JPH0136840B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an aqueous composition of crosslinked collagen applicable to compositions and methods for treating body tissues.
The present invention relates to aqueous compositions of crosslinked collagen useful as injectable collagen implants for soft tissue augmentation in mammals. Collagen is used as a pharmaceutical carrier, surgical prostheses (sutures and dressings), and implants. (Chvapil, etal, Intl Rev of Connective
Tissue Res (1973) 6:1) Collagen is often cross-linked by chemicals, radiation, or other means to improve mechanical properties, reduce immunogenicity, or increase absorption resistance. Ru. This known crosslinked collagen has solid properties. Implants made from solid cross-linked collagen were surgically implanted. (i.e., implanted by incision.) Oliver et al., Clinical Orthopedics & Related
Research (1976) 115:291-302, Br J Exp.
Path (1980) 61:544-549 and Conn Tissu
Res (1981) 9:59-62 describes implants obtained by treating the skin with trypsin and then crosslinking with aldehydes. The resulting solid collagen implants are reported to maintain their original size long after implantation. The major problem with such solid implants is that they must be surgically implanted. Other problems are that they are not as easily deformed as injectable implants and that residual glutaraldehyde can cause the implant to lose its flexibility due to continued crosslinking in situ. Schechter et al., r J Plas Surg (1975)
28:198-202 discloses skin crosslinked with glutaraldehyde that is soaked in L-alanine after crosslinking. This article postulates that exposing the skin to L-alanine blocks residual reactive groups of aldehydes, thus preventing the release of toxic molecules generated by such reactive groups. US Pat. No. 3,949,073 describes the use of collagen atelopeptide solutions as injectable implants for soft tissue augmentation. According to the description in this publication, collagen is reconstituted before transplantation, and once transplanted, it becomes a fibrous tissue. This publication also suggests adding insoluble collagen microfibrils to control the contraction of the fibrous body formed at the augmentation site. A commercial product of the material described in the above publication is ZYDERM™ collagen implant, which consists of a reconstituted aqueous atelopeptide sodium chloride solution containing a small amount of local anesthetic. Although this commercially available product is highly effective, it may shrink when implanted, primarily due to its liquid component being absorbed into body tissue. Therefore,
If volume constancy, sometimes referred to as "permanence," is important, replacement implant material must be added by injection (not necessarily once). The primary object of the present invention is to provide collagen useful for skin augmentation and (1) homogeneous, i.e., the collagen has only a single physical shape; (2) injectable; 3) To provide a cross-linked collagen implant with superior durability and resistance to physical deformation compared to ZYDERM collagen implants. One aspect of the invention is a novel chemically cross-linked atelopeptide collagen containing 20-50 mg/ml protein, being a viscous fluid at 22°C, and substantially free of residual cross-linking agents. It is an aqueous composition. The collagen aqueous composition generally has a viscosity of about 700 to about 3000 cp at 22°C and contains about 20 ppm or less of residual crosslinker. Another aspect of the invention is the reconstitution of atelopeptide collagen from an acidic aqueous solution by neutralizing the solution at reduced temperature and hypotonic ionic strength, and at 22°C to form a viscous fluid. crosslinking the reconstituted atelopeptide collagen in an aqueous medium with a crosslinking agent that covalently bonds to the collagen at concentrations and conditions sufficient to produce crosslinked collagen; and an agent that reacts with the crosslinking agent ( The chemically crosslinked atelopeptide described above is characterized by terminating the crosslinking reaction by adding a crosslinking terminator (hereinafter referred to as a crosslinking terminator) and separating the viscous crosslinked atelopeptide collagen from the reaction mixture. A method of preparing an aqueous composition of collagen. Cross-linked collagen for use in the present invention can be derived from collagen harvested from many mammalian sources. The donor need not be genetically similar to the recipient into which the collagen material will ultimately be implanted. Cow or pig dermis is usually used because it is readily available. The first step in making cross-linked collagen is to prepare an atelopeptide collagen solution from the dermis. The animal's skin is softened by soaking in mild acid and then scraping the skin to remove hair, epidermis and fat. The depilated skin is then soaked in medium acid again,
Pulverize by physical methods such as grinding, shredding, milling, etc. This powder prepares the skin for solubilization. Ground tissue can be solubilized without denaturation by dispersing it in an acidic aqueous medium and digesting it with proteolytic oxygen other than collagenase.
At low temperatures, dilute acid solutions are usually used to avoid denaturation. Mineral acids such as HCl and carboxylic acids such as acetic acid, malonic acid, lactic acid, etc. can be used in a PH range of about 1.5 to 5 and a temperature range of about 5 to 25°C. 20℃,
Preferably, the ground tissue is dispersed in HCl to a concentration of 1-5 g/m at about PH2. After dispersion of the tissue, oxygen is added and the mixture is incubated to allow the enzymes to digest the telopeptide portion of the collagen and other solubilized components of the tissue. The enzyme used is one that digests the telopeptide portion without denaturing the collagen helix. Examples of such enzymes are trypsin, pepsin, chymotrypsin and papain. Pepsin is preferred because it is relatively easy to deactivate and separate from solubilized collagen. Typically, the enzyme concentration will range from about 0.1 to 10% by weight based on collagen. Cultivation time is generally about 2 days ~
You can choose between two weeks. The progress of solubilization can be checked by determining the viscosity of the solution. The point at which the viscosity reaches a nearly constant value is the end point of solubilization, at which point the enzyme is deactivated (denatured) and separated. Inactivation of the enzyme can be accomplished by adding an alkaline substance such as sodium hydroxide to bring the pH of the solution to at least about 7. After the enzyme is inactivated (denatured), the solution is processed to separate the denatured enzyme and the tissue portion that was digested during solubilization. Various dialysis methods, sedimentation methods, and filtration methods can be applied to this separation. In a preferred embodiment, acid is first added to lower the pH and then the solution is clarified by diatomaceous earth precipitation. Filter the precipitate and concentrate the liquid. The concentrate is then fractionated by ion exchange chromatography and further concentrated to a substantially pure atelopeptide collagen solution that can be used to make crosslinked collagen. The next step in making cross-linked collagen is to reconstitute the atelopeptide collagen from solution. Reconstitution is preferably carried out by neutralizing the solution at a low temperature, preferably about 10°C to 25°C. Preferably, the ionic strength of the neutralizing solution is low compared to physiological conditions. about 0.03 to about 0.1,
Preferably an ionic strength of about 0.06 is commonly used. Neutralization involves raising the PH of the solution by adding a suitable base or buffer, such as Na ₂ HPO ₄ or NaOH, to a level where the collagen solution reassociates with the fibrils. The fiber is about 4.9
is formed under these conditions in the PH range of ~10.0. Preferably, the final PH is in the range of about 5-8. Within this range, if the pH is less than about 7, thin and soft fibrils are likely to be formed.
If the pH is 7 or higher, coarser fibrils are likely to be formed. Soft fibrils are more flexible and form dispersions with a creamier texture than dispersions of coarse fibrils. Such a structure makes it easier to inject soft fibril dispersions. The time required for the fibril formation step is usually about 1/2 to about 18 hours. The reconstituted atelopeptide fibrillar collagen gel suspension is crosslinked with a crosslinking agent that forms covalent bonds with collagen. Typically, crosslinkers are polyfunctional, although difunctionality is more common. The crosslinking conditions provide an implant that can be formulated as an injectable fluid and has superior permanence to implants made from comparable formulations of non-crosslinked fibrillar atelopeptide collagen. Conditions are such as to produce viscous, covalently cross-linked collagen. When crosslinking reaches this extent, the crosslinking reaction is terminated by adding a crosslinking terminator. The crosslink terminator forms a non-hazardous water-soluble adduct with the crosslinker. The concentration of the crosslinking agent and the duration of the crosslinking reaction are important process conditions in obtaining a degree of crosslinking that provides a viscous fluid product that is injectable as such. Termination is important to prevent further crosslinking and changes in product viscosity. Inactivation of the reactive groups of the crosslinker also causes such reactive groups to be removed from the product, leaving the product as
Reduces immunogenicity compared to unterminated cross-linked collagen. Aldehydes are suitable crosslinking agents. Examples of aldehydes used to crosslink collagen include formaldehyde, glutaraldehyde, acetaldehyde, glyoxalpyruvate aldehyde, and dialdehyde starch. Glutaraldehyde is particularly preferred. Compounds having functional groups that react with functional groups (eg, aldehyde groups) of the crosslinking agent to form water-soluble adducts can be used to terminate the crosslinking reaction. Crosslink terminators having free amino groups such as amino acids are preferred. Glycine is particularly preferred. The concentration of glutaraldehyde in the reaction mixture is generally about 0.001
~0.05% by weight. Glutaraldehyde is
Reacts with lysine residues in collagen fibers, reducing the amount of free lysine per 1000 amino acids in collagen. At the above-mentioned glutaraldehyde concentrations, the number of free lysine residues in the 1000 residues remaining after crosslinking is greater than about 15/1000, and generally greater than about 20/1000. The amount of lysine is determined by reducing cross-linked collagen with borohydride and removing the reduced product.
It can be measured by hydrolysis in 5.7NHCl under vacuum at 100°C for 24 hours. Amino acid analysis can be performed with commercially available analytical equipment (eg, Durrum model D-500 analyzer), and lysine residues are quantified by comparing the lysine/alanine ratio to a non-crosslinked comparison. The duration of the crosslinking reaction is generally from half an hour to about a week. The reaction is usually carried out at about 10°C to about 35°C.
The rapid crosslinking terminator is added in at least stoichiometric proportion to the crosslinking agent. It is preferable to add the crosslinking terminator in excess. A particularly preferred crosslinking and termination step regimen is as follows: crosslinking with about 0.01% by weight glutaraldehyde at about 22°C for about 16 hours, excess glycine to about 0.2M at about 22°C. Time~2
Finish it in time. After crosslink termination is complete, the crosslinked atelopeptide collagen product is washed with an aqueous buffer solution to remove the aldehyde-crosslink terminator adduct, unreacted aldehyde, aldehyde polymer, and unreacted crosslink terminator. A sodium phosphate-sodium chloride buffer solution with a pH of 6.9 to 7.4 is preferred. The washed product has a suitable protein concentration, typically about 20-50
mg/ml, preferably about 25-40 mg/ml.
Concentrate by filtration or centrifugation. The protein concentration can be adjusted to the above range by adding a buffer or further concentrating, depending on the case. The washed product contains about 20 ppm or less free aldehydes and has a steady flow, viscosity.
An oscillating disk viscosimeter (Nametre) that measures kinematic viscosity rather than
Co., Ltd., Type 7.006PBD) at 22°C in the range of about 700 to about 3000 cp. The final formulation of the aqueous suspension of terminated crosslinked collagen allows the ionic strength of the suspension to be adjusted to isotonicity (i.e., from about 0.15 to about 0.2) and to reduce local pain upon injection. and adding a local anesthetic such as ridecaine to a concentration of about 0.3% by weight. Next, fill a syringe with a #27 gauge or larger needle with the suspension. The term "injectable" as used herein means that the formulation can be dispensed from the syringe with normal manual pressure. The above steps in the process of preparing the new injectable crosslinked collagen are preferably performed under sterile conditions using sterile materials. The collagen transplantation material of the present invention described above can be injected intradermally or subcutaneously for the purpose of augmenting soft tissues, treating or correcting congenital abnormalities, acquired defects, and cosmetic defects. Examples of these include hemifacial hypoplasia, cheek and zygomatic hypoplasia, unilateral breast hypoplasia, funnel chest,
Congenital anomalies such as hypoplasia or agenesis of the pectoral muscles (Poland anomaly) and velopharyngeal dysfunction associated with cleft palate or submucosal cleft palate treatment (as a retropharyngeal graft), depressed scars, (e.g. DLE discs) submucosal atrophy (associated with lupus erythematosus), enucleated eye enophthalmos (superior temporal sulcus syndrome), acne scarring on the face, linear atrophy (scleroderma) with submucosal atrophy, saddle nose syndrome, Romberg disease, Acquired defects (after trauma, surgery, infection) such as unilateral vocal cord paralysis, and cosmetic defects such as glabellar frown lines, deep nasolabial folds, geometric wrinkles around the mouth, sunken cheeks and breast hypoplasia. There is a defect on the top. The following examples illustrate the advantages of crosslinked collagen having viscosity, a transplant composition made from the collagen, a method for using the transplant composition, and an implant made from the transplant composition. The present invention is not limited to these examples. Preparation of Bovine Atelopeptide Collagen Solution Bovine skin was softened and hair removed by HCl treatment. Next, crush the depilated skin and add HCl of PH2 to 8-11
It was dispersed in an amount of g/g. 0.1 for total protein
% by weight of pepsin was added to this dispersion and the mixture was
Culture was performed at 15-20°C for about 100-300 hours. next,
Raise the pH of the culture medium to approximately 7 by adding NaOH;
This stopped the digestion. Inactivated (denatured) enzyme was removed from the reaction mixture by precipitation at low pH. The solution was then clarified and concentrated by filtration and chromatography to produce a 3 mg/ml solution of bovine atelopeptide collagen in dilute hydrochloric acid (PH 1-4). Hereinafter, this solution will be referred to as CIS. Preparation of fibrillar collagen Add 0.02 MNa ₂ HPO ₄ to CIS at 18°C to 20°C,
The PH was raised to 7.4±0.2 or 5.8-6.5 to reconstitute fibrillar collagen from the CIS. Fibers were allowed to form for 1-2 hours. Preparation of cross-linked viscous collagen A: Using collagen reconstituted at PH7.4±0.2. Neutral reconstituted fibrous collagen suspension
1.0% glutaraldehyde aqueous solution with pH 3 in 160ml
Added 1.62ml. The glutaraldehyde solution was gradually added while stirring until the final concentration was 0.01%. After a reaction time of 16 hours, the reactants were
It was terminated by adding 3M glycine to 0.2M. The finishing time was one hour. The cross-linked collagen was then washed at pH 7.4 with centrifugation at 17,000 g for 5-7 min between each wash.
Approximately 100ml of buffer, 0.002M Na ₂ HPO ₄ , 0.13M
was washed three times with NaCl. The kinematic viscosity in collagen was measured using a vibrating disk viscometer (NammetreCo.,
7.006PBD at a shear rate of approximately 5000 sec ^-1 ) and was found to be approximately 700 cp at 22°C. After the final wash and centrifugation, the collagen was resuspended in buffer until the protein concentration was approximately 28-35 mg/ml. This dispersion was filled into a syringe fitted with a #27 gauge needle. This collagen preparation will be referred to as preparation C hereinafter. Preparation of injectable cross-linked collagen fluid
It was performed as described above using various crosslinking reaction times and glutaraldehyde concentrations. These preparations are listed in the table below.

[Table] B When using collagen reconstituted at PH5.8-6.5. A 1% aqueous glutaraldehyde solution at pH 3 was gradually added to the reconstituted fibrocollagen dispersion while stirring to give a final glutaraldehyde concentration of 0.005% to 0.010%. Crosslinking was allowed to proceed for 16 hours with stirring. 2M glycine was then added with stirring until the final concentration was 0.2M-0.3M to terminate. The completion required 2-3 hours with stirring. Next, the terminated cross-linked collagen was collected after centrifugation at 16000g for 5-10 minutes, and 0.02M Na ₂ HPO ₄ ,
Resuspend in 10-40 volumes of 0.13M Nacl, PH7.4. The suspension was centrifuged at 16000 g for 5-20 minutes to obtain the final, viscous, cross-linked collagen product. The protein concentration after the final centrifugation was 45±5 mg/ml. In vivo test of collagen preparation Sprague- 45 to 50 days old, weight 120 ± 20 g
Dawley female rats were used as transplant recipients. Groups of three rats were implanted with Preparations A, C, E, and G and ZYDERM collagen implants as a control agent. Each rat has two locations:
The cross-linked collagen preparation was implanted into the right dorsal cranial region, and the control agent was implanted into the left dorsal cranial region. Approximately 1 c.c. of the preparation was injected subcutaneously per site. All preparations were administered ex vivo 15 days after implantation.
The donor tissue was carefully dissected from the explant and its wet weight was recorded. Weight recovery rate (persistence) was then calculated from the implanted weight. The weighed specimens were then embedded, sectioned, and stained for histological examination. The dyes used were hematoxylin, eosin, and Gomori trichrome. Test Results A summary of the histological examination of Preparations A, C, E and G is provided below. Preparation A: This preparation was more cross-linked and had a much more uniform lace-like appearance compared to the implants of the non-glycine terminated preparation. Preparations that are more cross-linked and not glycine-terminated generally form larger, more closely packed areas with intervening clefts. Tears also occurred between the implants of Formulation A, but were smaller and fewer in number. Preparation agent A
There was good infiltration of fibroblasts within the implant material as well as within the small intervening fissures.
New collagen appeared to be synthesized within the fissure. Almost no round cells were observed. Vascularization was moderate in the peripheral half of the A preparation implant and was not confined to the new collagen synthesis zone. No evidence of encapsulation was seen.
There were no epithelioid cells and no multinucleations. Formulation C: This formulation was more lacy and porous than Formulation A. This preparation showed excellent dispersive cell infiltration with areas of new collagen synthesis. These properties, along with the generally small number of round cells, are the reasons why Preparation C is considered the best of the four preparations from a histological standpoint. Formulation E: This formulation was generally less lacy than the one crosslinked with 0.01% glutaraldehyde. Fibroblasts were distributed diffusely, but in small numbers, and angiogenesis was generally limited to the peripheral third of the implant. Areas of new collagen synthesis were also observed less frequently compared to other preparations. Formulation G: This formulation exhibited the most uniform lacy porous appearance of all formulations crosslinked with 0.05% glutaraldehyde. Although the migration of cells into this preparation was good, an increase in the number of multinucleated focal areas and round cells was also evident. This is 0.01%
This was not observed in the preparation cross-linked with glutaraldehyde. The following table reports the persistence (percent wet weight recovery of the carefully dissected explants relative to the wet weight of the implants) of the preparation and control agents. Preparation Persistence A 77% C 68±5% E 82±2% G 64±5% Control (average) 30-40%

Claims (1)

[Claims] 1 (a) Contains 20 to 50 mg/ml of protein and has a value of 700 to 3000 cp at 22°C as measured by a vibratory viscometer.
(b) substantially free of residual crosslinking agent. 2. The collagen aqueous composition according to claim 1, wherein the amount of residual crosslinking agent in the collagen is 20 ppm or less. 3. The collagen aqueous composition according to claim 1 or 2, wherein the crosslinking agent is glutaraldehyde. 4 Chemically crosslinked atelopeptide collagen, which is a viscous fluid containing 20 to 50 mg/ml of protein and having a viscosity of 700 to 3000 cp at 22°C as measured by an oscillatory viscometer and substantially free of residual crosslinking agents. A method for the preparation of an aqueous composition, characterized in that it consists of the following steps: (a) reconstituting atelopeptide collagen from an acidic aqueous solution by neutralizing the acidic aqueous solution at low temperature and low tonic ionic strength; (b) crosslinking the reconstituted atelopeptide collagen in an aqueous medium with a crosslinking agent that forms a covalent bond with the collagen at a concentration and under conditions sufficient to produce crosslinked collagen; ) terminating the crosslinking reaction by adding a crosslinking terminator that reacts with the crosslinking agent to maintain a viscous fluid at 22°C; and (d) removing other reaction products and unreacted crosslinker from the reaction mixture. to separate the cross-linked atelopeptide collagen as a viscous aqueous composition. 5. The method according to claim 4, wherein the residual amount of the reactive crosslinking agent is 20 ppm or less. 6 The temperature in step (a) is 10°C to 25°C, the ionic strength in step (a) is 0.03 to 0.1, and the step
The final pH of (a) is between 4.9 and 10.0, the crosslinking agent is glutaraldehyde, and the concentration of glutaraldehyde in the crosslinking reaction mixture is between 0.001% and 0.05% by weight. Range 5th
The method described in section. 7 Collagen is from cow dermis,
The final pH of step (a) is in the range of 5 to 8,
The crosslinking reaction is carried out for half an hour to one week, the crosslinking terminator is glycine, and the separation includes washing the viscous crosslinked atelopeptide collagen to remove other reaction products and unreacted reactants; 7. The method according to claim 6, wherein the content of reactive aldehyde in the aqueous composition is 20 ppm or less. 8. The method according to claim 4 or 5, wherein the reaction terminator is an amino acid.