RU2433828C1 - Injection heterogenic biopolymer hydrogel for substitutional and regenerative surgery and method of its obtaining - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: injection heterogeneous elastically-resilient biodegradable biopolymer hydrogel for substitutional and regenerative surgery, obtained from hydrolysate of embryonic or postnatal collagen-containing tissues of animal origin, excluding human, which consists of two constituents: hard - microparticles from linked hydrolysate and liquid - from initial hysrolysate, taken in specified ratio, with particle size not exceeding 100 mcm. Method of obtaining injection hydrogel includes crush of initial raw material, freezing obtained mass under definite conditions, following defrosting, processing with solution of icy acetic acid, separation of supernatant liquid, washing with water, processing with sodium hydroxide solution, centrifuging obtained hydrolysate of animal tissues in form of hydrogel mass with its further filtration, part of obtained hydrolysate is processed by γ-irradiartion in dose 1.0 kGy in gaseous medium and homogenised until particles with size not larger than 100 mcm are obtained, washed with phosphate buffer solution and mixed with remaining volume of initial hydrolysate of animal tissues, obtaining injection heterogeneous elastically-resilient biodegradable biopolymer hydrogel.

EFFECT: hydrogel possesses immunogenicity, prolonged effect of biostimulating properties.

5 cl, 4 dwg, 3 tbl, 6 ex

Description

The invention relates to medicine, in particular to a new biodegradable biopolymer hydrogel for replacement and regenerative surgery of soft tissues and a method for its preparation.

To restore the functions of damaged tissues and organs in a number of serious diseases and for plastic surgery, new materials are being actively developed, with the main emphasis on biopolymer materials being considered as the most biologically safe and functionally effective [Advances in replacement organs and tissue engineering. Technical Insights, Frost & Sullivan, 2008].

In this regard, one of the key problems is the development of biodegradable two-dimensional and three-dimensional materials with specified medical and technical properties both for repairing defects in soft and hard tissues, and for transplanting cells of organs and tissues.

In recent decades, there has been a growing interest in biodegradable natural (biological) polymers or biopolymers (alginates, collagen, gelatin, chitosan, silk fibroins, polyesters of bacterial origin - polyoxy butyrates and their copolymers). Natural polymers, in addition to a high degree of biocompatibility with the body, are highly effective biostimulants [Atala A., Lanza R., Thompson J., Nerem R. Principles of regenerative medicine. Academic Press is an imprint of Elsevier, First edition, 2008, 1473 p .; Shumakov V.I., Sevastyanov V.I. Biopolymer matrices for artificial organs and tissues. Health and medical technology. 2003, No. 4, p.30-33; Kim B.-S., Baez C.E., Atala A., Biomaterials for tissue engineering. World J. UroL, 2000, v. 18 (4), p. 2-9].

Intermediate products of biodegradation of such materials may be substances included in the metabolism of cells, for example, monosugar, lactic, glycolic and β-hydroxybutyric acids, or substances not metabolized by cells and tissues. Products of the complete degradation of biopolymers are carbon dioxide and water.

Of the listed biopolymers, the most common materials for the manufacture of medical devices, including implants, are collagen, which is a three-helix protein with a molecular weight of 300,000 Da.

Collagen is a protein that is the main fibrillar component of the extracellular matrix, as well as the most important component of connective tissue [Khilkin A.M., Shekhter A.B., Istranov L.P., Lemenev V.L. Collagen and its use in medicine, Moscow, "Medicine", 1976, 210 S.]. It makes up about 30% of the total protein mass in mammals and is present in almost every tissue, providing strength and structural stability. Although collagen is the predominant extracellular matrix glycoprotein, the extracellular matrix contains many other proteins: fibrin, elastin, fibronectins, laminins, and nidogens.

The various mechanical and physiological properties of various loose and dense types of connective tissue (dermis, tendons, cartilage, cornea, organ stroma, synovial membrane, etc.) are determined by quantitative and qualitative variations in the relationships between cells, fibers and the main substance.

All types of soluble collagen can be divided into two categories. The first category covers soluble collagen fractions obtained by direct extraction from connective tissue (native solutions), the second covers soluble collagen obtained after preliminary enzymatic, chemical or mechanical treatment of insoluble, mature collagen (forcibly dissolved or solubilized collagen). Extracted by acidic buffers (soluble) collagen is called procollagen. They differ only in metabolic activity, content in tissues and the degree of intramolecular binding of peptide chains.

Collagen has extremely weak antigenic properties, and also has extremely weak anaphylactogenic and toxic properties. When collagen is introduced into the body, it undergoes rapid resorption, splitting, it stimulates reparative processes, in particular the formation of the body’s own collagen, has hemostatic properties. In this regard, collagen and collagen-based products are widely used in cosmetology and medicine.

Mucopolysaccharides, or glycosaminoglycans, which are part of the extracellular matrix, are of the greatest importance in the formation of collagen structures. In the connective tissue, they exist in the form of complexes with proteins (proteoglycans) and are associated with the latter not only by electrovalent (ionic, hydrogen, etc.) bonds, but also by more durable covalent bonds. Collagen is the only animal protein that contains a large number of amino acids such as hydroxyproline, glycine and proline. The level of oxyproline characterizes the amount and purity of collagen. Collagen contains simple sugars: glucose, galactose, mannose, which provides cell nutrition.

The most important quality of collagen as a biopolymer is its biocompatibility, which is due to the structure of its molecule and physico-chemical properties. Collagen is able to be resorbed (absorbed) and utilized by the body, breaking down into simpler compounds that are excreted from the body or take part in biosynthesis at the cellular level.

Practical studies on the mechanism of biostimulating and therapeutic properties of collagen have shown its active effect on the implementation of many functions of connective tissue, especially morphogenetic and reparative. He actively participates in the interaction with cells that organize the reparative (restoring) process, which significantly reduces the healing time. However, along with the listed advantages, a serious drawback of collagen matrices is an unregulated biodegradation time and a limited period of functioning of collagen products (up to 1 month) in a living organism, which is insufficient for complete recovery and leads to the formation of scar tissue.

To reduce the rate of bioresorption, collagen is crosslinked using chemical agents, ultraviolet radiation or subjected to hydrothermal crosslinking. For example, chemical crosslinking can be accomplished using chondroitin-4-sulfate and / or chondroitin-6-sulfate. As an example, we can consider the patent of the Russian Federation No. 2188206, published in August 2002, which refers to the use of native collagen or such in a crushed and solubilized state, gradually heated with certain physicochemical properties. This drug can be used in pharmaceutical, medical, surgical, ophthalmic and cosmetic compositions. However, the resulting collagen gel cannot be injected through an injection needle.

Different collagen-containing substances and biocompositions that can be used for surgical and bioplastic purposes can be considered analogues of the present invention: for example, a method for producing a means stimulating reparative processes (RF patent No. 2065745, 08/27/1996); wound healing coating (RF patent No. 2085217, 1997), tissue plastic material (RF patent No. 2137441, 1999), a reinforced graft for scleroplastic operations (RF patent No. 2140242, 1999), release agent (RF patent No. 2155592, 2000), a biocompatible polymer material and a method for its preparation (RF patent No. 2162343, 2001), collagen-containing material for keratinoplasty (US patent No. 6197330, 2001), a method for surgical removal of cicatricial deformities of the skin (RF patent No. 2201146, March 2003), a method for producing collagen I and collagen II, capable of rac asyvaniyu collagen matrix intended to reconstruct the cartilage (RF patent №2323011, 27.04.2008 city).

However, the listed developments describe the use of homogeneous collagen or other components of the extracellular matrix, often in combination with other polymers, to replace soft tissue defects and create biological implants.

As the closest analogue (both for the hydrogel and the production method), we can consider the patent of the Russian Federation No. 2249462, 04/10/2005, where it is a universal heterogeneous collagen matrix for implantation, which is an elastic-elastic mass obtained from two sources of collagen, and one source is the tissue of a vertebrate animal of one class, and the second is an animal of another class, while collagen sources are animal tissues, for example, the class of mammals and the class of birds. The matrix consists of two phases: solid - in the form of microspheres from collagen tissue of mammals 100-300 microns in size, and liquid - from denatured collagen of bird tissue, with a phase ratio of (1-10): (1-10), the final products of matrix biodegradation are CO ₂ and H ₂ O.

The main disadvantages of this heterogeneous collagen matrix are:

- observed cases of immunogenicity caused by collagen microspheres;

- a relatively short-term effect of the manifestation of biostimulating properties due to the high rate of biodegradation of the liquid phase of the denatured collagen of bird tissue, which is responsible for biostimulating properties;

- the possibility of introducing a hydrogel through a thin insulin needle (diameter 0.45 mm) only at large dilutions of the solid phase, which significantly limits its scope.

The technical result achieved by using the proposed group of inventions is:

- the exclusion of immunogenicity due to the lack of native collagen in the composition of the proposed heterogeneous gel and the use of embryonic or postnatal collagen-containing tissues of animal origin (excluding humans) that do not have immunogenic activity, do not have immunogenic activity,

- an increase in the duration of the manifestation of the effect of biostimulating properties due to the use of extracellular matrix of animal origin (excluding humans) as a raw material for the extracellular matrix hydrolyzate, which is a unique complex of polypeptides, hexosamines, uronic acids and other biologically active substances,

- the possibility of introducing a hydrogel through a thin needle (with a diameter of less than 0.45 mm) due to the use of cross-linked and non-cross-linked components from the hydrolyzate of animal tissues (excluding humans) in the embryonic or postnatal period due to the smaller average microglobul size (50 μm versus 100 μm) and their greater elasticity due to the lower molecular weight of the components of the hydrolyzate. So, the molecular weight of pharmacopoeial collagen obtained by alkaline-salt treatment is ~ 450 kD, and the molecular weight of tropocollagen (acid-soluble collagen) is ~ 400 kD [Babloyan O.O., Golovanova P.M. // Experimental and clinical aspects of the use of biological polymers in medicine: Proceedings. - M., 1981. - S. 34-37], while the mass of the protein component of the hydrolyzate - polypeptides does not exceed 6,000 Da.

A priority area of application of the proposed heterogeneous biopolymer hydrogel is the use of soft tissues as an implantable material or an application wound healing coating for injuries and burns to replace defects in soft tissues.

The method is as follows.

To obtain a heterogeneous biopolymer hydrogel, raw materials of animal origin are used, mainly healthy chickens at 5-8 days of age or pig embryos at gestational periods of not more than 120 days. As a raw material, you can use other sources of collagen-containing extracellular matrix of immature freshly killed animals, with weak antigenic properties. The raw materials are cleaned, washed, crushed to particle sizes from 4 to 10 mm and the resulting mass is frozen at a temperature not exceeding minus 18 ° C for a period of 10 to 90 days. Then it is thawed and immersed in a 1% aqueous solution of glacial acetic acid in a ratio of 1: 1 by weight for 8-12 hours at a temperature not exceeding 60 ° C and periodic stirring. After this, the solution is drained from the mass (the supernatant is separated) and washed until clean water. The resulting mass is treated at room temperature with a 0.5% sodium hydroxide solution in a ratio of 1: 1 by weight for 30-40 minutes, concentrating the dissolved active peptides, amino acids and mineral compounds extracted from the extracellular matrix during hydrolysis, centrifuging (obtained hydrolyzed tissue in the form of a hydrogel mass), fat removal and solution filtration. Then, part of the obtained hydrolyzate of animal tissue is treated with γ-radiation at a dose of 10 kGy in a gaseous medium and homogenized to obtain microglobules (microparticles) of no more than 100 μm in size, then washed with a phosphate buffer solution and mixed with the remaining volume of the original hydrolyzate of collagen-containing animal tissues.

Thus, a hydrogel is obtained from a hydrolyzate of animal tissue. The hydrogel consists of two components: solid - microparticles from a cross-linked hydrolyzate of collagen-containing tissues and liquid - from the original hydrolyzate of collagen-containing tissues in a ratio of 1:10 to 10: 1. The size of the microparticles does not exceed 100 microns.

EXAMPLE 1

Tests in vitro.

Cytotoxicity: 1) a heterogeneous biopolymer hydrogel on a suspension culture of motile cells (cattle seed) was not detected. The toxicity index was 100 ± 10% with acceptable values of 70-120%; 2) hydrogel on mouse fibroblasts of the 3T3 line of clone SC-1. The morphology of the cells was similar to the control, the number of cells differed from the control within the margin of error. No toxic effects have been identified.

The hemolytic effect of the hydrogel was tested on isolated human red blood cells. The degree of hemolysis was 0.010 ± 0.002% with a permissible value of the indicator not more than 2%.

EXAMPLE 2

Tests in vivo.

There is no irritating effect with a single installation in the conjunctival sac of the rabbit's eye. On a rating scale, the reaction corresponded to degree zero.

A study of the reactions of general anaphylaxis and active cutaneous anaphylaxis in guinea pigs showed the absence of allergic reactions of the anaphylactic type.

There was no sensitizing effect on white rats, the mast cell degranulation reaction was negative.

Implantation was performed on rats (intramuscularly). The observation period is 3 months. Morphological studies revealed no pathological changes in the surrounding tissues.

Sterility: Tested samples are sterile.

Pyrogenicity: Extracts prepared with 0.9% sodium chloride solution for injection did not show pyrogenic reactions after intravenous administration to rabbits. The total temperature increase did not exceed 1.4 ° C.

The resulting product (matrix, hydrogel) can be placed in disposable syringes, for example, with a volume of 1 ml, 3 ml, 5 ml, 20 ml, etc., the syringe is sterilized, provided with instructions for use and a label that indicates the contents of the package production date.

The invention is illustrated by the following laboratory tests of an injectable heterogeneous biopolymer hydrogel.

Tests of the proposed inventions were carried out under the conditions of an acute experiment in laboratory animals for the purpose of repairing damage to the spinal cord and peripheral nervous system.

EXAMPLE 3

For experimental damage to the spinal cord, we used sexually mature outbred female rats weighing at least 300 g. In accordance with the objectives of the study on rats, we used an acute model of spinal cord damage by removing a section of the spinal cord after dissection at the level of 10 vertebra of the thoracic spine, S . Woerly et al. [S.Woerly, V.D. Doan, F. Evans-Martin, C.G. Paramore, J.D. Peduzzi, "Spinal cord reconstruction using NeuroGel implants and functional recovery after chronic injury", J. of Neurosciensce Res. 2001. Vol.66, P.1187-1197].

Of all the rats operated, 20 control animals (group I) did not transplant injection heterogeneous biopolymer hydrogel (IHBG) into the area of spinal cord injury.

In the experimental group, 30 animals (group II) were injected with IHBH in the area of damage to the spinal cord. The IHBH implant was formed so that it corresponded in volume and shape to the damaged spinal cord region and filled the wound cavity, providing the hydrogel in full contact with the surfaces of the cut spinal cord. From above, the area of hydrogel administration and the adjacent areas of the spinal cord were isolated from the surrounding tissues by subcutaneous fat (TFA), as in the control.

During the observation, changes in muscle tone and motor activity of the hind limbs of rats were recorded. The results of observations over 2 months showed: in the control group of 20 animals in 85% of cases (17 rats) hind limb paraplegia persisted. In 3 rats (15%) at 35 ± 17 days (n = 3), a positive dynamics in the restoration of motor activity was revealed. In all three cases, hypertonicity of the muscles of the hind limbs was observed.

In experimental group II, when IHBH was implanted with isolation of the pancreas, positive dynamics of the restoration of motor activity was detected on 28 ± 10 days in animals (57%) (n = 17). All 17 rats noted hind limb hypertonicity with positive dynamics at the end of the experiment. In the remaining 13 rats out of 30, hind limb paraplegia persisted throughout. From a comparison of the results of the control and experimental groups, we can conclude that there are bistimulating (regenerative) properties of IHBH in the experimental model of a complete spinal cord break. The difference in the experimental results is significant with a reliability of p <0.5.

EXAMPLE 4

The objective of this study was to evaluate the effect of IHBH on the formation of scar tissue in the area of intersection of peripheral nerves and on the processes of regeneration and degeneration of nerve tissue. The studies were performed on female non-linear rats weighing 200-250 g. 10 animals were used in the experiment (20 surgical interventions on both hind limbs) - sciatic nerves were intersected under peritoneal anesthesia (a mixture of ketamine and thiopental was anesthetized). The studied groups included 8 animals (16 nerves), which, during operations, IHBH was introduced into the nerve intersection area. The control group (without the use of biomaterials) included 2 animals (4 nerves). Animals were withdrawn from the experiment on days 21 and 101 by overdose of thiopental anesthesia. After opening, the area of the operated nerve with adjacent tissues was removed and subjected to morphological processing. The preparations were prepared according to standard methods (staining: hemotoxylin and eosin, according to Van Gieson, Kluver-Barrer).

In the control group, without the use of any materials in the sciatic nerve transection area, the replacement of nerve tissue with a dense scar, the formation of scar-adhesions in the surgical area were determined (Fig. 1. General view of the sciatic nerve in the transection area. 101 days after surgery: Van- Gizon × 60, 1 - scar tissue, 2 - epineuria, 3 - muscle tissue).

In the group of operations performed with the introduction of the IHB in the area of intervention in the intervention area, a formed connective tissue scar was identified, in which regenerating axons were noted (Fig. 2. Scar tissue in the area of sciatic nerve transection using Sphero® Gel. 101 days after surgery: Van -Gizon × 1000, 4 - connective tissue fibers, 5 - axial cylinders, 6 - fibroblast).

The results of an experimental study of the use of a new injectable heterogeneous biopolymer hydrogel suggest an increase in the effectiveness of surgical interventions for traumatic lesions of the peripheral nerves made using this material.

With the introduction of an injectable heterogeneous biopolymer hydrogel into the area of the nerve suture, prerequisites are created for more efficient regeneration of axons and their germination through the area of anatomical damage.

EXAMPLE 5

Assessment of the biodegradation rate of IHBH during implantation.

Experimental animals (10 Wistar rats) were divided into two groups. Samples of collagen (2%) obtained by alkaline-salt treatment of a bovine dermis split skin split (FS 42-2670-89, manufactured by the Belkozin plant in Luga) and IHBH were injected intramuscularly into the left and right femoral muscles, respectively, simultaneously for each to the animal. Material sampling in the first group of rats was carried out after 1 month, in the second - after 3 months with subsequent histological analysis.

In vivo experiments have confirmed that microheterogeneity of the gel leads to an increase in its lifetime. At a period of 1 month after implantation of IHPG in the subcutaneous fatty tissue of the rat, there were no signs of sample degradation (Fig.3a. Degradation of IHBH when implanted into the subcutaneous fatty tissue of the rat: the histological picture after implantation of IHBH in the subcutaneous fatty tissue for 1 month (uv .x200), hematoxylin-eosin staining). After three months of implantation, fragmentation of the sample is noted, but the weight loss is not more than one third of the original (Fig.3b. The histological picture after implantation of IHBH in the subcutaneous fat for a period of 3 months: staining of hematoxylin - eosin, UV.x200). It was not possible to detect collagen at the sites of introduction of collagen samples because of its complete resorption.

Example 6

Comparative analysis of the chemical composition of type I collagen and IHBH.

Objects for testing: collagen (2%) obtained by alkaline-salt treatment of the golina split dermis of cattle. FS 42-2670-89. Production plant "Belkozin" Luga; hydrolyzate obtained.

Comparative research results are summarized in tables 1-3.

Table 1. Comparative data on the amino acid composition of collagen (obtained from the dermis of cattle) and IHP No. Name of amino acid The amino acid content in 100 g of dry protein Collagen IGBG one Glycol (glycine) 26.57 23.41 2 Alanine 10.32 9.14 3 Valine 2.46 2.43 four Leucine 3.73 3.00 5 Isoleucine 1.88 1.76 6 Proline 14.42 11.83 7 Oxyproline 12.83 10.14 8 Aspartic acid 6.95 5.51 9 Glutamic acid 11.16 10.24 10 Arginine 8.22 8.01 eleven Lysine 3.96 3.80 12 Oxylysine 1.15 1.19 13 Histidine 0.70 0.81 fourteen Series 4.27 2.86 fifteen Threonine 2.26 2.26 16 Cystine - - 17 Methionine 0.97 1.24 eighteen Phenylalanine 2,35 2.23 19 Tyrosine 0.99 0.73 twenty Tryptophan -

According to the amino acid composition, IHPG is identical to collagen obtained by alkaline-salt treatment of the golina split of the cattle dermis.

Table 2. The content of trace elements in IGPG and collagen Name of chemical element Chemical symbol Content in mg per kg IGPG Collagen Arsenic As 0 0 Beryllium Be 0 0 Calcium Sa 452.00 442.00 Cadmium Kd 0 0 Cobalt With 0.60. 0.62 Chromium Cr 1.14 1.17 Copper Cu 1.39 1.4 Iron Fe 5.79 5,0 Potassium K 40.19 42.1 Lithium Li 0.12 0.16 Magnesium Mg 53.61 50.61 Manganese Mn 0.74 0.76 Sodium Na 165.12 162.2 Nickel Ni 3.48 3.8 Phosphorus R 143.44 141.44 Lead Pb 0 0 Selenium Se 0 0 Silicon Si 6.02 6.2 Tin Ol 0 0 Titanium Ti 0.44 0.34 Tungsten W 0 0 Zinc Zn 10.41 8.41

Both samples are identical in microelement composition.

The hydrolyzate is 2 times superior to collagen in the content of hexosamines, and uronic acids more than 15 times. It’s like in a known material (not heterogeneous) in exoline.

At room temperature, the IHPG is an elastic homogeneous gel (Figure 4. General view of an injectable heterogeneous biopolymer hydrogel (IHPG) from an animal tissue hydrolyzate), stable during long-term storage, and not prone to syneresis (liquid phase separation). At a temperature of 37 ° IHPG turns into a liquid that mixes in all proportions with water. The high water solubility of IGPG at body temperature and the presence of lower molecular weight residues increase its resorption and penetration into tissues.

Thus, the advantages of IGBG are:

1. Both components: solid and liquid, are formed from a hydrolyzate with bio-stimulating properties.

2. Due to the significantly lower molecular weight of the hydrolyzate (not more than 6000 Da), the average microparticle size is about 50 μm, which allows you to enter the finished product through an injection needle at any ratio of the components (phases) that make up the hydrogel.

3. The prolonged biostimulating (reparative, regenerative) effect, because biostimulating properties, in contrast to the closest analogue, RF patent No. 2249462, published in April 2005, also has a solid component of a heterogeneous gel (in the form of microparticles), whose biodegradation rate during implantation or skin application is significantly lower than the rate of biodegradation of a liquid (homogeneous) component , which, accordingly, determines the prolongation of the biostimulating effect of IHBH.

4. The lack of allergenicity of IHBH due to the use of embryonic or postnatal raw materials of animal origin (excluding humans) that do not have immunogenic activity

Claims (5)

1. Injection heterogeneous elastically elastic biodegradable biopolymer hydrogel for replacement and regenerative surgery, which is obtained from a hydrolyzate of embryonic or postnatal collagen-containing tissues of animal origin, excluding humans, and it consists of two components: solid - microparticles from a crosslinked hydrolyzate and liquid - from the ratio of the original hydrolyzate from 1:10 to 10: 1, and the size of the microparticles does not exceed 100 microns. 2. Injection heterogeneous elastically elastic biodegradable biopolymer hydrogel for replacement and regenerative surgery according to claim 1, characterized in that the tissue of healthy chickens at 5-8 days of age or pig embryos for gestation no more than 120 days are used as collagen-containing tissues. 3. A method for producing an injectable heterogeneous elastically biodegradable biopolymer hydrogel for replacement and regenerative surgery, including grinding of raw materials - embryonic or postnatal collagen-containing tissues of animal origin, excluding humans, after which the resulting mass is frozen at a temperature of no higher than minus 18 ° C for a period of 10 to 90 days, then thawed, treated with a 1% solution of glacial acetic acid with a ratio of 1: 1 by weight at a temperature of not more than 60 ° C, separating t of supernatant, washing with water and subsequent re-treatment at room temperature with a 0.5% sodium hydroxide solution in a ratio of 1: 1 by weight for 30-40 minutes, then centrifuging the obtained hydrolyzate of animal tissues in the form of a hydrogel mass followed by by filtration, a part of the obtained hydrolyzate is treated with γ-radiation at a dose of 1.0 kGy in a gas medium and homogenized to obtain microparticles with a size of not more than 100 μm, washed with phosphate buffer solution and mixed with the remaining I starting volume of hydrolyzate of animal tissue, prepared injectable biodegradable biopolymer heterogeneous elastic hydrogel. 4. The method according to claim 3, characterized in that the resulting heterogeneous hydrogel is sterilized by γ-radiation at a dose of 15 kGy. 5. The method according to claim 3, characterized in that the tissue of healthy chickens at 5-8 days of age or pig embryos at gestational periods of not more than 120 days are used as collagen-containing tissues.

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