Soluble col lagen can be prepared by enzymic treatment of col lagen from those sources. Suitable enzymes include pepsin. The collagen may also be derived from the culture medium of cells, tissues or organs grown in cell- or tissue- culture. The culture medium used to produce the collagen may be a culture medium from cell or tissue culture derived from a person for whom a synthetic body part is to be produced; it is believed that doing this wil l substantially reduce the l ikelihood of rejection. Further, it is also possible that a substrate may be introduced into the culture medium such that collagen and other components ωil l be directly produced thereon. Such a substrate may have the form of a particular body part or biomedical product desired. The substrate may take the form of a matrix. The substrate may take the form of a plastic or other synthetic surface in the form of a sheet, tube or mesh, onto ωhich the col lagen and other components are directly deposited forming a collagenous coating. The substrate may be formed from aggregates of tactoidal collagen of this invention. The ωater soluble or miscible polymer is preferably a neutral polymer. Such polymers may be at least one of the synthetic polymers polyvinyl alcohol, polyethylene oxide, po 1 y v iny 1 pyrro 1 idinone , po 1 y a cr y 1 amide , polyethylene glycol, polypropylene glycol, polyvinyl methyl ether, maleic anhydride copolymers and the like; or at least one of the modified, natural, neutral polymers hydroxyethyl starches, methyl cel lulose, h y d r o x y m e t hy 1 cellulose, hydroxyethyl cellulose, hy droxy rop 1 cellulose or the like; or at least one of the natural neutral polymers agarose, dextrins, dextrans , starches , pectins , alginates and the like. Mixtures of such polymers may be used and the molecular weight of the polymer or polymers can vary over a wide range provided the polymer remains soluble or miscible ωith ωater. This l ist of po lymers is not exhaustiv e as the important factor is the use of a water soluble polymer or polymers to precipitate the collagen. Neutral ωater soluble or miscible polymers are preferable but charged, ωater soluble polymers may also be used particularly if they are only mildly charged. The precipitate of collagen is generally found to be improved if it is allowed to stand in said solution. Such standing is preferable for a period of one hour to six months ωith one day to one month being more preferred. Such standing is effected at temperatures between the denaturation temperature of the col lagen and the freezing point of the solution; preferably at between zero and 20°C; more preferably between zero and 10°C. I f d e s i r e d , a d d e d m a t e r i a l s s u c h a s p l asticisers, col ourants, bio l ogica l ly activ e m a t e r i a l s s u c h a s p r o t e o g l y c a n s o r g 1 y c o s a m i n o g 1 c a n s , proteins, other extracel lular products, hormones, growth factors, antibiotics and agents which affect wound healing or have other beneficial effects, ionic strength modifiers such as salts, or solids such as insoluble collagen or the like may be inc l ude d wit h th e s o pr ec ip i t at e d c o l l a ge n a nd incorporated into material made from the collagen. These added materials may also be incorporated into the solution of soluble collagen before addition of the polymer or otherwise incorporated into material made from the col lagen. Charged, water soluble or water miscible polymers may be used as part of a mixture ωith the neutral polymer or polymers and added to the soluble col lagen ωith the neutral polymer solution. These charged polymers may be used to modify the properties of the soluble collagen solution or the material made from the precipitated collagen. The col l agen product of this in v ention may be chemical l y or biochemical l y stabi l ised. Biochemica l stabilisa ion may be effected by enzymes such as lysyl oxidase. Chemical sta ilisation may be effected by tanning agents, syntans, other c ros s - 1 inking agents or chemica l modif iers of col lagen. Of particular interest are stabilisers ωhich limit proteolysis or the immunogenicity of the co l l agen. G 1 u t a r a 1 d eh y d e is a stabiliser of particular interest. The product may also be stabilised by dehydration by mild heat, water miscible solvents, critical point drying or the like. Such stabilisation may be performed before or after a shaping operation. The col lagen product of this invention may be sterilised chemically or by irradiation. Chemical sterilisation may be conducted by means of suitable solutions of sterilising materials such as g 1 uta a 1 dehy de from between 0.5. to 5 . concentration. The product may be stored in solutions of sterilant until required f or use. Steril isation by means of irradiation can be conducted by exposing the col lagen product of this invention to gamma rays from a suitable source. From 0.5 to 5 Clrads of irradiation may be used, preferably 2.5 Clrads of gamma ray irradiation is suitable for satisfactory sterilisation of the product. The tactoids f ormed by precipitation of the soluble col lagen in this invention are useful in production of synthetic body parts, and other materials for medical or veterinary applications. The collagen tactoids or tactold assemblages could be stabilised by chemical or biochemical techniques or could be formed into various useful shapes and then stabilised. The tactoidal collagen has potential application in many areas such as the manufacture of collagen sponges or haemostatic agents , of dressings, of membranes, of skin, of tubes and the like and in the treatment of disease such as peridonta l disease. The tactoida l col lagen can also be used in coπjuction with other structural type materials to form composite materials with different properties. For example, a tube of tactoidal collagen can be covered ωith a woven or knitted mesh of fibre such as Dacron to give the tube additional strength. Alternatively, the tactoidal collagen can be formed into a tube surrounding the mesh to give a more intimate contact with the mesh and better properties. To better "utilise the properties of the tactoidal collagen in the formation of artificial body parts it is possible to arrange the tactoids in a preferred orientation by the application of an electric field o'r by means of mechanical action. materials made from the oriented tactoids may have beneficial effects in the healing of wounds. Claπy other methods of utilising the " tactoidal col lagen in a variety of shapes and forms and in conjuction with diverse other materials can be envisaged. The product of this invention also has application in areas outside medical and veterinary products including plastics, fabric, leather or as composites or the like. The present invention a lso includes such collagen products and articles produced therefrom. The col lagen products of this inv ention ha v e advantages over presently available products. These include, low i munogenicity, ease of preparation, high collagen content, and strength. The following examples illustrate the invention. EXAMPLE 1 Type I collagen was solubilised and extracted from foeta l ca l fskin by pepsin digestion and purified by fractional salt precipi ation according to the method of Trelstad et al.(1967). This purified collagen was dissol ved in 200 m_ Tris-HCl buffer pH 7.5 at 4°C and at a concentration of 10 mg/ml. Polyethylene glycol (PEG) 4000 was than added to produce a final concentration of 2.5{ (ω/v). A precipitate of tactoidal collagen formed ωhich settled to the bottom of the container after standing at 4°C for a feω hours or could be concentrated by filtration or cen rifuga ion. EXAMPLE 2 As for Example 1 except that the concentration of the collagen ωas 1 mg/ml. EXAMPLE 3 As for Examp l e 2 except that PEG 400 to a fina l concentration of 3.5 . (ω/v) ωas used to precipitate the collagen. EXAMPLE 4 Type III col lagen, solubilised and extracted as in Example 1 , ωas dissol ved at a concentration of 1 mg/ml in 200mM Tris- HC1 buffer pH7.6 at 4°C. PEG 400 was added to the solution to a final concentration of 4.0 . (w/v) and the ' precipitate of tactoidal collagen formed. EXAMPLE 5 As for Example 4 except that a final concentration of 2.5. (ω/v ) PEG 4000 was used. EXAMPLE 6 Typ e I I co l l agen was iso l ated by the me tho d o f Tre l stad et a l . ( 1976 ) f rom bo v in e articu l ar cartilage by pepsin so 1 ub i 1 is a t io π and fractional salt precipitation. The purified type II collagen ωas dissol ved in 200 mM Tris- HC1 buffer at pH 7.6 at 4°C and at a concentration of 1 mg/ml. PEG 400 ωas then added to produce a f inal concentration of 3.0 . (ω/v). The precipitate of tactoidal col lagen formed as in Exampl es above . EXAMPLE 7 As for Example 6 except that PEG 4000 was added to a final concentration of 2.0. (ω/v). EXAMPLE 8 As f or Exampl e 1 except that PEG 1000 to a f ina l c oncent ratio n of 5. (ω/v ) ωa s us ed to precipitate the collagen. EXAMPLE 9 As for Examp le 1 except that PEG 10000 to a f ina l concentr ati on o f 5 % (ω/ v ) ωas us ed to precipitate the col lagen. EXAMPLE 10 The suspension of tactoidal collagen from Example 1 ωas stored at 4°C for 4 weeks and co l l ected on Idh a tma n No. 1 filter paper in a 125 mm diameter basket centrifuge rotating at 4000 rp . The resulting collagen sheet was removed from the centrifuge and separated from the filter paper. The collagen sheet ωas found to have properties similar to those of a thick, ωet paper tissue and to be suitable for assisting in the healing of open skin ωounds. EXAMPLE 11 The collagen sheet, prepared as in Example 10, ωas tanned using a solution of 0.01. g 1 ut a ra 1 de h yd e for 18 hours " . After drying the sheet was found to have a tensile strength of 6.2N/sq cm and an elongation of 12. at a moisture content of 15JC. EXAMPLE 12 The collagen sheet, prepared as in Example 10 was sealed in a polyethylene bag and subjected to 2.5Mrads of gamma ray irradiation. The sheet was found to have been sterilised and to have improved tensile properties over those of the sheet in Example 10. EXAMPLE 13 As for Example 2 except that the buffer was at pH5. EXAMPLE 14 As for Example 1 except that the collagen extracted from foetal calfskin was not purified by fraction salt precipitation but was used as a crude extract and that 5. PEG 4000 was used. EXAMPLE 15 As for Example 14 except that 5. polyvinyl alcohol was used . EXAMPLE 16 As for Example 14 except that 5. dextran of 10,000 average molecular weight ωas used. EXAMPLE 17 As for Example 14 except that 5 % dextran of 40,000 average molecular weight ωas used. EXAMPLE 18 A collagen sheet prepared as in Example 10 was rolled into a tube and then stabilized by tanning using a solution of 0.01!. g 1 utara 1 dehyde for 18 hours. EXAMPLE 19 A col lagen sheet prepared as in Example 10 was dried by critical point drying using liquid carbon dioxide. BIBLIOGRAPHY Ch v api l , M . ( 1979 ) - I n "Fibrous P roteins , Scientif ic, Industrial and Medical As ects", Vol. 1 (Eds Parry, D.A.D. and Creamer L.K.) Academic Press, London pp 247-269. Bornst ein , P. and Traub , li) . ( 1979 ) I n "The Pr otein s" V o l 4 (Eds N eura th , H. and Hi l l , R.L.) Acade~mic Press, New York pp411 -632. Linsenm eyer, T.F. (1982) In "Col lagen in Health and Disease" (Eds tileiss, J.B. and Jayson, M.I.V.) Churchil l Livingston, Edinburgh pp244-268. Leibo v ich. S.J. and 111 e i s s , J.B. ( 1970 ) Biochim. Biophys. Acta 214:445-465. Electron microscope studies of the effects of endo- and e x o - e p t i d a s e digestion on tropocollagen. Lee, S.L. and Piez, K.A. (1983) Col lagen Rel. Res. 3:98-103. Type II collagen from Lathyritic rat ch ondr osarcoma : preparation and in vitro fibril formation. Trelstad-, R.L., Catanese, V.M. and Rubin, D.F. (1976) Ana l . Biochem. 71 :114-118. Col l agen f ractioπation : Separation of native types I, II and III by differentia l precipitation. Modifications and adaptations may be made to the above described without departing from the spirit and scope " of this in vention which includes every no vel feature and combination of features disclosed herein.

Claims: The claims defining the invention are as fol lows: 1. Col lagen in tactoid form obtained by forming an aqueous solution containing dissolved collagen and a water soluble or miscible polymer adapted to precipitate collagen out of solution in the form of tactoids. 2. A method of producing a col lagen product comprising forming an aqueous solution containing dissolved col lagen and a wa ter so l ub l e or miscib l e po l ymer adapted to precipitate the col l agen out of solution in the f orm of tactoids. 3. A method of producing a collagen product as claimed in claim 2, wherein the pH of said solution is 3.5 - 10. 4. A method of producing a collagen product as claimed in claim 2, wherein the pH of said solution is 7 - 8. 5. A method of producing a collagen product as claimed in any one of claims 2 - 4, including forming the thus formed precipitate to a shape. 6. A method of producing a collagen product as claimed in any one of claims 2 - 5, including precipitating the collagen onto a pre-shaped substrate. 7. A method of producing a collagen product as claimed in claim 6, wherein the substrate has the form of a body part. 8. A method of producing a collagen product as claimed in claim 6, ωherein the substrate is itself formed of collagen in the form of tactoids. 9. A method of producing a collagen product as claimed in claim 5, ωherein prior to forming said precipitate to a shape the precipitate is permitted to stand in said solution for a period of greater than 1 hour. 10. A method of producing a collagen product as claimed in claim 9, ωherein the temperature of standing is from 0 - 20°C. 11. A method of producing a collagen product as claimed in any one of c l aims 2 - 10 , and inc l udin g the st ep of chemica l ly or biochemical ly stabilizing the col lagen so formed. 12. Α method of producing a collagen product as claimed in any one of claims 2 - 11 , wherein the dissolved collagen is derived from cell or tissue culturing. 13. A method of producing a collagen product as claimed in any one of claims 2 - 12, ωherein said ωater soluble or miscible polymer is selected from polyvinyl alcohol , polyethylene oxide, po1yv iny 1 yrro 1 idinone, po1 yaery 1 amide, polyethylene glycol, polypropylene glycol, polyvinyl methyl ether, maleic anhydride copolymers and the like. 14. A method of producing a collagen product as claimed in any one of claims 2 - 12, wherein said water soluble or miscible polymer is selected from hydroxyethyl starches, methyl cellulose, h dr o x y me thy 1 cellulose, hydroxyethyl cellulose, hydroxypropy 1 cellulose or the like. 15. A method of producing a collagen product as claimed in any one of claims 2 - 12, wherein said water soluble or miscible polymer is selected from agarose, dextrins, dextrans, starches, pectins, alginates and the like. 16. Collagen as claimed in claim 1 and in admixture with a biologically active material. 17. Col lagen as claimed in claim 1 and in the form of a synthetic body part. 18. Collagen as claimed in claim 1 and precipitated onto a shaped substrate. 19. Col lagen as claimed in claim 17 and in the form of a sheet or tube. 20. Col lagen as claimed in claim 1 and in the form of a slurry or paste. 21. Collagen as claimed in claim 20 and containing at least 10 mgm/ml of collagen. 22. A method of producing a collagen product substantially as hereinbefore described with reference to any one of the Examples . 23. Collagen in tactoid form substantially as hereinbefore described with reference to any one of the Examples. 24. The articles, things, parts, elements, steps, features, methods, processes, compounds and compositions referred to or indicated in the specification and/or claims of the applfcation individually or collectively, and any and all combinations of any two or more of such.

Inventors: BATEMAN JOHN FRANCIS, PETERS DAVID EDWARD, RAMSHAW JOHN ALAN MAURICE, TULLOCH PETER ARCHIBALD, BATEMAN, JOHN, FRANCIS, PETERS, DAVID, EDWARD, RAMSHAW, JOHN, ALAN, MAURICE, TULLOCH, PETER, ARCHIBALD
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1987
Main classification: A61L27
IPC codes: A61F2, A61L15, A61L26, A61L27, C08H1, C08L89