CA1260650A - Process for preparing malleable collagen and product thereof - Google Patents
Process for preparing malleable collagen and product thereofInfo
- Publication number
- CA1260650A CA1260650A CA000485572A CA485572A CA1260650A CA 1260650 A CA1260650 A CA 1260650A CA 000485572 A CA000485572 A CA 000485572A CA 485572 A CA485572 A CA 485572A CA 1260650 A CA1260650 A CA 1260650A
- Authority
- CA
- Canada
- Prior art keywords
- collagen
- solution
- nucleation
- preparation
- precipitate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
- C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
- A61F2310/00005—The prosthesis being constructed from a particular material
- A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S128/00—Surgery
- Y10S128/08—Collagen
Abstract
Abstract A collagen preparation having unique properties of elasticity and malleability which is associated with a microscopic structure having an intertwining rope-like characteristics, and a process for preparing same are disclosed. The collagen thus formed is the second nucleation product obtained upon incubation of a supernatant from a quick harvested first stage nucleation product.
Description
~26V6S~) Technical Field The present invention relates to preparation of collagen for medical or other associated uses. More specifically, the invention relates to preparation of a collagen composition which is malleable and which has desirable physical properties.
Background Art Preparations of collagen have found use in a variety of therapeutic and reconstructive contexts in both soft tissue and bone, as well as in the preparation of surgical sponges and associated accessories for medical use.
Collagen is the major protein of the skeletal system, and is found in bone, skin and cartilage.
Native collagen consists in large part of a triple helical structure which appears to be a consequence of repeating triplet sequences composed of glycine linked to two additional amino acids, commonly proline and hydroxy-proline, the glycine being in every third posi-tion in the chain. In addition, all collagen chainscontain regions at each end which do not have the triplet glycine sequence and are thus not helical.
These regions are thought to be responsible for the immunogenicity associated with most collagen prepara-tions, and this property can, in large part, be miti-gated by removal of these regions to produce "atelo-peptide" collagen. The removal can be accomplished by digestion with proteolytic enzymes such as trypsin or pepsin. These non-helical telopeptide regions are however, required to form the cross-links which are responsible for stability of the fibrillar structure ~6C~ O
in native collagen, since they contain aldehydes capable of cross-linkage; atelopeptide collagen must be cross-linked artificially if it i5 desired to obtain this characteristicO
S Starting from the native material, derived either from bone or from skin, a variety of approaches to the preparation of pure collagenous materials has been disclosed. For example, Battista, U.S. 3,471,598 and 3,632,361 discloses the preparation of a collagen sponge which is a partial salt formed by preparing a dispersion of the collagen salt in an aqueous medium, casting into a mold and freeze drying. This approach is different from that disclosed in U.S. 3,157,524 which describes a collagen reconstituted by repreci-pitation of a solublized tropocollagen (the basicmolecular triple helical unit) from a solution. In addition, commercial preparations commonly known as "Collagenfleece" (U.S. Patent 4,066,083) and Avitene provide relatively pure, contiguous collagen prepara-tions which, however, contain telopeptides and areoften immunogenic.
Other preparations of collagen are available commercially. Prominent among these is Zyderm* col-lagen implant (ZCI) which is a reconstituted fibrillar suspension of atelopeptide collagen. This prepara-tion is usable in augmenting soft tissue (U.S. Patent 4,424,208) and for cosmetic purposes (when provided as a suspension (U.S. Patent 4,140,537)). The nature of the application to which the collagen preparation is to be put is, of course, instrumental in determining the form of collagen which is most desirable. Certain properties such as, for example, non-immunogenicity are common as desirable to all medical applications. However, other desirable properties vary. For construction of bone re-placement material, for example, it would be preferable *Registered Trade Mark ~26~6SO
to have a malleable contiguous mass, whereas for cosmetic"wrinkle-smoothing" applications, an injectable suspension would be preferred. Thus, it is beneficial to provide an arsenal of collagen preparation types whose characteristics offer a spectrum of physical properties.
The present invention adds to the repertoire o~
available physical properties associated ~ith non-immunogenic collagen preparations.
Disclosure of the Invention The present invention provides a malleable, contiguous, elastic form of non-immunogenic collagen which is suitable for a variety of applications. The preparation is characterized on a microscopic scale by interlocking fibrillar ropes having a mean diameter of the 15 order of 170-300 nm, and a microstructure which, within this context, is highly ordered. These physical properties result from a preparation process which employs a secondary nucleation from a collagen solution under specified conditions.
Accordingly, in one aspectr the invention concerns a process for preparing a second nucleation form of collagen which process comprises separating a first nucleation product formed by rapid, low temperature mixing of a collagen solution with an insolubilizin~
buffer, and incubating the remaining collagen in solution to encourage the formation of the desired second nucleation product. The invention, in other aspects, relates to the product of this process, and to collagen preparations having the characteristics associated with it. Included among these characteristics is a microstructure which is predominately formed of interlocking rope-like fibers.
l260650 - 3a -According to a further broad aspect of the present invention, there is provided a process for preparing a second nucleation collagen which comprises mixing at a temperature of about l-10C a preparation of collagen in solution with ~ solution effective in rendering the collagen insoluble to give a mixture.
Immediately thereafter, the mixture is subjected to a force of 8,000-13,000 x g to give a precipitate of first nucleation collagen and a supernatant frac-tion. The supernatant fraction is then removed andthe supernatant fraction is incubated at about 15-25C
for a period effective to form a precipitate of second nucleation collagen. The second nucleation collagen is then recovered.
~..
~6~6S~
Brief Description of the Drawin -Figure 1 shows a scanning electron micrograph (reflectance) of the collagen preparation under three different (1000 x, 6000 x, and 12,000 x) magnifications.
Figure 2 shows a transmission electron micro-graph at 30,000 x magnification of the collagen preparation of the invention.
Modes of Carrying Out the Invention A. Definitions "Second nucleation collagen preparation" refers to a collagen precipitated from solution after a first stage precipitation has already taken place. The collagen preparation of the invention is, indeed, formed by a process which involves pre-precipitation of a "first nucleation" preparation followed by treatment of the resulting supernatant to yield the desired product.
A solution "effective in rendering collagen insoluble" refers to a solution which may be added to a solution containing solubilized collagen which will cause the collagen, in principle, to be unstable in solution, and ultimately to precipitate. The precipitation may not be immediate, due to, for example, formation of a supersaturated solution or other metastable condition.
In the process of the present invention, the collagen in solution i~ typically in a solution of approximately pH 2-3 and the protein is soluble indefinitely in the concentrations used at that pH. However, for example, addition of a solution which converts the pH to approximate neutrality will cause the protein eventually to precipitate. However, the precipitation is a relatively slow process, and depends on the conditions of mixing, ~emperature, and external forces, such as that lX6~)650 exerted by centrifugation, in order for the precipitate actually to appear.
B. Detailed Description The starting material for the preparation of the collage~ of the invention is solubilized collagen in a concentration range of 2 mg/ml to 4 m~/ml. A
suitable form o$ this material is an atelopeptide form of bovine skin collagen, which is commercially available from Collagen Corporation, Palo Alto, California, zygen~
collagen in solution (CIS). This material is a solution containing 3 mg/ml of solubilized collagen at a pH of approximately 2Ø Any solubilized form of collagen can, of course, be used but modifications to the protocols set forth below will undoubtedly be necessary to accomodate to alterations in the starting material.
From the standpoint of the physical properties obtained, it may be possible to use collagen which still contains telopeptides; however it is desirable to use an atelopeptide form of solubilized collagen as there would be no particular advantage in using collagen which is known to be immunogenic for medical purposes~
The crux of the process is to form the first nucleation product quickly, under gravitational pressure, and at a temperature gradient in order to provide a supernatant fraction which has the remaining collagen in the correct form for proper formation of the second nucleation batch. To do this, the CIS is rapidly mixed wi~h a solution effective in insolubilizing the collagen -- most appropriately a solution which will abruptly raise the p~ to approximate neutrality, such as a phosphate buffer of pH above 8. The concentration of the buffer will be compatible with that of the acid components in the CIS so as to result in a final pH of ' 1~60~
approximately 7 when the appropriate volume is added. It is preEerred that the volume ratio of the CIS to that of the added solution be between about 95:5 to 1:1. Each solution is precooled to between 1C and 10C, preferably around 4C, the two solutions are mixed quickly and the mixture immediately subjected to centrifugation at about 4000 x g - 16000 x g, preferably around 8000 x 9 - 13000 x g for 1-2 hrs. Thus, during the centrifugation the temperature slowly rises to about room temperature.
As used above, "immediately~ refers to a time frame which is in the early lag phase of the fiber formation process. (Collagen fiber formation from solution has been shown to follow a sinusoidal pattern, and it is desired to subject the mixture to the gravitational force before the main thrust of growth phase.) The centrifugation takes place at between about 1C to 25C, preferably around 20C, and results in a precipitate of between 25~ to 60~ of the collagen contained in the solution. Optimally, only about 25% of the collagen will precipitate, and the remaining 75% will be available for the second nucleation.
After removal from the centrifuge, the supernatant is gently decanted or otherwise gently removed and incubated at about 15C-30C, preferably around 20C from about 4 hrs to about 24 hrs, preferably overnightO During this incubation, the second nucleation preparation forms and i5 harvested by centrifugation at about 9000 rpm (13,000 x g) for about 10 min. Depending on the amount of collagen precipitated during the first nucleation, about 25-60% will be harvested in this second nucleation step. The remaining supernatant will have a concentration of 0.4-0.7 mg/ml, approximately, of collagen, or ahout 15~ of the total.
~L26~SO
The desired second nucleation product has a set of physical properties useful in applications where malleable or moldable collagen materials are needed.
Specifically, precipitate is characterized by a putty-like texture which is cohesive, and which permits moldingwith only slight resilience and "memory". There are a number of diagnostic characteristics which indicate the presence of these properties. When examined under a scanning electron microscope as shown in Figure 1, the molecular structure appears to be composed of intertwining rope-like fibers which have diameters in the range of 170-300 nm. This is especially apparent in Fiyures lb and lc which show 6,000 and 12,000 fold magnifications respectively. When subjected to transmission electron microscopy the preparation appears as shown in Figure 2.
These properties appear to result from the manner of handling the first and second nucleations. In a different, but commercially useful process for preparing Zyderm0 collagen implant (ZCI) dispersions, the same starting material can be used. $hese dispersions, which are prepared at 35 mg/ml (ZCI-I) and 65 mg/ml (ZCI-II) lack the cohesiveness of the present preparation, and do not show the characteristic rope-like structure. While the chemical conditions of precipita-tion to form ZCI and the preparation of the invention can be identical, the physical conditions are quite different. The ZCI is precipitated at approximately room temperature, by gradual mixing of the insolubilizing buffer and the fibers are permitted to form without centrifugation. The precipitate harvested from the ZCI
precipitation yields approximately 85~ of the collagen in the precipitate leaving about 15% in the supernatant, approximately the amount found in t~e supernatant from 65~
the second nucleation product of the invention. ThuS, ZCI is a first nucleation product and presumably includes additional collagen which would, in the process of the invention, be left to form the desired product. The precipitate in the ZCI dispersion is harvested, homogenized, and formulated to give the commercially available Zyderm~ collagen implant product. It may also be possible to homogenize or otherwise process and formulate the first nucleation product in the process of the invention; thus providing a convenient utility for a major by-product of the invention.
C. Example The following specific preparation procedure illustrates one embodiment of the invention. It is not intended to limit the scope of conditions under which the process can be carried out.
90 ml of Zygen0 CIS was cooled to 4C, and quickly mixed in a beaker resting in a 4C bath with 10 ml 0.2 M Na2 HP04 solution which has also been precooled to 4C. The CIS and phosphate were rapidly thoroughly mixed, and transferred to a centrifuge bottle. The mixture was centrifuged at 10,000 x 9 for 1.5 hrs at 20C, and the precipitate thus obtained was separated from the supernatant by gentle decantation. The super-natant was then incubated overnight without stirring at20C, and the resulting new precipitate harvested by centrifugation at 13,000 x g for 10 min. The precipitate was harvested by decanting the supernatant.
The precipitated second nucleation product was then subjected to scanning electron microscopy and transmission electron microscopy, giving the results shown in Figures 1 and 2 respectively. It was also tested for melting point and showed a higher melting ' ~26~0 point than ZCI using diffeeential calorimetry. A
dispersion of the resulting collagen was capable, at a concentration of 35 mg/ml to be injected with a number 22 needle.
. .
,, '
Background Art Preparations of collagen have found use in a variety of therapeutic and reconstructive contexts in both soft tissue and bone, as well as in the preparation of surgical sponges and associated accessories for medical use.
Collagen is the major protein of the skeletal system, and is found in bone, skin and cartilage.
Native collagen consists in large part of a triple helical structure which appears to be a consequence of repeating triplet sequences composed of glycine linked to two additional amino acids, commonly proline and hydroxy-proline, the glycine being in every third posi-tion in the chain. In addition, all collagen chainscontain regions at each end which do not have the triplet glycine sequence and are thus not helical.
These regions are thought to be responsible for the immunogenicity associated with most collagen prepara-tions, and this property can, in large part, be miti-gated by removal of these regions to produce "atelo-peptide" collagen. The removal can be accomplished by digestion with proteolytic enzymes such as trypsin or pepsin. These non-helical telopeptide regions are however, required to form the cross-links which are responsible for stability of the fibrillar structure ~6C~ O
in native collagen, since they contain aldehydes capable of cross-linkage; atelopeptide collagen must be cross-linked artificially if it i5 desired to obtain this characteristicO
S Starting from the native material, derived either from bone or from skin, a variety of approaches to the preparation of pure collagenous materials has been disclosed. For example, Battista, U.S. 3,471,598 and 3,632,361 discloses the preparation of a collagen sponge which is a partial salt formed by preparing a dispersion of the collagen salt in an aqueous medium, casting into a mold and freeze drying. This approach is different from that disclosed in U.S. 3,157,524 which describes a collagen reconstituted by repreci-pitation of a solublized tropocollagen (the basicmolecular triple helical unit) from a solution. In addition, commercial preparations commonly known as "Collagenfleece" (U.S. Patent 4,066,083) and Avitene provide relatively pure, contiguous collagen prepara-tions which, however, contain telopeptides and areoften immunogenic.
Other preparations of collagen are available commercially. Prominent among these is Zyderm* col-lagen implant (ZCI) which is a reconstituted fibrillar suspension of atelopeptide collagen. This prepara-tion is usable in augmenting soft tissue (U.S. Patent 4,424,208) and for cosmetic purposes (when provided as a suspension (U.S. Patent 4,140,537)). The nature of the application to which the collagen preparation is to be put is, of course, instrumental in determining the form of collagen which is most desirable. Certain properties such as, for example, non-immunogenicity are common as desirable to all medical applications. However, other desirable properties vary. For construction of bone re-placement material, for example, it would be preferable *Registered Trade Mark ~26~6SO
to have a malleable contiguous mass, whereas for cosmetic"wrinkle-smoothing" applications, an injectable suspension would be preferred. Thus, it is beneficial to provide an arsenal of collagen preparation types whose characteristics offer a spectrum of physical properties.
The present invention adds to the repertoire o~
available physical properties associated ~ith non-immunogenic collagen preparations.
Disclosure of the Invention The present invention provides a malleable, contiguous, elastic form of non-immunogenic collagen which is suitable for a variety of applications. The preparation is characterized on a microscopic scale by interlocking fibrillar ropes having a mean diameter of the 15 order of 170-300 nm, and a microstructure which, within this context, is highly ordered. These physical properties result from a preparation process which employs a secondary nucleation from a collagen solution under specified conditions.
Accordingly, in one aspectr the invention concerns a process for preparing a second nucleation form of collagen which process comprises separating a first nucleation product formed by rapid, low temperature mixing of a collagen solution with an insolubilizin~
buffer, and incubating the remaining collagen in solution to encourage the formation of the desired second nucleation product. The invention, in other aspects, relates to the product of this process, and to collagen preparations having the characteristics associated with it. Included among these characteristics is a microstructure which is predominately formed of interlocking rope-like fibers.
l260650 - 3a -According to a further broad aspect of the present invention, there is provided a process for preparing a second nucleation collagen which comprises mixing at a temperature of about l-10C a preparation of collagen in solution with ~ solution effective in rendering the collagen insoluble to give a mixture.
Immediately thereafter, the mixture is subjected to a force of 8,000-13,000 x g to give a precipitate of first nucleation collagen and a supernatant frac-tion. The supernatant fraction is then removed andthe supernatant fraction is incubated at about 15-25C
for a period effective to form a precipitate of second nucleation collagen. The second nucleation collagen is then recovered.
~..
~6~6S~
Brief Description of the Drawin -Figure 1 shows a scanning electron micrograph (reflectance) of the collagen preparation under three different (1000 x, 6000 x, and 12,000 x) magnifications.
Figure 2 shows a transmission electron micro-graph at 30,000 x magnification of the collagen preparation of the invention.
Modes of Carrying Out the Invention A. Definitions "Second nucleation collagen preparation" refers to a collagen precipitated from solution after a first stage precipitation has already taken place. The collagen preparation of the invention is, indeed, formed by a process which involves pre-precipitation of a "first nucleation" preparation followed by treatment of the resulting supernatant to yield the desired product.
A solution "effective in rendering collagen insoluble" refers to a solution which may be added to a solution containing solubilized collagen which will cause the collagen, in principle, to be unstable in solution, and ultimately to precipitate. The precipitation may not be immediate, due to, for example, formation of a supersaturated solution or other metastable condition.
In the process of the present invention, the collagen in solution i~ typically in a solution of approximately pH 2-3 and the protein is soluble indefinitely in the concentrations used at that pH. However, for example, addition of a solution which converts the pH to approximate neutrality will cause the protein eventually to precipitate. However, the precipitation is a relatively slow process, and depends on the conditions of mixing, ~emperature, and external forces, such as that lX6~)650 exerted by centrifugation, in order for the precipitate actually to appear.
B. Detailed Description The starting material for the preparation of the collage~ of the invention is solubilized collagen in a concentration range of 2 mg/ml to 4 m~/ml. A
suitable form o$ this material is an atelopeptide form of bovine skin collagen, which is commercially available from Collagen Corporation, Palo Alto, California, zygen~
collagen in solution (CIS). This material is a solution containing 3 mg/ml of solubilized collagen at a pH of approximately 2Ø Any solubilized form of collagen can, of course, be used but modifications to the protocols set forth below will undoubtedly be necessary to accomodate to alterations in the starting material.
From the standpoint of the physical properties obtained, it may be possible to use collagen which still contains telopeptides; however it is desirable to use an atelopeptide form of solubilized collagen as there would be no particular advantage in using collagen which is known to be immunogenic for medical purposes~
The crux of the process is to form the first nucleation product quickly, under gravitational pressure, and at a temperature gradient in order to provide a supernatant fraction which has the remaining collagen in the correct form for proper formation of the second nucleation batch. To do this, the CIS is rapidly mixed wi~h a solution effective in insolubilizing the collagen -- most appropriately a solution which will abruptly raise the p~ to approximate neutrality, such as a phosphate buffer of pH above 8. The concentration of the buffer will be compatible with that of the acid components in the CIS so as to result in a final pH of ' 1~60~
approximately 7 when the appropriate volume is added. It is preEerred that the volume ratio of the CIS to that of the added solution be between about 95:5 to 1:1. Each solution is precooled to between 1C and 10C, preferably around 4C, the two solutions are mixed quickly and the mixture immediately subjected to centrifugation at about 4000 x g - 16000 x g, preferably around 8000 x 9 - 13000 x g for 1-2 hrs. Thus, during the centrifugation the temperature slowly rises to about room temperature.
As used above, "immediately~ refers to a time frame which is in the early lag phase of the fiber formation process. (Collagen fiber formation from solution has been shown to follow a sinusoidal pattern, and it is desired to subject the mixture to the gravitational force before the main thrust of growth phase.) The centrifugation takes place at between about 1C to 25C, preferably around 20C, and results in a precipitate of between 25~ to 60~ of the collagen contained in the solution. Optimally, only about 25% of the collagen will precipitate, and the remaining 75% will be available for the second nucleation.
After removal from the centrifuge, the supernatant is gently decanted or otherwise gently removed and incubated at about 15C-30C, preferably around 20C from about 4 hrs to about 24 hrs, preferably overnightO During this incubation, the second nucleation preparation forms and i5 harvested by centrifugation at about 9000 rpm (13,000 x g) for about 10 min. Depending on the amount of collagen precipitated during the first nucleation, about 25-60% will be harvested in this second nucleation step. The remaining supernatant will have a concentration of 0.4-0.7 mg/ml, approximately, of collagen, or ahout 15~ of the total.
~L26~SO
The desired second nucleation product has a set of physical properties useful in applications where malleable or moldable collagen materials are needed.
Specifically, precipitate is characterized by a putty-like texture which is cohesive, and which permits moldingwith only slight resilience and "memory". There are a number of diagnostic characteristics which indicate the presence of these properties. When examined under a scanning electron microscope as shown in Figure 1, the molecular structure appears to be composed of intertwining rope-like fibers which have diameters in the range of 170-300 nm. This is especially apparent in Fiyures lb and lc which show 6,000 and 12,000 fold magnifications respectively. When subjected to transmission electron microscopy the preparation appears as shown in Figure 2.
These properties appear to result from the manner of handling the first and second nucleations. In a different, but commercially useful process for preparing Zyderm0 collagen implant (ZCI) dispersions, the same starting material can be used. $hese dispersions, which are prepared at 35 mg/ml (ZCI-I) and 65 mg/ml (ZCI-II) lack the cohesiveness of the present preparation, and do not show the characteristic rope-like structure. While the chemical conditions of precipita-tion to form ZCI and the preparation of the invention can be identical, the physical conditions are quite different. The ZCI is precipitated at approximately room temperature, by gradual mixing of the insolubilizing buffer and the fibers are permitted to form without centrifugation. The precipitate harvested from the ZCI
precipitation yields approximately 85~ of the collagen in the precipitate leaving about 15% in the supernatant, approximately the amount found in t~e supernatant from 65~
the second nucleation product of the invention. ThuS, ZCI is a first nucleation product and presumably includes additional collagen which would, in the process of the invention, be left to form the desired product. The precipitate in the ZCI dispersion is harvested, homogenized, and formulated to give the commercially available Zyderm~ collagen implant product. It may also be possible to homogenize or otherwise process and formulate the first nucleation product in the process of the invention; thus providing a convenient utility for a major by-product of the invention.
C. Example The following specific preparation procedure illustrates one embodiment of the invention. It is not intended to limit the scope of conditions under which the process can be carried out.
90 ml of Zygen0 CIS was cooled to 4C, and quickly mixed in a beaker resting in a 4C bath with 10 ml 0.2 M Na2 HP04 solution which has also been precooled to 4C. The CIS and phosphate were rapidly thoroughly mixed, and transferred to a centrifuge bottle. The mixture was centrifuged at 10,000 x 9 for 1.5 hrs at 20C, and the precipitate thus obtained was separated from the supernatant by gentle decantation. The super-natant was then incubated overnight without stirring at20C, and the resulting new precipitate harvested by centrifugation at 13,000 x g for 10 min. The precipitate was harvested by decanting the supernatant.
The precipitated second nucleation product was then subjected to scanning electron microscopy and transmission electron microscopy, giving the results shown in Figures 1 and 2 respectively. It was also tested for melting point and showed a higher melting ' ~26~0 point than ZCI using diffeeential calorimetry. A
dispersion of the resulting collagen was capable, at a concentration of 35 mg/ml to be injected with a number 22 needle.
. .
,, '
Claims (9)
1. A process for preparing a second nucleation collagen which comprises:
(a) mixing at a temperature of about 1°-10°C
a preparation of collagen in solution with a solution effective in rendering the collagen insoluble to give a mixture;
(b) immediately subjecting the mixture to a force of 8,000-13,000 x g to give a precipitate of first nucleation collagen and a supernatant fraction;
(c) removing the supernatant fraction and incu-bating said supernatant fraction at about 15-25°C
for a period effective to form a precipitate of second nucleation collagen; and (d) recovering the second nucleation collagen.
(a) mixing at a temperature of about 1°-10°C
a preparation of collagen in solution with a solution effective in rendering the collagen insoluble to give a mixture;
(b) immediately subjecting the mixture to a force of 8,000-13,000 x g to give a precipitate of first nucleation collagen and a supernatant fraction;
(c) removing the supernatant fraction and incu-bating said supernatant fraction at about 15-25°C
for a period effective to form a precipitate of second nucleation collagen; and (d) recovering the second nucleation collagen.
2. The process of claim 1 wherein the collagen in solution has a concentration range of 2 mg/ml to 4 mg/ml.
3. The process of claim 1 wherein the collagen in solution is an atelopeptide collagen.
4. The process of claim 3 wherein the collagen in solution is solubilized atelopeptide bovine skin collagen in solution.
5. The process of claim 1 wherein the per-centage of total collagen removed in the first nuclea-tion is about 25% to 60%.
6. The process of claim 1 wherein the percen-tage of total collagen removed in the second nucleation is about 25% to 60%.
7. The process of claim 1 wherein the percen-tage of total collagen remaining in solution after the second nucleation is about 15%.
8. A malleable, elastic, non-immunogenic form of collagen characterized on a microscopic scale by interlocking rope-like fibers having an average diameter of 170-300 nm.
9. A collagen preparation comprising inter-locking rope structures having average diameter of approximately 170-300 nm.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/648,001 US4557764A (en) | 1984-09-05 | 1984-09-05 | Process for preparing malleable collagen and the product thereof |
US648,001 | 1984-09-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1260650A true CA1260650A (en) | 1989-09-26 |
Family
ID=24599038
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000485572A Expired CA1260650A (en) | 1984-09-05 | 1985-06-27 | Process for preparing malleable collagen and product thereof |
Country Status (5)
Country | Link |
---|---|
US (1) | US4557764A (en) |
EP (1) | EP0174175A3 (en) |
JP (1) | JPS61137826A (en) |
AU (1) | AU579538B2 (en) |
CA (1) | CA1260650A (en) |
Families Citing this family (55)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61122222A (en) * | 1984-11-19 | 1986-06-10 | Koken:Kk | Hemostatic agent composed of collagen or gelatin and protamine |
US4600533A (en) * | 1984-12-24 | 1986-07-15 | Collagen Corporation | Collagen membranes for medical use |
JPS61230728A (en) * | 1985-04-06 | 1986-10-15 | Koken:Kk | Surfactant comprising acylated collagen or acylated gelatine and its preparation |
JPS61234918A (en) * | 1985-04-10 | 1986-10-20 | Koken:Kk | Surfactant consisting of acylated collagen and acylated gelatin and its production |
US4776890A (en) * | 1985-12-18 | 1988-10-11 | Collagen Corporation | Preparation of collagen hydroxyapatite matrix for bone repair |
EP0250571B1 (en) * | 1986-01-06 | 1991-05-22 | The University Of Melbourne | Precipitation of collagen in tactoid form |
BR8707660A (en) * | 1987-02-12 | 1989-08-15 | Univ Melbourne | COLLAGEN PRODUCTS |
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US3131130A (en) * | 1961-07-31 | 1964-04-28 | Ethicon Inc | Method of producing soluble collagen |
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US4295894A (en) * | 1979-11-19 | 1981-10-20 | Seton Company | Method of preparing soluble collagen fibers |
US4420339A (en) * | 1981-03-27 | 1983-12-13 | Kureha Kagaku Kogyo Kabushiki Kaisha | Collagen fibers for use in medical treatments |
US4424208A (en) * | 1982-01-11 | 1984-01-03 | Collagen Corporation | Collagen implant material and method for augmenting soft tissue |
US4505855A (en) * | 1982-09-30 | 1985-03-19 | Massachusetts General Hospital | Transparent non-fibrilized collagen material by ultracentrifugation |
-
1984
- 1984-09-05 US US06/648,001 patent/US4557764A/en not_active Expired - Fee Related
-
1985
- 1985-06-27 CA CA000485572A patent/CA1260650A/en not_active Expired
- 1985-09-02 EP EP85306214A patent/EP0174175A3/en not_active Withdrawn
- 1985-09-03 AU AU47013/85A patent/AU579538B2/en not_active Ceased
- 1985-09-05 JP JP60195010A patent/JPS61137826A/en active Pending
Also Published As
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EP0174175A2 (en) | 1986-03-12 |
JPS61137826A (en) | 1986-06-25 |
AU579538B2 (en) | 1988-11-24 |
EP0174175A3 (en) | 1987-07-29 |
US4557764A (en) | 1985-12-10 |
AU4701385A (en) | 1986-03-13 |
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