CA1340222C - Preparations of placenta collagen, their extraction method and their applications - Google Patents
Preparations of placenta collagen, their extraction method and their applicationsInfo
- Publication number
- CA1340222C CA1340222C CA 517280 CA517280A CA1340222C CA 1340222 C CA1340222 C CA 1340222C CA 517280 CA517280 CA 517280 CA 517280 A CA517280 A CA 517280A CA 1340222 C CA1340222 C CA 1340222C
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- Prior art keywords
- collagen
- solution
- fibres
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- acid
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
- C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
Abstract
Human or animal placenta is subjected to moderate enzymatic digestion, in particular with pepsin at an acid pH, then, after separation of the residual tissue the impurities are separated at a moderately acid pH, the collagen is precipitated at a neutral pH, redissolved and theresidual impurities precipitated at an acid pH. The collagen obtained can be brought back to a neutral pH and dried in the form of fibres.
Completely transparent, physiological and hemocompatible gels and solutions can be prepared. Applications for the fabrication of contact lenses and implants.
Completely transparent, physiological and hemocompatible gels and solutions can be prepared. Applications for the fabrication of contact lenses and implants.
Description
1340~22 .
NEW PREPARATIONS OF PLACENTA COLLAGEN, THEIR EXTRACTION METHOD AND THEIR
APPLICATIONS
This invention relates to the preparation of a gel or a physio-logical solution from unmodified, transparent and hemocompatible collagen derived from placenta.
In general it covers a method for the extraction of collagen from human and animal placenta enabling type IV enricned collagen to be isolated and used industrially.
It relates more precisely to a method for the preparation of acid or neutral collagen solutions or gels that are completely transpa-rent at a great ~hickness.
Attempts have been made for a number of years to develop trans-parent collagen gels for the fabricaticn of contact lenses. More recently their utilization has been sought for grafting to or inserting these pro-ducts in the cornea itself, and even in the eye. The importance of having a collagen identical with human collagen or as near to it as possible for surgical use in the human species will be understood if any infiamma-tory or im.nunological reaction is tc be avoided. But today the only colla-gen preparations available in commerce in sufficient quantity and at an acceptable price are those obtained from rat tails or tendons or fromthe skins of yourlg animals (c~lf, for example). They consist almost solely of type I collagen. The other types, II, III, IV, V and others are prepared only by specialized research workers at prices that are too high to allow their being use(l on a large scale.
It has been stated in numerous publications that animal collag-en molecules are not very antigenic or not antigenic when they are obtained by a method using pepsin which removes the non-helical terminal peptides (teloptides). The tripie helix structure of collagen, resistant to the action of pepsin, being similar in all animal species is said to be not very antigenic. But, it seems that this non-antigenic character of collagen that has been subjected to the action of pepsin must be involved. For it is now known that these structures are antigenic in an other species, which has enabled specific antisera to be prepared from a particular or other type of bovine or human collagen, in the rabbit for example. In 13l~0222 addition, account must be taken of the always possible presence of anti-genic animal protein impurities in such preparations.
Since some years ago the injection of collagen I of bovine origin has been practiced intensively in the human species to close up scars or in repair surgery techniques. These tests revealed a number of disadvantages of which two are important :
- the occurrence of immunological reactions in 1 to 3 % of subjects, - the oe~ ~cl~ce of local blood coagulation reactions when the product has unfortunately been injected into a blood vessel or capillary.
These facts therefore underline how very important it would be to obtain hurnan collagen gels whicn were not only transparent for use in ophthalmology in t,he human species without risk of any occurrence of undesirable immunological reactions but also hemocompatible and physiolo-gical.
Type I, II or III human cr animal collagens, even after the action of pepsin, have a characteristic fibrillar structure under an elec-tron microscope, which make them powerful platelet aggregating agents.
But as these collagens are not hemocompatible, it is preferable to avoid using them whenever the physician concerned does not require a contact coagulant effect.
Since these collagens are practically insoluble at a neutral pH, only opaque suspensions can be obtained and it is impossible to use them for the preparation of transparent, physiological gels (isotonic at a neutral pH), without chemically modifyir.g the collagen molecules 25 resulting in the appearance of antigenic determinants. US Patent 3,553,299, THIELE, has already described the fabrication of contact lerlses with a gel obtained from the crystallline lens of the eye, and consisting there-fore ~ssentially of collagen. US Patents 4,223,984 and 4,268,131, MIYAYA, also deal with the fabrication of contact lenses from a type I collagen gel which, to be soluble at a neutral pH, must be modified chemically.
The methods described in these patents give relatively trans-parent gels of only s,nall thicknesses that are not very strong mechani-cally. It is not clear when reading them whether the transparency is ob-tained because of a subsequent chemical conversion (succinylation or methy-lation).
US Patent 4,388,428, KUZMA, describes gels in which the col-3 13~0222 lagen is mixed with chemical polymers to improve their chemical properties.The use of such products is not desirable because of the difficulties in removing all traces of toxic products from viscous or solid gels.
R.R. BRUNS and J. GROSS (US Patent 4,505,855) confirm the difficulty of obtaining completely transparent gels from animal collagen by prior art methods. They suggest an ultra-centrifuging method, but this metnod is very costly and difficult to reconci e with industrial usage.
In addition, consideration has already been given to enzymatic extractions of placenta collagen with pepsin, but the collagen preparations obtained do not have the transparency properties required for use in the lhe field of ophthalmology.
Thus M.Z. ABEDIN and R. RIEMSCHNEIDER (Die Angewandte Makro-molekulare Chemie, Vol. 11, N~ 1701, January 1983, pages 107-122, Huthig & Wepf Verlag, Heidelberg) describe a method for the digestion of placenta witn pepsin which results in the contamination of type IV collagen by types I and III, requiring precipitation and redissolution at a high NaCl concentration indicating the intensive character of the digestion.
These same authors (Die Ar.gewandte Makromolekulare Chemie, Vol. 82, N~ 1276, 1979, pages 171-186)describe a method for a long-duration treatment of placenta with ether, followed by freeze-drying and then treat-ment with sodium acetate or caustic soda before digestion with ?epsin and then dialysis of the supernatant layer in order to precipitate col-lagen. The collagen, after having been taken up, must also be precipitated at a high NaCl concentration. These ccnditions, which moreover preclude application on an industrial scale, also bear witness to the excessively intensive character of the digestion and the non-transparent product ob-tained appears to be especially type I + III collagen.
Th. F. KRESINA and Ed. J. MILLER (isolation and characteriza-tion of basement membrane collagen from human placental tlssue. Evidence for the presence of two genetically distinct col agen chains. Biochemistry, Vol. 18, N~ 14, 1979, pages 3089-3097, American Chemical Society) alsodescribe a total pepsin digestion of placental tissue resulting in heavy contamination by type I and III collagens. After fractionation, the colla-gen must be treated at an acid pH at high NaCl concentrations. The product obtained is not suitable for ophthamological uses. b¦ish~ ~ay 6~
.~ German Patent Application 2,462,2221~describes a pepsin treat-.
i - 13~022~
ment consisting of successive extractions of the supernatant layer of placental tissues giving a cloudy solution of collagen.
European Patent Application 0,080,956 published June 8, 1983 describes a pepsin digestion of alkaline extracts of placenta giving an opaque, denatured collagen.
The aim of this invention is to overcome these various disadvantages by providing unmodified, non-antigenic, completely transparent, physiological and hemocompatible preparations of collagen, free from striated fibres observable under an electron microscope, it being possible to provide these preparations in the form of gel or solution.
The invention provides a placenta collagen preparation obtained from human or animal collagen, enriched with, or consisting essentially of, type IV collagen, characterized in that the collagen is non-denatured, unmodified, completely transparent, physiologically acceptable, and hemocompatible and free of striated fibres visible under an electron microscope, soluble in both acid and physiological solutions, said preparation giving, in a physiological solution, either a solution up to a collagen concentration at most equal to 2% or a gel with a collagen concentration of between 2% and 30%.
The invention also provides a transparent collagen gel obtained from human or animal collagen enriched with, or consisting essentially of, type IV collagen, characterized in that it comprises between 2 to 30% of collagen which is non-.~
Ei - 13~022~
4a denatured, unmodified, completely transparent having transparency, at a thickness of between 0.1 and 20 mm, comparable to that of glass or an aqueous solution of the same _ ~ 13~222 thlckness, free from strlated flbres vlslble under an electron mlcroscope and soluble ln both acld solutlons and physlologlcal sallne solutlons.
The lnventlon further provldes a transparent collagen solutlon obtalned from human or anlmal collagen enriched wlth, or conslstlng essentially of, type IV collagen, characterized in that it comprises not more than 2% of collagen which is non-denatured, unmodlfled, completely transparent, free from strlated flbres vlslble under an electron mlcroscope and havlng an acld or neutral pH and whlch, at a neutral pH, can be rendered lsotonlc, physlologlcally acceptable and hemocompatlble.
The collagen solutlons or gels obtalned are transparent ln great thlcknesses, capable easlly of attalnlng 20 mm, a thlckness whlch ls largely sufflclent to meet the needs of the market (contact lenses, lntra-ocular or corneal lmplants).
An extractlon method has been developed by the appllcant startlng from human placenta, the only source that can be consldered for the preparatlon of human collagen on an lndustrlal scale.
The knowledge of thls new method suitable for human placenta has enabled the applicant to extend lt to animal placentas. For anlmal collagens can be useful ln veterlnary medlclne, for example, or acceptable for use as a non-lmplanted external devlce ln man such as contact lenses.
.
llD '' 5a The method accordlng to the lnvention conslsts therefore:
- flrst, ln extracting collagens from human and animal placenta and then lsolatlng a type IV enrlched collagen;
- secondly, ln preparlng solutlons or gels from thls type IV enrlched collagen having the previously descrlbed propertles.
The method for the extractlon of placenta collagens according to the invention is characterized in that lt comprises the following steps:
- startlng wlth human or animal placenta ln deep-frozen form and grinding lt;
- washing the ground placental tlssue, for example, by successlve washlngs at a neutral pH and then at an acid pH;
- sub~ectlng sald placental tlssue to enzymatlc dlgestion in order to extract type IV collagen, practlcally uncontamlnated by type I or III collagens. Dlgestlon ls carrled out wlth a sultable enzyme (wlth the exceptlon of collagenase), preferably pepsin. In that case, digestlon is preferentlally carrled out at an acld pH, preferably between 2 and 3.5 at a temperature preferably of between 0 and 20~C for a sultable time, ln partlcular of the order of 8 to 24 h, the quantlty of pepsln per kg of deep-frozen placenta belng between 0.5 and 2 g, preferably between 0.5 and 1 g or 1.5 g, that ls between 0.5 and 1 or 1.5 per mll.
.~
13~û222 5b In particular the invention provides a method for the extraction of placenta collagen in which the placenta is digested enzymatically, comprising grinding and washing a deep-frozen form of human or animal placenta;
subjecting the washed placental tissue to a first enzymatic digestion to obtain a type IV collagen substantially uncontaminated by type I or III collagen wherein the enzymatic digestion is effected with pepsin at a pH of between 2 and 3.6, at a temperature between 0~C and 20~C for a duration of between 8 and 24 h, wherein the pepsin content with respect to the deep-frozen placenta is between 0.5 and 1.5 per mil and, after separation of the residual tissue from the suspension derived from the enzymatic digestion;
effecting precipitation at a moderately acid pH to remove impurities diluting said suspension with water;
adjusting the pH to between 7 and 10;
maintaining said suspension for 1 to 24 h at a temperature of between 0 and 20~C;
separating the residual tissue from a supernatant layer of the first digestion, said residual tissue containing non-solubilized type I and type III collagens whereas the supernatant contains type IV collagen substantially free from type I and type III collagens;
adjusting the pH of the supernatant layer to a pH of between 4.5 and 5.5;
;
$~
I .~ .~ ~, , .
13~0222 5c maintalnlng sald supernatant layer for 1 to 24 h at a temperature of between 0 an 20~C; and removlng the lnsoluble preclpltate;
collectlng the supernatant layer and preclpltatlng the collagen contalned ln the supernatant layer wlth a salt at a neutral pH;
redlssolvlng the preclpltated collagen ln a weak acld solutlon whlch ls neutrallzed;
removlng lnsoluble lmpurltles; and preclpltatlng the collagen by acld preclpltatlon wlth a salt.
The tlssue resldue havlng reslsted the flrst treatment may lf necessary be sub~ected to a second dlgestlon wlth pepsln, thls second dlgestlon belng carrled out under the same condltlons as the flrst dlgestlon, enabllng type I and type III collagens practically free from type IV collagen, to be recovered subsequently by known methods.
The placenta suspenslon havlng undergone the flrst enzymatlc dlgestlon ls then treated by the followlng method, conslstlng successlvely:
- after separatlng the resldual tlssue from the placenta suspenslon derlved from the enzymatlc dlgestlon, - ln preclpltatlng at a moderately acld pH to remove a preclpltate of lmpurltles, - preclpltatlng the collagen from the supernatant layer by preclpltatlon wlth a salt at a neutral pH, r 13~0 22~
5d - redlssolvlng the preclpltated collagen and neutrallzlng the solutlon obtalned, - removlng the lnsoluble lmpurltles, and - preclpltatlng the collagen wlth a salt at an acld pH.
Thus the placenta suspenslon derlved from the enzymatlc dlgestlon ls advantageously sub~ected to the followlng steps:
- dllutlng sald suspenslon, ln partlcular by a volume of water;
- ad~ustlng the pH to a value between 7 and 9 or 10;
- allowlng sald suspenslon to rest for 1 to 24 hours at a temperature of between 0 and 20~C;
- separatlng the resldual tlssue from the supernatant layer of the flrst dlgestlon;
- ad~ustlng the supernatant layer to a moderate pH, for example, LD' I _ _ 6 of between 4.5 and 5.5 ;
- allowing said supernatant layer to rest, for example, for 1 to 24 h at a temperature between 0 and 20~C ;
- removing the insoluble part containing numerous impurities ;
- adjusting the clear supernatant layer to an about neutral pH of the order of 7.5 ;
- adding a salt, such as for example NaCl, until a final concentration of about 1.2 M is obtained :
- allowing the suspension obtained to rest, for example, for 1 to 24 h at a temperature between 10 and 25~C ;
- recovering the collagen precipitate formed ;
- dissolving the precipitate in a weakly acid solution ;
- neutralizing the solution to a pH of 7.5 ;
- allowing the suspension obtained to rest for 1 to 24 hours at a temperature between 0 and 20~C ;
- removing the insoluble part, thereby also removing the impurities that might affect transparency adversely:
- acidifying the supernatant layer to an acid pH of, for example, between 2 and 3.5 or 4 ;
- precipitating the collagen with a salt, such as NaCl, until a final cor.centration of between 0.4 and 0.8 M is obtained ;
- allowing the suspension obtained to rest for 1 to 24 h at a tempe-rature of between 0 and 25~C ;
- recovering the precipitate of purified a~id collagen.
The purified collagen precipitate obtained on completion of the op-ration is taken up with acetone or an equivalent volatile organic solvent, which results in the formation of acid collagen fibres that are recovered by filtration through a sieve. These fibres are dried under a current of lukewarm, sterile air.
By fibres are here understood to mean the appearance the solid collagen assumes after drying, and which in no way corresponds to thc striated fibrous structure as such observable under an electron microscope with known collagens in solution.
In a variant of the embodiment of the invention, the purified collagen precipitate is redissolved in water. The pH of the solution is 1~40222 adjusted to 7.5 and a salt, such as NaCl, added to it until a final concen-tration of the order of 1.2 M is obtained. The suspension obtained is allowed to rest for 1 to 24 h and the neutral collagen precipitate reco-vered.
The precipitate of neutral collagen fibres obtained is taken up with acetone or an equivalent volatile organic solvent, which results in the formation cf neutral collagen f'ibres which are recovered by filtra-tion through a sieve. These fibres are dried under a current of lukewarm, sterile air.
It is these acid or neutral collagen fibres in their dry state (< 10 %) which are used as a source of coliagen for the preparation of solutions or gels according to the invention.
Gels from acid or neutral collagen fibres obtained after treat-ment with acetone can be prepared as follows :
- the fibres are brought into contact with a volume of water determined so as to obtain a collagen con_entration of be-tween 2 ard 30 % ;
- the fibres are allowed to swell in the water, for example, for 1 h ;
- the whole is subjected to moderate heating ;
- the viscous, transparent solution is filtered thrGugh mem-branes of pore size between 0.2 and 8 ~ ;
- the solution is allowed to cool.
A solution of collagen from type IV enriched acid collagen fibres is prepared by the following method :
- the collagen fibres are brought into contact with a deter-mined volume of water ;
- the collagen fibres are allowed to swell in the water, for example, for about 1 h ;
- a solutioh of a salt, such as for example, sodium acetate at a concentration of less than or equal to 0.1 M is added to initiate neutralization.
The volumes of water are determined so as to obtain collagen concentration of at the most 2 %.
- the solution is adjusted to a pH of about 7.5 and if neces-_ 8 13~0222 sary made isotonic by addition of NaCl ;
- the solution is subjected to moderate heating at a tempera-ture of between 30 and 80~C for 10 to 60 minutes. As a va-riant, the collagen solution may be filtered in the acid state through membranes of pore size between 0.2 and 8 ~.
Neutralization takes place only after this, if necessary under bacterially sterile conditions, to ensure that the product prepared is sterile, while facilitating the filtra-tion operation as a collagen solution is less viscous at an acid pH. Another variant consists in filtering the solu-tion while subjecting it to ultrasonic vibrations to reduce the viscosity temporarily.
- the solution is filtered through membranes of pore size between 0.2 and 8 ~ ;
- and the solution allowed to cool.
On completion of these operations a solution is obtained which has these characteristics :
- it is very viscous, - it contains at the most 2 % of non-denatured, collagen, 20 - it is completely transparent, - it is isotonic, physiological and hemocompatible, - it is free from striated fibres visible under an electron microscope.
Such a solution may be used as an implant in human and animal medicine, in the fields of ophthalmology, bone joints, treatment of burns and dermatology.
When acid collagen fibres are no longer used as a source of collagen but neutral type IV enriched neutral collagen obtained according to a variant of the embodiment of the invention, the same successive opera-tiGns as those previously described are performed to obtain solutions from collagen fibres, neutralization no longer being necessary.
To prepare a collagen solution from type IV enriched neutral collagen fibres, the following procedure is followed :
- the neutral collagen fibres are dissolved in a volume of water determined so as to obtain a collagen concentration of at the most ~ - 9 - 13~0222 2 % ;
- to this solution is added a salt such as NaCl until a concentration of the order of 0.15 M is obtained ;
- said solution is subjected to moderate heating at a temperature of between 30 and 80~C for 10 to 60 minutes, preferably at a tempe-rature of about 60~C for some ten minutes ;
- the solution is filtered through membranes of pore size between 0.2 and 8 ~ ;
- and the solution allowed to cool.
A solution lS obtained which has the same ~haracteristics as the collagen solutions prepared from acid collagen.
Other advantages and features of the invention will become apparent on reading the non-limitative examples of embodiment of the inven-tlon given hereinafter.
Example 1 illustrates the r-~ethod for the extraction of type IV enriched collagen from human placentas and the preparation of acid collagen fibres.
Example 2 relates to tne preparation of neutral collagen f'ibres according to the invention.
Example 3 illustrates the method for the extraction of type IV enriched collagen from animal placentas.
Examples 4 and 5 describe the preparation of 15 % collagen gels.
Examples 6, 7,8 and 9 describethe preparation of collagen solu tions~
Example 1 300 kg of deep-frozen placenta were ground to give lumps of a few cm3. The ground material was then mixed with 300 l of an aqueous solution containing 8 % ethanol, 6 g/l of NaCl and 10 kg of cellulose.
After stirring at 10~C, the whole was pressed in a MABILLE press to sepa-rate the blood from the placental tissue. 102 kg of placental tissue con-taining 65 % water were obtained.
The tissue extracted from the press was stirred in 500 l of 0.05 M sodium citrate at a pH of 7.2 for 30 minutes at 10~C and then pres-13~0222 sed to remove the washing solution and recover the tissue. A second washing operation was performed with 500 l of 0.05 M sodium citrate at a pH of 7.2 with addition of 30 g/l of NaCl. A third washing operation was per-formed with 500 l of 0.05 M of sodium citrate at a pH of 7.2. The tissue washed in this way at a neutral pH was then subjected to three succes-sive washing sequences at an acid pH at 10~C :
- with 500 l of 0.05 M citric acid at a final pH adjusted to 2.8 by addition of 2 N HCl ; stirring for 30 minutes before the pressing operation ;
- with 500 l of 0.5 M formic acid for 15 h ;
- with 500 l of 0.05 M citric acid for 30 mn, with addition of 20 g/l.
The placental tissue washed in this way has a white appea-rance which is evidence of a good removal of the red pigments from the initial placental blood. The weight obtained was 82 kg.
It was then subjected to enzymatic digestion with pepsin in 500 l of 0.05 M citric acid at a pH of 2.8 containing 300 g of pepsin for 15 h at 10~C. The suspension was then diluted by addition of 500 l of water at 10~C. The pH was adjusted to 7.5 by addition of 4 N NaOH in order to denature the pepsin and suppress its protease action. After a waiting period of 15 h at 10~C, the tissue residue which contained the essential Gf the non-solubilized collagens I and III was separated conti-nuously by means of a centrifuge (Westfalia KG 10006). The weight of this residue was 103 kg ; it could be subjected to a second enzymatic digestion identical with the first one, followed by extraction and separation of each of the collagens I and III by the methods described in the literature.
The supernatant layer corresponding to the first enzymatic digestion contains the essential of the collagens and other macromolecules soluble at a neutral pH after action of the pepsin. In particular it con-tains type IV collagen.
The pH of the supernatant layer was adjusted to 5 with 2N
HCl and, after a waiting period of 15 h at 10~C, the precipitate formed removed by continuous centrifuging in an Alfa Laval "Bactofuge" centrifuge.
The pH of the supernatant layer, which was very clear, was adjusted to 7.5 by addition of 4 N NaOH and at a final concentration of 1.2 M in NaCl. After 15 h at 16~C, the collagen precipitate formed was _ 134~222 recovered by continuous centrifuging in a "Bactofuge" centrifuge.
The precipitate of 13 kg was then dissolved in 600 l of 0.01 N
HCl and the pH adjusted to 7.5 by addition of 4 N NaOH. The precipitate formed after a waiting period of 15 h at 4~C was removed in the "Bactofuge"
centrifuge (weight obtained 10 kg).
The clear supernatant layer was acidifed to a pH of 2.8 with 2 N HCl until a final concentration of 0.6 M was obtained at 4~C.
After 15 h, the collagen precipitate was collected in a "Bacto-fuge" centrifuge. The precipitate whose weight was 6.5 k r had a fluidaspect. 7 l of acetone were gradually added to it, which resulted in the formation of collagen fibres that were recovered by filtration through a sieve. ~Jashing these fibres Dy several treatments with acetone gave, after drying under a lukewarm, sterile current of air, 180 g of dry fibres of final product. The analysis of tne composition of the amino acids o these fibres showed a structure similar to that of type IV collagen and characteristic of the collagen family (~ 30 % gly-clne, about 10 % glutamic acid, proline and hydroxyproline).
The following Table snows the resu_ts of the analysis of the composition of the amino acids of four different preparations of human placenta collagen obtained by application of the method given in this example (the results are expressed in numbers of molecules of each amino acid per 1000 residues).
- __ 13~0222 ¦ ~tch N~ ¦ 1022 ¦ 1023 ¦ 1025 ¦ 1027 ~SP I ~6 1 42 1 41 1 ~2 I ilYP I ~30 1 91 1 ~35 1 86 ¦ T13R ¦ 19 ¦ 18 ¦ 18 ¦ 1', S~ 17 1 15 1 17 1 lf l~
¦ ~SN ¦ 28 ¦ 30 ¦ 31 ¦ 25 __ I _.. _ ___. I ._...... I ._ . . . I __ _ _ I
¦ GLU ¦ 10-7 ¦ 105 ¦ 105 ¦ 1() 7 l__ ~ l l l _. _ _l ¦ Gr-i~ ¦ 3 ¦ trClccs ¦ 1 ¦ trc~ s ¦
I Pr~O I92 1 91 1 9f~ 1 98 ¦ GLY ¦317 ¦ 327 313 ¦ 324 __ _ l__ J~ I ~1 139 1 39 1 ~13 ¦V.~L ¦ 33 ¦29 ¦ 3~1 ¦ 34 _ ICYS I9 1 6 1 9 1 (~ I .
l_ l ¦ CYST~ ¦ 1 ¦ 1 ¦ 1 ¦ 1 ¦
~ r,'r Il 5 1 26 1 16 1 l f:~
l_ l IL,~ I3f~ 1 36 1 37 1 3f I_ 1 1~.
¦~ EU ¦57 ¦ 56 ¦ 57 ¦ 5~ l _ _ _ _ l _ _ _ _ __ l ¦TYR ¦ ln ¦ 7 ¦ 12 ¦ 1(' __ l l ~ _ 13 13~0 222 BatCh N~ ¦ 1()22 ¦1()23 ¦1U25 ¦1()2 7 P~IE I 33 I 31 I 39 I 34 ¦ ORN ¦ 3 ¦LYS I 9 I 8 I 12 I 12 ¦IRY ¦ 0 ¦ 0 ¦ 0 ¦ () I
¦'rOtdl ¦ 999 ¦~96 1()()0 ¦] O()U
Example 2 The method described in Example 1 was applied in a strictly identical manner to a second batch of 300 kg of human placentas.
On completion of this operation, the weight of the precipitate obtained at a pH of 2.8 in the presence of 0.6 M NaCl at 4~C was 6.5 kg.
This precipitate was redissolved in 200 l of water. The pH of the solution obtained was adjusted to 7.5 by addition of 4 N NaOH at +16~C and NaCl added to obtain a final concentration of 1.2 M.
The precipitate formed after 15 h was recovered by centrifuging in a "Bactofuge". The weight obtained was 3450 g. The precipitate, dried with acetone as in Example 1, gave 200 g of dry neutral collagen fibres, enriched essentially with type IV collagen.
Example 3 The method described in Example 1 was applied in a strictly identical manner to 180 kg of cow placentas. Successive washings were carried out with volumes of 300 litres. The first digestion with pepsin was carried out by addition of 180 g of pepsin in 300 l of 0.05 M citric -- 14 13~222 acid adjusted to a pH of 2.8. The other selective collagen precipitation steps were identical with those of Example 1.
After final drying with acetone, 130 g of dry bovine collagen fibres were recovered. An analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) witl1 or without reductant (dithiothreitol) demonstrated that this preparation was rich in type IV
collagen.
Dissolution of these bovine collagen fibres in water gave an acid solution due to the final precipitation being made to take place at an acid pH.
Example 4 1.5 g of dry, human collagen fibres prepared according to Example 1 were brought into contact with 10 ml of distilled water. After the fibres had swelled for 1 h, the whole was placed in a water-bath at 70~C for 30 minutes. A very viscous, transparent solution was obtained which could be filtered easily through membranes of pore size between 0.8 and 8 ~ at this same temperature. After cooling, the gel obtained was colourless, acid and completely transparent, like filtered water.
Checks by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with or without dithiothreitol demonstrated the complete preservation of the collagen IV molecules during the heating stage.
This gel could then~be subjected to known operations of shaping into contact lens forms or any other device with optical properties. Moul-ding and cross-linking methods by chemical agents (aldehydes) or physical agents (y,~, X-ray or UV radiation) can be applied perfectly well.
Example 5 The bovine collagen fibres prepared according to Example 3 were dissolved according to the protocol in Example 4. A gel with physical properties comparable to that of Example 4 was obtained.
Example 6 1 g of neutral collagen fibres obtained in Example 2 were dissolved in 90 ml of distilled water. To the final solution of pH 7.5 adjusted to 100 ml was added sodium chloride so as to obtain a final con--~ 15 13~222 centration of 0.15 M taking the sodium chloride already present in the initial precipitate into account.
The solution obtained contained about 1 % protein, principally type IV human collagen.
Placed in a water-bath at 60~C for 10 minutes, this solution could be filtered through membranes of pore size between 0.2 and 8 ~.
Filtration could be facilitated by subjecting the solution to ultrasonic vibrations. After cooling, a very viscous, completely transparent, sterile solution of human collagen was obtained.
This solution was hemocompatible. Thus after dilution in nine volumes of human blood drawn off over citrate, it did not cause any blood coagulation, platlet aggregation ? or activation of prothrombin into throm-bin.
Example 7 1 g of human collagen fibres prepared according to Example 1 were placed in 50 ml of water to swell for 1 h and 50 ml of 0.1 M of sodium acetate added. The final solution obtained contained about 1 %
of proteins, principally type IV human collagen. Its pH was adjusted to 7.5 and if necessary the solution could be made isotonic by addition of sodium chloride.
Placed in a water bath at 60~C for 10 minutes, this solution could be filtered through membranes of pore size between 0.2 and 8 ~.
A very viscous, physiological, completely transparent, sterile solution of human collagen was obtained.
This solution, like the preceding one, was hemocompatible.
Example 8 The method described in Example 7 was repeated in exactly the same manner with the exception of the addition of 50 ml of 0.1 M sodium acetate, which was replaced by the addition of 50 ml of 0.05 M disodium phosphate.
The solution obtained was hemocompatible.
Example 9 1 g of collagen fibres prepared according to Example 1 were 16 13~0222 placed in 50 ml of water to swell for 1 h. The solutlon obtained was heated for 10 mn at 30~C and filtered through membranes of pore size 0.2 ~.
The use of ultrasonics can accelerate such filtration, as aiso heating to 37~C. To the sterile solution obtalned were then added 50 ml of 0.1 M sodium acetate. The pH was adjusted to 7.5 with addition if necessary of sodium chloride to obtain a physiological aqueous solution.
All these operations after filtration may/performed protected from bacte-rial contamination.
A solution identical with the preceding solutions was obtained.
.
.
NEW PREPARATIONS OF PLACENTA COLLAGEN, THEIR EXTRACTION METHOD AND THEIR
APPLICATIONS
This invention relates to the preparation of a gel or a physio-logical solution from unmodified, transparent and hemocompatible collagen derived from placenta.
In general it covers a method for the extraction of collagen from human and animal placenta enabling type IV enricned collagen to be isolated and used industrially.
It relates more precisely to a method for the preparation of acid or neutral collagen solutions or gels that are completely transpa-rent at a great ~hickness.
Attempts have been made for a number of years to develop trans-parent collagen gels for the fabricaticn of contact lenses. More recently their utilization has been sought for grafting to or inserting these pro-ducts in the cornea itself, and even in the eye. The importance of having a collagen identical with human collagen or as near to it as possible for surgical use in the human species will be understood if any infiamma-tory or im.nunological reaction is tc be avoided. But today the only colla-gen preparations available in commerce in sufficient quantity and at an acceptable price are those obtained from rat tails or tendons or fromthe skins of yourlg animals (c~lf, for example). They consist almost solely of type I collagen. The other types, II, III, IV, V and others are prepared only by specialized research workers at prices that are too high to allow their being use(l on a large scale.
It has been stated in numerous publications that animal collag-en molecules are not very antigenic or not antigenic when they are obtained by a method using pepsin which removes the non-helical terminal peptides (teloptides). The tripie helix structure of collagen, resistant to the action of pepsin, being similar in all animal species is said to be not very antigenic. But, it seems that this non-antigenic character of collagen that has been subjected to the action of pepsin must be involved. For it is now known that these structures are antigenic in an other species, which has enabled specific antisera to be prepared from a particular or other type of bovine or human collagen, in the rabbit for example. In 13l~0222 addition, account must be taken of the always possible presence of anti-genic animal protein impurities in such preparations.
Since some years ago the injection of collagen I of bovine origin has been practiced intensively in the human species to close up scars or in repair surgery techniques. These tests revealed a number of disadvantages of which two are important :
- the occurrence of immunological reactions in 1 to 3 % of subjects, - the oe~ ~cl~ce of local blood coagulation reactions when the product has unfortunately been injected into a blood vessel or capillary.
These facts therefore underline how very important it would be to obtain hurnan collagen gels whicn were not only transparent for use in ophthalmology in t,he human species without risk of any occurrence of undesirable immunological reactions but also hemocompatible and physiolo-gical.
Type I, II or III human cr animal collagens, even after the action of pepsin, have a characteristic fibrillar structure under an elec-tron microscope, which make them powerful platelet aggregating agents.
But as these collagens are not hemocompatible, it is preferable to avoid using them whenever the physician concerned does not require a contact coagulant effect.
Since these collagens are practically insoluble at a neutral pH, only opaque suspensions can be obtained and it is impossible to use them for the preparation of transparent, physiological gels (isotonic at a neutral pH), without chemically modifyir.g the collagen molecules 25 resulting in the appearance of antigenic determinants. US Patent 3,553,299, THIELE, has already described the fabrication of contact lerlses with a gel obtained from the crystallline lens of the eye, and consisting there-fore ~ssentially of collagen. US Patents 4,223,984 and 4,268,131, MIYAYA, also deal with the fabrication of contact lenses from a type I collagen gel which, to be soluble at a neutral pH, must be modified chemically.
The methods described in these patents give relatively trans-parent gels of only s,nall thicknesses that are not very strong mechani-cally. It is not clear when reading them whether the transparency is ob-tained because of a subsequent chemical conversion (succinylation or methy-lation).
US Patent 4,388,428, KUZMA, describes gels in which the col-3 13~0222 lagen is mixed with chemical polymers to improve their chemical properties.The use of such products is not desirable because of the difficulties in removing all traces of toxic products from viscous or solid gels.
R.R. BRUNS and J. GROSS (US Patent 4,505,855) confirm the difficulty of obtaining completely transparent gels from animal collagen by prior art methods. They suggest an ultra-centrifuging method, but this metnod is very costly and difficult to reconci e with industrial usage.
In addition, consideration has already been given to enzymatic extractions of placenta collagen with pepsin, but the collagen preparations obtained do not have the transparency properties required for use in the lhe field of ophthalmology.
Thus M.Z. ABEDIN and R. RIEMSCHNEIDER (Die Angewandte Makro-molekulare Chemie, Vol. 11, N~ 1701, January 1983, pages 107-122, Huthig & Wepf Verlag, Heidelberg) describe a method for the digestion of placenta witn pepsin which results in the contamination of type IV collagen by types I and III, requiring precipitation and redissolution at a high NaCl concentration indicating the intensive character of the digestion.
These same authors (Die Ar.gewandte Makromolekulare Chemie, Vol. 82, N~ 1276, 1979, pages 171-186)describe a method for a long-duration treatment of placenta with ether, followed by freeze-drying and then treat-ment with sodium acetate or caustic soda before digestion with ?epsin and then dialysis of the supernatant layer in order to precipitate col-lagen. The collagen, after having been taken up, must also be precipitated at a high NaCl concentration. These ccnditions, which moreover preclude application on an industrial scale, also bear witness to the excessively intensive character of the digestion and the non-transparent product ob-tained appears to be especially type I + III collagen.
Th. F. KRESINA and Ed. J. MILLER (isolation and characteriza-tion of basement membrane collagen from human placental tlssue. Evidence for the presence of two genetically distinct col agen chains. Biochemistry, Vol. 18, N~ 14, 1979, pages 3089-3097, American Chemical Society) alsodescribe a total pepsin digestion of placental tissue resulting in heavy contamination by type I and III collagens. After fractionation, the colla-gen must be treated at an acid pH at high NaCl concentrations. The product obtained is not suitable for ophthamological uses. b¦ish~ ~ay 6~
.~ German Patent Application 2,462,2221~describes a pepsin treat-.
i - 13~022~
ment consisting of successive extractions of the supernatant layer of placental tissues giving a cloudy solution of collagen.
European Patent Application 0,080,956 published June 8, 1983 describes a pepsin digestion of alkaline extracts of placenta giving an opaque, denatured collagen.
The aim of this invention is to overcome these various disadvantages by providing unmodified, non-antigenic, completely transparent, physiological and hemocompatible preparations of collagen, free from striated fibres observable under an electron microscope, it being possible to provide these preparations in the form of gel or solution.
The invention provides a placenta collagen preparation obtained from human or animal collagen, enriched with, or consisting essentially of, type IV collagen, characterized in that the collagen is non-denatured, unmodified, completely transparent, physiologically acceptable, and hemocompatible and free of striated fibres visible under an electron microscope, soluble in both acid and physiological solutions, said preparation giving, in a physiological solution, either a solution up to a collagen concentration at most equal to 2% or a gel with a collagen concentration of between 2% and 30%.
The invention also provides a transparent collagen gel obtained from human or animal collagen enriched with, or consisting essentially of, type IV collagen, characterized in that it comprises between 2 to 30% of collagen which is non-.~
Ei - 13~022~
4a denatured, unmodified, completely transparent having transparency, at a thickness of between 0.1 and 20 mm, comparable to that of glass or an aqueous solution of the same _ ~ 13~222 thlckness, free from strlated flbres vlslble under an electron mlcroscope and soluble ln both acld solutlons and physlologlcal sallne solutlons.
The lnventlon further provldes a transparent collagen solutlon obtalned from human or anlmal collagen enriched wlth, or conslstlng essentially of, type IV collagen, characterized in that it comprises not more than 2% of collagen which is non-denatured, unmodlfled, completely transparent, free from strlated flbres vlslble under an electron mlcroscope and havlng an acld or neutral pH and whlch, at a neutral pH, can be rendered lsotonlc, physlologlcally acceptable and hemocompatlble.
The collagen solutlons or gels obtalned are transparent ln great thlcknesses, capable easlly of attalnlng 20 mm, a thlckness whlch ls largely sufflclent to meet the needs of the market (contact lenses, lntra-ocular or corneal lmplants).
An extractlon method has been developed by the appllcant startlng from human placenta, the only source that can be consldered for the preparatlon of human collagen on an lndustrlal scale.
The knowledge of thls new method suitable for human placenta has enabled the applicant to extend lt to animal placentas. For anlmal collagens can be useful ln veterlnary medlclne, for example, or acceptable for use as a non-lmplanted external devlce ln man such as contact lenses.
.
llD '' 5a The method accordlng to the lnvention conslsts therefore:
- flrst, ln extracting collagens from human and animal placenta and then lsolatlng a type IV enrlched collagen;
- secondly, ln preparlng solutlons or gels from thls type IV enrlched collagen having the previously descrlbed propertles.
The method for the extractlon of placenta collagens according to the invention is characterized in that lt comprises the following steps:
- startlng wlth human or animal placenta ln deep-frozen form and grinding lt;
- washing the ground placental tlssue, for example, by successlve washlngs at a neutral pH and then at an acid pH;
- sub~ectlng sald placental tlssue to enzymatlc dlgestion in order to extract type IV collagen, practlcally uncontamlnated by type I or III collagens. Dlgestlon ls carrled out wlth a sultable enzyme (wlth the exceptlon of collagenase), preferably pepsin. In that case, digestlon is preferentlally carrled out at an acld pH, preferably between 2 and 3.5 at a temperature preferably of between 0 and 20~C for a sultable time, ln partlcular of the order of 8 to 24 h, the quantlty of pepsln per kg of deep-frozen placenta belng between 0.5 and 2 g, preferably between 0.5 and 1 g or 1.5 g, that ls between 0.5 and 1 or 1.5 per mll.
.~
13~û222 5b In particular the invention provides a method for the extraction of placenta collagen in which the placenta is digested enzymatically, comprising grinding and washing a deep-frozen form of human or animal placenta;
subjecting the washed placental tissue to a first enzymatic digestion to obtain a type IV collagen substantially uncontaminated by type I or III collagen wherein the enzymatic digestion is effected with pepsin at a pH of between 2 and 3.6, at a temperature between 0~C and 20~C for a duration of between 8 and 24 h, wherein the pepsin content with respect to the deep-frozen placenta is between 0.5 and 1.5 per mil and, after separation of the residual tissue from the suspension derived from the enzymatic digestion;
effecting precipitation at a moderately acid pH to remove impurities diluting said suspension with water;
adjusting the pH to between 7 and 10;
maintaining said suspension for 1 to 24 h at a temperature of between 0 and 20~C;
separating the residual tissue from a supernatant layer of the first digestion, said residual tissue containing non-solubilized type I and type III collagens whereas the supernatant contains type IV collagen substantially free from type I and type III collagens;
adjusting the pH of the supernatant layer to a pH of between 4.5 and 5.5;
;
$~
I .~ .~ ~, , .
13~0222 5c maintalnlng sald supernatant layer for 1 to 24 h at a temperature of between 0 an 20~C; and removlng the lnsoluble preclpltate;
collectlng the supernatant layer and preclpltatlng the collagen contalned ln the supernatant layer wlth a salt at a neutral pH;
redlssolvlng the preclpltated collagen ln a weak acld solutlon whlch ls neutrallzed;
removlng lnsoluble lmpurltles; and preclpltatlng the collagen by acld preclpltatlon wlth a salt.
The tlssue resldue havlng reslsted the flrst treatment may lf necessary be sub~ected to a second dlgestlon wlth pepsln, thls second dlgestlon belng carrled out under the same condltlons as the flrst dlgestlon, enabllng type I and type III collagens practically free from type IV collagen, to be recovered subsequently by known methods.
The placenta suspenslon havlng undergone the flrst enzymatlc dlgestlon ls then treated by the followlng method, conslstlng successlvely:
- after separatlng the resldual tlssue from the placenta suspenslon derlved from the enzymatlc dlgestlon, - ln preclpltatlng at a moderately acld pH to remove a preclpltate of lmpurltles, - preclpltatlng the collagen from the supernatant layer by preclpltatlon wlth a salt at a neutral pH, r 13~0 22~
5d - redlssolvlng the preclpltated collagen and neutrallzlng the solutlon obtalned, - removlng the lnsoluble lmpurltles, and - preclpltatlng the collagen wlth a salt at an acld pH.
Thus the placenta suspenslon derlved from the enzymatlc dlgestlon ls advantageously sub~ected to the followlng steps:
- dllutlng sald suspenslon, ln partlcular by a volume of water;
- ad~ustlng the pH to a value between 7 and 9 or 10;
- allowlng sald suspenslon to rest for 1 to 24 hours at a temperature of between 0 and 20~C;
- separatlng the resldual tlssue from the supernatant layer of the flrst dlgestlon;
- ad~ustlng the supernatant layer to a moderate pH, for example, LD' I _ _ 6 of between 4.5 and 5.5 ;
- allowing said supernatant layer to rest, for example, for 1 to 24 h at a temperature between 0 and 20~C ;
- removing the insoluble part containing numerous impurities ;
- adjusting the clear supernatant layer to an about neutral pH of the order of 7.5 ;
- adding a salt, such as for example NaCl, until a final concentration of about 1.2 M is obtained :
- allowing the suspension obtained to rest, for example, for 1 to 24 h at a temperature between 10 and 25~C ;
- recovering the collagen precipitate formed ;
- dissolving the precipitate in a weakly acid solution ;
- neutralizing the solution to a pH of 7.5 ;
- allowing the suspension obtained to rest for 1 to 24 hours at a temperature between 0 and 20~C ;
- removing the insoluble part, thereby also removing the impurities that might affect transparency adversely:
- acidifying the supernatant layer to an acid pH of, for example, between 2 and 3.5 or 4 ;
- precipitating the collagen with a salt, such as NaCl, until a final cor.centration of between 0.4 and 0.8 M is obtained ;
- allowing the suspension obtained to rest for 1 to 24 h at a tempe-rature of between 0 and 25~C ;
- recovering the precipitate of purified a~id collagen.
The purified collagen precipitate obtained on completion of the op-ration is taken up with acetone or an equivalent volatile organic solvent, which results in the formation of acid collagen fibres that are recovered by filtration through a sieve. These fibres are dried under a current of lukewarm, sterile air.
By fibres are here understood to mean the appearance the solid collagen assumes after drying, and which in no way corresponds to thc striated fibrous structure as such observable under an electron microscope with known collagens in solution.
In a variant of the embodiment of the invention, the purified collagen precipitate is redissolved in water. The pH of the solution is 1~40222 adjusted to 7.5 and a salt, such as NaCl, added to it until a final concen-tration of the order of 1.2 M is obtained. The suspension obtained is allowed to rest for 1 to 24 h and the neutral collagen precipitate reco-vered.
The precipitate of neutral collagen fibres obtained is taken up with acetone or an equivalent volatile organic solvent, which results in the formation cf neutral collagen f'ibres which are recovered by filtra-tion through a sieve. These fibres are dried under a current of lukewarm, sterile air.
It is these acid or neutral collagen fibres in their dry state (< 10 %) which are used as a source of coliagen for the preparation of solutions or gels according to the invention.
Gels from acid or neutral collagen fibres obtained after treat-ment with acetone can be prepared as follows :
- the fibres are brought into contact with a volume of water determined so as to obtain a collagen con_entration of be-tween 2 ard 30 % ;
- the fibres are allowed to swell in the water, for example, for 1 h ;
- the whole is subjected to moderate heating ;
- the viscous, transparent solution is filtered thrGugh mem-branes of pore size between 0.2 and 8 ~ ;
- the solution is allowed to cool.
A solution of collagen from type IV enriched acid collagen fibres is prepared by the following method :
- the collagen fibres are brought into contact with a deter-mined volume of water ;
- the collagen fibres are allowed to swell in the water, for example, for about 1 h ;
- a solutioh of a salt, such as for example, sodium acetate at a concentration of less than or equal to 0.1 M is added to initiate neutralization.
The volumes of water are determined so as to obtain collagen concentration of at the most 2 %.
- the solution is adjusted to a pH of about 7.5 and if neces-_ 8 13~0222 sary made isotonic by addition of NaCl ;
- the solution is subjected to moderate heating at a tempera-ture of between 30 and 80~C for 10 to 60 minutes. As a va-riant, the collagen solution may be filtered in the acid state through membranes of pore size between 0.2 and 8 ~.
Neutralization takes place only after this, if necessary under bacterially sterile conditions, to ensure that the product prepared is sterile, while facilitating the filtra-tion operation as a collagen solution is less viscous at an acid pH. Another variant consists in filtering the solu-tion while subjecting it to ultrasonic vibrations to reduce the viscosity temporarily.
- the solution is filtered through membranes of pore size between 0.2 and 8 ~ ;
- and the solution allowed to cool.
On completion of these operations a solution is obtained which has these characteristics :
- it is very viscous, - it contains at the most 2 % of non-denatured, collagen, 20 - it is completely transparent, - it is isotonic, physiological and hemocompatible, - it is free from striated fibres visible under an electron microscope.
Such a solution may be used as an implant in human and animal medicine, in the fields of ophthalmology, bone joints, treatment of burns and dermatology.
When acid collagen fibres are no longer used as a source of collagen but neutral type IV enriched neutral collagen obtained according to a variant of the embodiment of the invention, the same successive opera-tiGns as those previously described are performed to obtain solutions from collagen fibres, neutralization no longer being necessary.
To prepare a collagen solution from type IV enriched neutral collagen fibres, the following procedure is followed :
- the neutral collagen fibres are dissolved in a volume of water determined so as to obtain a collagen concentration of at the most ~ - 9 - 13~0222 2 % ;
- to this solution is added a salt such as NaCl until a concentration of the order of 0.15 M is obtained ;
- said solution is subjected to moderate heating at a temperature of between 30 and 80~C for 10 to 60 minutes, preferably at a tempe-rature of about 60~C for some ten minutes ;
- the solution is filtered through membranes of pore size between 0.2 and 8 ~ ;
- and the solution allowed to cool.
A solution lS obtained which has the same ~haracteristics as the collagen solutions prepared from acid collagen.
Other advantages and features of the invention will become apparent on reading the non-limitative examples of embodiment of the inven-tlon given hereinafter.
Example 1 illustrates the r-~ethod for the extraction of type IV enriched collagen from human placentas and the preparation of acid collagen fibres.
Example 2 relates to tne preparation of neutral collagen f'ibres according to the invention.
Example 3 illustrates the method for the extraction of type IV enriched collagen from animal placentas.
Examples 4 and 5 describe the preparation of 15 % collagen gels.
Examples 6, 7,8 and 9 describethe preparation of collagen solu tions~
Example 1 300 kg of deep-frozen placenta were ground to give lumps of a few cm3. The ground material was then mixed with 300 l of an aqueous solution containing 8 % ethanol, 6 g/l of NaCl and 10 kg of cellulose.
After stirring at 10~C, the whole was pressed in a MABILLE press to sepa-rate the blood from the placental tissue. 102 kg of placental tissue con-taining 65 % water were obtained.
The tissue extracted from the press was stirred in 500 l of 0.05 M sodium citrate at a pH of 7.2 for 30 minutes at 10~C and then pres-13~0222 sed to remove the washing solution and recover the tissue. A second washing operation was performed with 500 l of 0.05 M sodium citrate at a pH of 7.2 with addition of 30 g/l of NaCl. A third washing operation was per-formed with 500 l of 0.05 M of sodium citrate at a pH of 7.2. The tissue washed in this way at a neutral pH was then subjected to three succes-sive washing sequences at an acid pH at 10~C :
- with 500 l of 0.05 M citric acid at a final pH adjusted to 2.8 by addition of 2 N HCl ; stirring for 30 minutes before the pressing operation ;
- with 500 l of 0.5 M formic acid for 15 h ;
- with 500 l of 0.05 M citric acid for 30 mn, with addition of 20 g/l.
The placental tissue washed in this way has a white appea-rance which is evidence of a good removal of the red pigments from the initial placental blood. The weight obtained was 82 kg.
It was then subjected to enzymatic digestion with pepsin in 500 l of 0.05 M citric acid at a pH of 2.8 containing 300 g of pepsin for 15 h at 10~C. The suspension was then diluted by addition of 500 l of water at 10~C. The pH was adjusted to 7.5 by addition of 4 N NaOH in order to denature the pepsin and suppress its protease action. After a waiting period of 15 h at 10~C, the tissue residue which contained the essential Gf the non-solubilized collagens I and III was separated conti-nuously by means of a centrifuge (Westfalia KG 10006). The weight of this residue was 103 kg ; it could be subjected to a second enzymatic digestion identical with the first one, followed by extraction and separation of each of the collagens I and III by the methods described in the literature.
The supernatant layer corresponding to the first enzymatic digestion contains the essential of the collagens and other macromolecules soluble at a neutral pH after action of the pepsin. In particular it con-tains type IV collagen.
The pH of the supernatant layer was adjusted to 5 with 2N
HCl and, after a waiting period of 15 h at 10~C, the precipitate formed removed by continuous centrifuging in an Alfa Laval "Bactofuge" centrifuge.
The pH of the supernatant layer, which was very clear, was adjusted to 7.5 by addition of 4 N NaOH and at a final concentration of 1.2 M in NaCl. After 15 h at 16~C, the collagen precipitate formed was _ 134~222 recovered by continuous centrifuging in a "Bactofuge" centrifuge.
The precipitate of 13 kg was then dissolved in 600 l of 0.01 N
HCl and the pH adjusted to 7.5 by addition of 4 N NaOH. The precipitate formed after a waiting period of 15 h at 4~C was removed in the "Bactofuge"
centrifuge (weight obtained 10 kg).
The clear supernatant layer was acidifed to a pH of 2.8 with 2 N HCl until a final concentration of 0.6 M was obtained at 4~C.
After 15 h, the collagen precipitate was collected in a "Bacto-fuge" centrifuge. The precipitate whose weight was 6.5 k r had a fluidaspect. 7 l of acetone were gradually added to it, which resulted in the formation of collagen fibres that were recovered by filtration through a sieve. ~Jashing these fibres Dy several treatments with acetone gave, after drying under a lukewarm, sterile current of air, 180 g of dry fibres of final product. The analysis of tne composition of the amino acids o these fibres showed a structure similar to that of type IV collagen and characteristic of the collagen family (~ 30 % gly-clne, about 10 % glutamic acid, proline and hydroxyproline).
The following Table snows the resu_ts of the analysis of the composition of the amino acids of four different preparations of human placenta collagen obtained by application of the method given in this example (the results are expressed in numbers of molecules of each amino acid per 1000 residues).
- __ 13~0222 ¦ ~tch N~ ¦ 1022 ¦ 1023 ¦ 1025 ¦ 1027 ~SP I ~6 1 42 1 41 1 ~2 I ilYP I ~30 1 91 1 ~35 1 86 ¦ T13R ¦ 19 ¦ 18 ¦ 18 ¦ 1', S~ 17 1 15 1 17 1 lf l~
¦ ~SN ¦ 28 ¦ 30 ¦ 31 ¦ 25 __ I _.. _ ___. I ._...... I ._ . . . I __ _ _ I
¦ GLU ¦ 10-7 ¦ 105 ¦ 105 ¦ 1() 7 l__ ~ l l l _. _ _l ¦ Gr-i~ ¦ 3 ¦ trClccs ¦ 1 ¦ trc~ s ¦
I Pr~O I92 1 91 1 9f~ 1 98 ¦ GLY ¦317 ¦ 327 313 ¦ 324 __ _ l__ J~ I ~1 139 1 39 1 ~13 ¦V.~L ¦ 33 ¦29 ¦ 3~1 ¦ 34 _ ICYS I9 1 6 1 9 1 (~ I .
l_ l ¦ CYST~ ¦ 1 ¦ 1 ¦ 1 ¦ 1 ¦
~ r,'r Il 5 1 26 1 16 1 l f:~
l_ l IL,~ I3f~ 1 36 1 37 1 3f I_ 1 1~.
¦~ EU ¦57 ¦ 56 ¦ 57 ¦ 5~ l _ _ _ _ l _ _ _ _ __ l ¦TYR ¦ ln ¦ 7 ¦ 12 ¦ 1(' __ l l ~ _ 13 13~0 222 BatCh N~ ¦ 1()22 ¦1()23 ¦1U25 ¦1()2 7 P~IE I 33 I 31 I 39 I 34 ¦ ORN ¦ 3 ¦LYS I 9 I 8 I 12 I 12 ¦IRY ¦ 0 ¦ 0 ¦ 0 ¦ () I
¦'rOtdl ¦ 999 ¦~96 1()()0 ¦] O()U
Example 2 The method described in Example 1 was applied in a strictly identical manner to a second batch of 300 kg of human placentas.
On completion of this operation, the weight of the precipitate obtained at a pH of 2.8 in the presence of 0.6 M NaCl at 4~C was 6.5 kg.
This precipitate was redissolved in 200 l of water. The pH of the solution obtained was adjusted to 7.5 by addition of 4 N NaOH at +16~C and NaCl added to obtain a final concentration of 1.2 M.
The precipitate formed after 15 h was recovered by centrifuging in a "Bactofuge". The weight obtained was 3450 g. The precipitate, dried with acetone as in Example 1, gave 200 g of dry neutral collagen fibres, enriched essentially with type IV collagen.
Example 3 The method described in Example 1 was applied in a strictly identical manner to 180 kg of cow placentas. Successive washings were carried out with volumes of 300 litres. The first digestion with pepsin was carried out by addition of 180 g of pepsin in 300 l of 0.05 M citric -- 14 13~222 acid adjusted to a pH of 2.8. The other selective collagen precipitation steps were identical with those of Example 1.
After final drying with acetone, 130 g of dry bovine collagen fibres were recovered. An analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) witl1 or without reductant (dithiothreitol) demonstrated that this preparation was rich in type IV
collagen.
Dissolution of these bovine collagen fibres in water gave an acid solution due to the final precipitation being made to take place at an acid pH.
Example 4 1.5 g of dry, human collagen fibres prepared according to Example 1 were brought into contact with 10 ml of distilled water. After the fibres had swelled for 1 h, the whole was placed in a water-bath at 70~C for 30 minutes. A very viscous, transparent solution was obtained which could be filtered easily through membranes of pore size between 0.8 and 8 ~ at this same temperature. After cooling, the gel obtained was colourless, acid and completely transparent, like filtered water.
Checks by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with or without dithiothreitol demonstrated the complete preservation of the collagen IV molecules during the heating stage.
This gel could then~be subjected to known operations of shaping into contact lens forms or any other device with optical properties. Moul-ding and cross-linking methods by chemical agents (aldehydes) or physical agents (y,~, X-ray or UV radiation) can be applied perfectly well.
Example 5 The bovine collagen fibres prepared according to Example 3 were dissolved according to the protocol in Example 4. A gel with physical properties comparable to that of Example 4 was obtained.
Example 6 1 g of neutral collagen fibres obtained in Example 2 were dissolved in 90 ml of distilled water. To the final solution of pH 7.5 adjusted to 100 ml was added sodium chloride so as to obtain a final con--~ 15 13~222 centration of 0.15 M taking the sodium chloride already present in the initial precipitate into account.
The solution obtained contained about 1 % protein, principally type IV human collagen.
Placed in a water-bath at 60~C for 10 minutes, this solution could be filtered through membranes of pore size between 0.2 and 8 ~.
Filtration could be facilitated by subjecting the solution to ultrasonic vibrations. After cooling, a very viscous, completely transparent, sterile solution of human collagen was obtained.
This solution was hemocompatible. Thus after dilution in nine volumes of human blood drawn off over citrate, it did not cause any blood coagulation, platlet aggregation ? or activation of prothrombin into throm-bin.
Example 7 1 g of human collagen fibres prepared according to Example 1 were placed in 50 ml of water to swell for 1 h and 50 ml of 0.1 M of sodium acetate added. The final solution obtained contained about 1 %
of proteins, principally type IV human collagen. Its pH was adjusted to 7.5 and if necessary the solution could be made isotonic by addition of sodium chloride.
Placed in a water bath at 60~C for 10 minutes, this solution could be filtered through membranes of pore size between 0.2 and 8 ~.
A very viscous, physiological, completely transparent, sterile solution of human collagen was obtained.
This solution, like the preceding one, was hemocompatible.
Example 8 The method described in Example 7 was repeated in exactly the same manner with the exception of the addition of 50 ml of 0.1 M sodium acetate, which was replaced by the addition of 50 ml of 0.05 M disodium phosphate.
The solution obtained was hemocompatible.
Example 9 1 g of collagen fibres prepared according to Example 1 were 16 13~0222 placed in 50 ml of water to swell for 1 h. The solutlon obtained was heated for 10 mn at 30~C and filtered through membranes of pore size 0.2 ~.
The use of ultrasonics can accelerate such filtration, as aiso heating to 37~C. To the sterile solution obtalned were then added 50 ml of 0.1 M sodium acetate. The pH was adjusted to 7.5 with addition if necessary of sodium chloride to obtain a physiological aqueous solution.
All these operations after filtration may/performed protected from bacte-rial contamination.
A solution identical with the preceding solutions was obtained.
.
.
Claims (22)
1. A placenta collagen preparation obtained from human or animal collagen, enriched with, or consisting essentially of, type IV collagen, characterized in that the collagen is non-denatured, unmodified, completely transparent, physiologically acceptable, and hemocompatible and free of striated fibres visible under an electron microscope, soluble in both acid and physiological solutions, said preparation giving, in a physiological solution, either a solution up to a collagen concentration at most up to 2% or a gel with a collagen concentration of between 2% and 30%.
2. A method for the extraction of placenta collagen in which the placenta is digested enzymatically, comprising grinding and washing a deep-frozen form of human or animal placenta;
subjecting the washed placental tissue to a first enzymatic digestion to obtain a type IV collagen substantially uncontaminated by type I or III collagen wherein the enzymatic digestion is effected with pepsin at a pH of between 2 and
subjecting the washed placental tissue to a first enzymatic digestion to obtain a type IV collagen substantially uncontaminated by type I or III collagen wherein the enzymatic digestion is effected with pepsin at a pH of between 2 and
3.6, at a temperature between 0°C and 20°C for a duration of between 8 and 24 h, wherein the pepsin content with respect to the deep-frozen placenta is between 0.5 and 1.5 per mil and, after separation of the residual tissue from the suspension derived from the enzymatic digestion;
effecting precipitation at a moderately acid pH to remove impurities diluting said suspension with water;
adjusting the pH to between 7 and 10;
maintaining said suspension for 1 to 24 h at a temperature of between 0 and 20°C;
separating the residual tissue from a supernatant layer of the first digestion, said residual tissue containing non-solubilized type I and type III collagens whereas the supernatant contains type IV collagen substantially free from type I and type III collagens;
adjusting the pH of the supernatant layer to a pH of between 4.5 and 5.5;
maintaining said supernatant layer for 1 to 24 h at a temperature of between 0 an 20°C; and removing the insoluble precipitate;
collecting the supernatant layer and precipitating the collagen contained in the supernatant layer with a salt at a neutral pH;
redissolving the precipitated collagen in a weak acid solution which is neutralized;
removing insoluble impurities; and precipitating the collagen by acid precipitation with a salt.
3. A method according to claim 2 comprising subjecting the tissue residue remaining after the first treatment with pepsin to a second digestion, said second digestion being effected under the same conditions as the first digestion, to obtain type I and type III collagen substantially free of type IV collagen.
effecting precipitation at a moderately acid pH to remove impurities diluting said suspension with water;
adjusting the pH to between 7 and 10;
maintaining said suspension for 1 to 24 h at a temperature of between 0 and 20°C;
separating the residual tissue from a supernatant layer of the first digestion, said residual tissue containing non-solubilized type I and type III collagens whereas the supernatant contains type IV collagen substantially free from type I and type III collagens;
adjusting the pH of the supernatant layer to a pH of between 4.5 and 5.5;
maintaining said supernatant layer for 1 to 24 h at a temperature of between 0 an 20°C; and removing the insoluble precipitate;
collecting the supernatant layer and precipitating the collagen contained in the supernatant layer with a salt at a neutral pH;
redissolving the precipitated collagen in a weak acid solution which is neutralized;
removing insoluble impurities; and precipitating the collagen by acid precipitation with a salt.
3. A method according to claim 2 comprising subjecting the tissue residue remaining after the first treatment with pepsin to a second digestion, said second digestion being effected under the same conditions as the first digestion, to obtain type I and type III collagen substantially free of type IV collagen.
4. A method according to claim 2 wherein said neutral pH precipitation is effected by:
adjusting the pH of the supernatant layer to 7.5;
adding a salt to obtain a concentration of 1.2 M;
maintaining the supernatant layer for 1 to 24 h at a temperature of between 10 and 25°C; and recovering the collagen precipitate.
adjusting the pH of the supernatant layer to 7.5;
adding a salt to obtain a concentration of 1.2 M;
maintaining the supernatant layer for 1 to 24 h at a temperature of between 10 and 25°C; and recovering the collagen precipitate.
5. A method according to claim 2 wherein after the neutralization in weak acid solution the removal of impurities is effected by:
dissolving the precipitate in a weak acid solution;
neutralizing the solution to a pH of 7.5;
maintaining the solution for 1 to 24 h at a temperature of between 0 and 20°C; and removing the insoluble precipitate.
dissolving the precipitate in a weak acid solution;
neutralizing the solution to a pH of 7.5;
maintaining the solution for 1 to 24 h at a temperature of between 0 and 20°C; and removing the insoluble precipitate.
6. A method according to claim 2 wherein after the neutralization in weak acid solution and removal of impurities, the collagen is precipitated by:
acidifying the supernatant layer to a pH of between 2 and 4;
adding a salt to obtain a final concentration of between 0.5 M and 0.8 M to precipitate the collagen;
maintaining the solution for 1 to 24 h at a temperature of between 0 and 25°C; and recovering the purified collagen precipitate.
acidifying the supernatant layer to a pH of between 2 and 4;
adding a salt to obtain a final concentration of between 0.5 M and 0.8 M to precipitate the collagen;
maintaining the solution for 1 to 24 h at a temperature of between 0 and 25°C; and recovering the purified collagen precipitate.
7. A method according to claim 2 comprising:
precipitating the collagen with a salt at a neutral pH after redissolving the precipitate of purified collagen.
precipitating the collagen with a salt at a neutral pH after redissolving the precipitate of purified collagen.
8. A method according to claim 7, comprising:
redissolving the final precipitate of purified acid collagen in water;
adjusting the pH of the solution to neutral;
adding a salt to the solution to obtain a concentration of 1.2 M;
allowing the solution to stand for 1 to 24 h; and recovering the collagen precipitate.
redissolving the final precipitate of purified acid collagen in water;
adjusting the pH of the solution to neutral;
adding a salt to the solution to obtain a concentration of 1.2 M;
allowing the solution to stand for 1 to 24 h; and recovering the collagen precipitate.
9. A method according to claim 2 wherein:
the acid or neutral purified collagen precipitate is contacted with a volatile, organic solvent to form collagen fibres;
the fibres are recovered and dried for use in the preparation of a solution or gel.
the acid or neutral purified collagen precipitate is contacted with a volatile, organic solvent to form collagen fibres;
the fibres are recovered and dried for use in the preparation of a solution or gel.
10. A method according to claim 9 to obtain a collagen gel, the method further comprising:
contacting the collagen fibres with a volume of water sufficient to obtain a collagen concentration of between 2 and 30%;
allowing the fibres to swell;
heating the fibres at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the transparent, viscous solution obtained through a membrane of pore size between 0.8 and 8 µ, and allowing the solution to cool.
contacting the collagen fibres with a volume of water sufficient to obtain a collagen concentration of between 2 and 30%;
allowing the fibres to swell;
heating the fibres at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the transparent, viscous solution obtained through a membrane of pore size between 0.8 and 8 µ, and allowing the solution to cool.
11. A method according to claim 9 to obtain a collagen solution, the method further comprising:
contacting the acid collagen fibres with water;
allowing the fibres to swell in the water;
adding a 0.1 M solution of a salt, the volume of water being selected to obtain a collagen concentration not exceeding 2%;
adjusting the pH to about 7.5;
heating the solution at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the solution through membranes of pore size between 0.2 and 8 µ; and cooling the solution.
contacting the acid collagen fibres with water;
allowing the fibres to swell in the water;
adding a 0.1 M solution of a salt, the volume of water being selected to obtain a collagen concentration not exceeding 2%;
adjusting the pH to about 7.5;
heating the solution at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the solution through membranes of pore size between 0.2 and 8 µ; and cooling the solution.
12. A method according to claim 9 to obtain a collagen solution further comprising:
dissolving the neutral collagen precipitate in sufficient water to obtain a collagen concentration not exceeding 2%;
adding a salt to the solution to obtain a concentration of about 0.15 M;
heating the solution at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the solution through a membrane of pore size between 0.2 and 8 µ; and allowing the solution to cool.
dissolving the neutral collagen precipitate in sufficient water to obtain a collagen concentration not exceeding 2%;
adding a salt to the solution to obtain a concentration of about 0.15 M;
heating the solution at a temperature of between 30 and 80°C for 10 to 60 minutes;
filtering the solution through a membrane of pore size between 0.2 and 8 µ; and allowing the solution to cool.
13. A method according to claim 10 wherein the solution is subjected to ultrasonic vibrations during filtration.
14. A method according to claim 4, 6, 8 or 12, wherein said salt is NaCl.
15. A method according to claim 9, wherein said organic solvent is acetone.
16. A method according to claim 10 or 11, wherein the fibres are allowed to swell for 1 hour.
17. A method according to claim 10, wherein the fibres are heated at about 70°C for about 30 minutes.
18. A method according to claim 11, wherein said salt is sodium acetate.
19. A method according to claim 11 or 12, wherein the solution is heated at about 60°C for about 10 minutes.
20. A transparent collagen gel obtained from human or animal collagen enriched with, or consisting essentially of, type IV collagen, characterized in that it comprises between 2 to 30% of collagen which is non-denatured, unmodified, completely transparent having transparency, at a thickness of between 0.1 and 20 mm, comparable to that of glass or an aqueous solution of the same thickness, free from striated fibres visible under an electron microscope and soluble in both acid solutions and physiological saline solutions.
21. A transparent collagen solution obtained from human or animal collagen enriched with, or consisting essentially of, type IV collagen, characterized in that it comprises not more than 2% of collagen which is non-denatured, unmodified, completely transparent, free from striated fibres visible under an electron microscope and having an acid or neutral pH
and which, at a neutral pH, can be rendered isotonic, physiologically acceptable and hemocompatible.
and which, at a neutral pH, can be rendered isotonic, physiologically acceptable and hemocompatible.
22. A collagen preparation according to claim 1, 20 or 21, which, when subjected to heating at a temperature of between 30 and 80°C for 10 to 60 minutes, resists denaturation as determined by a polyacrylamide gel electrophoresis with sodium dodecyl sulfate showing an absence of any molecular weight change.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8513004 | 1985-09-02 | ||
| FR8513004A FR2586703B1 (en) | 1985-09-02 | 1985-09-02 | PROCESS FOR EXTRACTING PLACENTAL COLLAGENS, COLLAGENS OBTAINED IN PARTICULAR IN THE FORM OF GELS OR SOLUTIONS AND THEIR APPLICATIONS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340222C true CA1340222C (en) | 1998-12-15 |
Family
ID=9322543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 517280 Expired - Fee Related CA1340222C (en) | 1985-09-02 | 1986-09-02 | Preparations of placenta collagen, their extraction method and their applications |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0214035B1 (en) |
| JP (1) | JP2563232B2 (en) |
| CA (1) | CA1340222C (en) |
| DE (1) | DE3682551D1 (en) |
| ES (1) | ES2002127A6 (en) |
| FR (1) | FR2586703B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8138147B2 (en) | 1999-05-14 | 2012-03-20 | Skinmedica, Inc. | Conditioned cell culture medium compositions and methods of use |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1214505B (en) * | 1987-03-12 | 1990-01-18 | Gentili Ist Spa | PROCEDURE FOR THE PREPARATION OF COLLAGEN AND PRODUCTS SO OBTAINED. |
| FR2617763B1 (en) * | 1987-07-07 | 1989-12-01 | Essilor Int | METHOD OF MANUFACTURING CONTACT LENS IN A NATURAL PROTEIN POLYMER, BY MOLDING BEFORE CROSS-LINKING |
| US4876332A (en) * | 1987-10-08 | 1989-10-24 | Regents Of The Univeristy Of Minnesota | Polypeptides with type IV collagen activity |
| FR2715405B1 (en) * | 1994-01-24 | 1996-04-05 | Imedex | Process for the elimination of prions in collagens and collagens thus obtained. |
| FR2720945B1 (en) | 1994-06-08 | 1996-08-30 | Coletica | Post-operative anti-adhesion collagen membrane. |
| FR2777284B1 (en) * | 1998-04-10 | 2000-05-26 | Hamza Mansour | PROCESS FOR THE STERILIZATION OF A NATIVE COLLAGEN IN A LIQUID MEDIUM, A STERILE NATIVE COLLAGEN OBTAINED, COMPOSITIONS CONTAINING THE SAME AND APPLICATIONS |
| RU2715715C1 (en) * | 2019-03-06 | 2020-03-03 | Общество с ограниченной ответственностью фирмы "Имтек" | Sterile transparent concentrated solution of biocompatible collagen, method for production and use thereof |
| CN111592592A (en) * | 2020-03-02 | 2020-08-28 | 内蒙古林宁医疗科技有限公司 | Selective extraction preparation method and application of different types of collagen of human placenta |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2462222A1 (en) * | 1974-02-02 | 1976-05-06 | Trommsdorff Fa H | Soluble human collagen free of non-helical peptides - prepd by treating purified collagen with ficin or pepsin, used to accelerate wound or burn healing etc. |
| DE2616939C3 (en) * | 1976-04-17 | 1981-10-15 | Böttger GmbH Pharmazeutische und Kosmetische Präparate, 1000 Berlin | Use of placenta extract residues and process for the production of a collagen-containing placenta extract |
| US4268131A (en) * | 1979-04-11 | 1981-05-19 | Opticol Corporation | Fiber collagen contact lens |
| DE2929144A1 (en) * | 1979-07-19 | 1981-02-12 | Behringwerke Ag | METHOD FOR PRODUCING COLLAGEN |
| FR2516927B1 (en) * | 1981-11-26 | 1986-05-23 | Merieux Fond | PROCESS FOR THE INDUSTRIAL PREPARATION OF COLLAGENIC MATERIALS FROM HUMAN PLACENTARY TISSUES, HUMAN COLLAGENIC MATERIALS OBTAINED, THEIR APPLICATION AS BIOMATERIALS |
| US4505855A (en) * | 1982-09-30 | 1985-03-19 | Massachusetts General Hospital | Transparent non-fibrilized collagen material by ultracentrifugation |
-
1985
- 1985-09-02 FR FR8513004A patent/FR2586703B1/en not_active Expired
-
1986
- 1986-08-11 DE DE8686401794T patent/DE3682551D1/en not_active Expired - Fee Related
- 1986-08-11 EP EP19860401794 patent/EP0214035B1/en not_active Expired - Lifetime
- 1986-09-01 ES ES8601542A patent/ES2002127A6/en not_active Expired
- 1986-09-02 JP JP61206674A patent/JP2563232B2/en not_active Expired - Lifetime
- 1986-09-02 CA CA 517280 patent/CA1340222C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8138147B2 (en) | 1999-05-14 | 2012-03-20 | Skinmedica, Inc. | Conditioned cell culture medium compositions and methods of use |
| US8476231B2 (en) | 1999-05-14 | 2013-07-02 | Allergan, Inc. | Conditioned cell culture medium compositions and methods of use |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2002127A6 (en) | 1988-07-16 |
| EP0214035A1 (en) | 1987-03-11 |
| DE3682551D1 (en) | 1992-01-02 |
| EP0214035B1 (en) | 1991-11-21 |
| FR2586703A1 (en) | 1987-03-06 |
| JP2563232B2 (en) | 1996-12-11 |
| FR2586703B1 (en) | 1989-12-01 |
| JPS6253927A (en) | 1987-03-09 |
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