DE102011077809A1 - Biocompatible adhesive and process for its preparation - Google Patents

Abstract

The present invention relates to an adhesive comprising or consisting of 1 to 20% by weight collagen, 0.5 to 15% by weight hyaluronic acid or hyaluronate and 65 to 98.5% by weight water; the data in% by weight refer to the total weight of the adhesive; and a method for producing such an adhesive.

Description

Biocompatible adhesives or adhesives are used in many areas of medicine. It is not only important that the adhesive has a high adhesive effect, but it is also important that the adhesive is well-tolerated and thus biocompatible. For this reason, efforts are made to provide adhesives, which preferably consist of well-tolerated components.

For example, out US 5,209,776 . US 2008/0208114 A1 or US 4,280,954 Adhesives are known which may contain the components collagen and hyaluronic acid.

A problem of these adhesives is e.g. in that these adhesives either contain denatured collagen, a covalent bond between collagen and hyaluronic acid is produced and / or catalysts or other constituents are used which bring about an increased allergy potential.

The object of the present invention is to provide improved adhesives and processes for their preparation. In particular, it is an object of the present invention to provide adhesives and processes for their preparation which combine a sufficient adhesive effect with a high biocompatibility and a low allergy potential.

The object is achieved by providing a method for producing an adhesive, characterized in that an aqueous collagen solution having a dissolved collagen concentration of 0.01 to 5% (w / v) and a pH of 2 to 5 with a aqueous hyaluronate solution having a hyaluronate or hyaluronic acid concentration of 0.02 to 5% (w / v) and a pH of 6 to 7 is mixed and the resulting precipitate is separated from the liquid phase. Preferably, the mixture of the aqueous collagen solution and the aqueous hyaluronate solution is stirred until the precipitate has formed.

The inventive method is characterized in particular by the fact that the preparation of the adhesive requires no addition of a catalyst.

The resulting precipitate is a biocompatible adhesive with good adhesion, high biocompatibility and low allergy potential.

The adhesive according to the invention contains or consists of 1 to 20% by weight of collagen, 0.5 to 15% by weight of hyaluronic acid or hyaluronate and 65 to 98.5% by weight of water; the data in% by weight relate to the total weight of the adhesive.

After thorough mixing of the two components, aqueous collagen solution and aqueous hyaluronate solution, the starting polymers are structured as a polyionic complex (PIK) of positively charged collagen and negatively charged hyaluronic acid. Between the polymers arise ionic interactions and no covalent bonds. By means of a polyion complex, an improved sustainability of the adhesive properties is achieved via the interaction mechanism. The functional groups of the polymers are reversibly associated with each other via ionic interactions and are therefore fully reactive again after a temporary separation. The interaction of the two polymers results in a liquid phase and a gelatinous, sticky solid phase as a precipitate (also referred to herein as a coprecipitate or adhesive). The biocompatible adhesive (ie the solid, sticky phase, precipitate or coprecipitate) is recovered by separation from the liquid phase. To separate the two phases, various methods such as centrifugation, filtration or simple decanting can be used. The formed PIKs of the precipitate further possess functional, in particular nucleophilic groups, which can ensure a continuous interaction with surrounding cellular systems or tissues. The free functional groups of the polymers in the precipitate (adhesive) can thus bonds with the environment, eg. B. in the form of tissues or frameworks, received. The adhesive is particularly suitable for use in the medical field.

In the method according to the invention, an aqueous collagen solution is used which has a concentration of dissolved collagen of 0.01% to 5% (w / v), based on the total volume of the aqueous collagen solution. The aqueous collagen solution preferably has a concentration of dissolved collagen of from 0.1% to 1% (w / v), in each case based on the total volume of the aqueous collagen solution.

The aqueous collagen solution has a pH in the acidic range of from pH 2 to pH 5, preferably a pH of from 2.3 to pH 4.5.

For the purposes of the present invention, dissolved collagen is understood as meaning collagen which is present dissolved in the aqueous collagen solution and is essentially nonhydrolyzed and / or undenatured. The collagen may, for example, have been present as a native collagen or renatured. Preferably, the dissolved collagen is present in the aqueous collagen solution in native triple helical structure. The recovery of such collagen is known to those skilled in principle. Preferably, the collagen can be obtained from the skin, especially from the skin of animals such as pigs.

The collagen used according to the invention contains or consists preferably of type I collagen or of a mixture of different types of collagen, with type I collagen in the mixture preferably making up the largest proportion by mass in comparison to every other type of collagen. Other collagen types may be, for example, Type II, III, V collagen. However, other types of collagen may also be present in the mixture. The collagen according to the invention is preferably collagen type I in substantially pure form or a mixture of different types of collagen, type I collagen making up at least 30%, particularly preferably at least 50%, of the total collagen mass. The collagen may contain or consist of atelocollagen and / or tropocollagen. Atelocollagen is characterized by the fact that it is dissolved collagen without telopeptides.

The aqueous collagen solution is mixed in the process according to the invention with an aqueous hyaluronate solution. Hyaluronic acid or hyaluronate is preferably used here, which has an average molecular weight of 1,400 to 2,400 kDa. The aqueous hyaluronate solution has a hyaluronic acid concentration or a hyaluronate concentration of from 0.02% to 5% (w / v), preferably from 0.2% to 1% (w / v), based on the total volume of the hyaluronate aqueous solution. The hyaluronate used is a salt of hyaluronic acid with various cations, preferably the sodium salt of hyaluronic acid.

The aqueous hyaluronate solution, prior to mixing with the aqueous collagen solution, has a pH of from pH 6 to pH 7, preferably from pH 6.3 to pH 6.5.

The concentration data for collagen and hyaluronic acid or hyaluronate and the pH ranges for the aqueous collagen solution and the aqueous hyaluronate solution before the mixture of the two aqueous solutions are essential components for the fact that the precipitate and thus the desired adhesive can form without the use of a catalyst. This ensures that the adhesive on the one hand collagen in native or renatured form and thus allows the formation of a polyionic complex with hyaluronic acid and on the other hand, the use of catalysts that may have allergenic potential or are only slightly biocompatible, can be avoided.

The allergy potential of the resulting adhesive can be further reduced if, for example, hyaluronate or hyaluronic acid of non-animal origin, preferably of bacterial origin, is used. This can be obtained, for example, biotechnologically. It has proved to be particularly favorable when the hyaluronate or hyaluronic acid is peptide-free.

In the method of the present invention, the aqueous collagen solution and the hyaluronate aqueous solution are mixed together to produce a precipitate which, separated from the aqueous phase, is an adhesive of the present invention. Particularly favorable results are achieved when the aqueous collagen solution and the aqueous hyaluronate solution are mixed with each other in a fixed mass ratio. Preferably, the aqueous collagen solution and the aqueous hyaluronate solution are mixed together in a mass ratio of 1: 0.5 to 1: 1.5, more preferably in a mass ratio of 1: 0.7 to 1: 1.2.

The efficiency of the preparation of the adhesive with the method according to the invention can be further increased by stirring the mixture until a precipitate is formed.

After formation of the precipitate, the precipitate is separated from the aqueous phase and can be used as the adhesive of the present invention. In this case, the separation of the precipitate from the aqueous phase of the mixture can be achieved by conventional methods, for example by centrifugation, filtration and / or decantation.

Due to the choice of collagen in a native or renatured state and hyaluronic acid or hyaluronate as components of the adhesive, the resulting adhesive is characterized by a high biocompatibility. In principle, aqueous collagen solution and / or aqueous hyaluronate solution and thus if appropriate, the resulting adhesive also has further constituents, in which case preferably biocompatible constituents are used. For example, biocompatible ions, salts, organic or inorganic acids and / or bases, sugars, proteins, telopeptides and / or gelatin and mixtures thereof may be used.

The present invention also relates to an adhesive comprising or consisting of:
1 to 20% by weight of collagen,
0.5 to 15% by weight of hyaluronic acid or hyaluronate and
65 to 98.5% by weight of water;
the data in% by weight relate to the total weight of the adhesive.

In a preferred embodiment, the adhesive according to the invention contains or consists of:
5 to 15% by weight of collagen,
3 to 10 wt .-% hyaluronic acid or hyaluronate and
75 to 92% by weight of water;
the data in% by weight relate to the total weight of the adhesive.

The adhesive according to the invention is distinguished by a particularly favorable adhesive effect if collagen and hyaluronic acid or hyaluronate are present in a weight ratio of 0.5: 1 to 5: 1 in the adhesive.

The adhesive of the present invention may be prepared by the above-described process of the present invention for producing an adhesive.

The invention will be explained in more detail with reference to subsequent embodiments.

Characters:

1 Figure 4 shows results from the SDS-PAGE study of native collagen extracted with acid solutions, where in lane 1 a molecular weight standard (band at 64, 98, 148 and 250 kDa), in lane 2 a collagen standard (mixture of collagen type I and collagen type II) and in tracks 3-7 extracted with acid solutions native collagens are shown. It means: Col I = type I collagen (with α1 / α2 chains at about 100 kDa, β11 / β12 chains at about 200 kDa and γ chains at about 300 kDa); Col III = collagen type III; Col V = collagen type V.

2 Figure 4: shows the IR spectrum of a coprecipitate of atelocollagen and Na-hyaluronate of biotechnological origin.

3 : shows the results of a determination of adhesiveness in vital cartilage (A) and bone tissue (B). Shown are the average values from 5 measurements with the standard deviation as error bars.

Examples:

• – Vorreinigung: Abtrennung von Schmutz- und Blutresten sowie gegebenenfalls zur NaCl-Entfernung; zur Hydratisierung und Auflockerung des Gewebes wird die geschnittene Haut mit der 5-fachen Wassermenge dreimal unter Rühren gewaschen (jeweils 2 Stdn. Behandlungszeit).
• – Hautentfettung: die gewaschene Haut wird dreimal mit reinem Aceton (jeweils 2 Stdn. Behandlungszeit, Haut-Aceton-Verhältnis 1:5 w/v) unter Rühren extrahiert.
• – Aufquellen der Haut: zur nochmaligen Hydratisierung der Haut wird diese nach der Acetonextraktion erneut dreimal mit reinem Wasser (jeweils 2 Stdn. Behandlungszeit, Haut-Wasser-Verhältnis 1:5 w/v) unter Rühren extrahiert.
• – Eiweißabtrennung: nicht kollagene Eiweiße sind aus der Haut abzutrennen. Zu diesem Zweck wird eine Lösung, die 6% NaCl (w/v) und 1% Ameisensäure (w/v) enthält, bereitet. Die aufgequollene Haut wird mit dieser Lösung zweimal unter Rühren extrahiert (jeweils 24 Stdn. Behandlungszeit, Haut-Lösung-Verhältnis 1:5 w/v).
• – nochmaliges Aufquellen der Haut – dieser Schritt erfolgt analog. Wie bei allen o. g.

First, the preparation of pig skin for collagen production is described in detail: Raw, depilated and mechanically largely defatted pig skin (1.5 to 2.5 mm thick), which was optionally stored in a NaCl solution for preservation, is in pieces (about 2 to 4 mm ² surface) cut. Then proceed in the following steps.

• - Pre-cleaning: separation of dirt and blood residues and, where appropriate, for NaCl removal; for hydration and loosening of the tissue, the cut skin is washed with 5 times the amount of water three times with stirring (each 2 hours treatment time).
• - Skin degreasing: the washed skin is extracted three times with pure acetone (each 2 hours treatment time, skin-acetone ratio 1: 5 w / v) with stirring.
• - Swelling of the skin: for renewed hydration of the skin, this is extracted again with pure water after the acetone extraction three times (each 2 hours treatment time, skin-water ratio 1: 5 w / v) with stirring.
• - Protein separation: non-collagenous proteins are to be separated from the skin. For this purpose, a solution containing 6% NaCl (w / v) and 1% formic acid (w / v) is prepared. The swollen skin is extracted with this solution twice with stirring (each 24 hours treatment time, skin-to-solution ratio 1: 5 w / v).
• - re-swelling of the skin - this step is analogous. As with all the above

Treatments, after the lapse of time, the skin is separated from the extractant. After pretreatment, collagen extraction from the pigskin can be done in different ways.

Example 1: Recovery of tropocollagen

In the extraction by means of acetic acid, 20 g of the treated skin are mixed with 125 ml of acetic acid (0.1M to 1.0M, preferably 0.5M) and extracted with stirring for 48 hours at 25 ° C. Subsequently, the liquid phase (collagen solution) is separated from the solid phase (skin residue). The collagen solution is then purified by ultrafiltration (cross-flow filtration on a UF membrane of polyethersulfone, MWCO 50 kDa) and concentrated. The amount of collagen can be determined by an amino acid analysis or a hydroxyproline determination according to the method of Stegemann and Stalder. The determination of the proportion of native collagen can be carried out by means of CD spectroscopy and SDS-PAGE electrophoresis. The collagen extracted in this way is present as tropocollagen (see Tables 1 and ).

In the extraction by means of urea, 20 g of the treated skin are mixed with 125 ml of urea solution (4 M to 8 M, preferably 6 M) and extracted with stirring for 48 hours at 25 ° C. Subsequently, the collagen solution is separated from the skin residue and purified by dialysis (dialysis tube of regenerated cellulose, pore size 8-15 kDa) against 0.1 M acetic acid. The collagen is also present as tropocollagen, here renatured before (see Table 1).

Example 2: Preparation of Atelocollagen from Porcine Skin

20 g of the treated skin are mixed with 125 ml of a 0.5 M acetic acid solution containing 5% pepsin (w / v) and extracted with stirring for 48 hours at 5 ° C. Subsequently, 1 l of a 0.1 M acetic acid solution is added and the mixture for further enzyme action for three weeks at 25 ° C allowed to stand. After addition of 0.1 M acetic acid to a total volume of 3 l and adjustment of the pH to 8 to 9 by means of 10 M NaOH, the pepsin is precipitated and separated by centrifugation from the supernatant (collagen solution). To purify the collagen solution obtained, the pH is adjusted to 3 to 4 with acetic acid and the collagen is precipitated with NaCl (2.5 M NaCl in the total mixture) and then centrifuged off. There are two further cleaning steps. In each of these steps, the collagen is redissolved in 0.1 M acetic acid, reprecipitated with NaCl (1.8 M NaCl in the total mixture) and then removed by centrifugation. To remove any NaCl, the purified collagen is finally dialysed against 0.1 M acetic acid (regenerated cellulose dialysis tube, pore size 8-15 kDa). The collagen is present as atelocollagen (see Table 1). Table 1: Amino acid composition of tropocollagen (Example 1) and atelocollagen (Example 2) collagen product tropocollagen tropocollagen atelocollagen example 1 1 2 extracting agent acid solution Urea solution Acetic acid-pepsin solution Hyp [number per 1000 amino acids] 80.06 84.56 88.81 Asp [number per 1000 amino acids] 46.31 42.71 46.28 Thr [number per 1000 amino acids] 15.71 17.16 16.34 Ser [number per 1000 amino acids] 29,47 34.12 35.72 Glu [number per 1000 amino acids] 76,80 74.93 74.41 Pro [number per 1000 amino acids] 134.90 135.32 130.94 Gly [number per 1000 amino acids] 331.87 336.85 337.22 Ala [number per 1000 amino acids] 111.29 109.37 108.23 Val [number per 1000 amino acids] 25.89 22.97 23.55 Met [number per 1000 amino acids] 4.23 3.03 4.86 Ile [number per 1000 amino acids] 10.30 9.69 10.34 Leu [number per 1000 amino acids] 25.47 24.52 22.32 Tyr [number per 1000 amino acids] 1.69 3.12 1.45 Phe [number per 1000 amino acids] 13.54 13.67 12.44 His [number per 1000 amino acids] 5.92 5.46 7.57 Hyl [number per 1000 amino acids] 5.77 6.30 6.11 Lys [number per 1000 amino acids] 28.74 25.99 27.33 Arg [number per 1000 amino acids] 52.04 50.76 46.08 Hyp / Pro 0.59 0.62 0.68 Hyp / (Hyp + Pro) 0.37 0.38 0.40 Hyl / Lys 0.20 0.24 0.22 Hyl / (Hyl + Lys) 0.17 0.20 0.18 HYL / Hyp 0.07 0.07 0.07 μg of protein inj 3.17 10.18 2.78 μg protein / sample 3,959.32 2,544.37 2,782.96 μg of collagen 3,251.17 2,216.06 2,550.18 % Collages 82,10 87.10 91,60

Example 3: Preparation of an adhesive of collagen based on tropocollagen

A collagen solution obtained as in Example 1, which mainly contains tropocollagen molecules (collagen concentration 0.1 to 1.0% w / v) and has a pH of 2.3 to 4.5, is mixed with an already prepared solution of Na Hyaluronate of biotechnological origin (pH 6.3 to 6.5, concentration 0.2 to 1.0% w / v, MG of hyaluronate 1,400 to 2,400 kDa) in a mass ratio of 1: 1 (collagen solution to

Hyaluronate solution). The mixture is then stirred until a coprecipitate is formed. This is separated from the liquid phase by centrifuging or decanting. The coprecipitate is highly adhesive and constitutes the adhesive. The coprecipitate contains 5.0 to 15.0% (w / w) collagen, 3.0 to 10.0% (w / w) hyaluronic acid and 75.0 to 92.0 % Water.

Example 4: Preparation of an adhesive of collagen and hyaluronic acid using atelocollagen

Example 4 describes the preparation of an adhesive based on atelocollagen. A collagen solution obtained as in Example 2, which contains mainly atelocollagen molecules (collagen concentration 0.1 to 1.0% w / v) and has a pH of 2.3 to 4.5, is mixed with an already prepared solution of sodium Hyaluronate of biotechnological origin (pH 6.3 to 6.5, concentration 0.2 to 1.0% w / v, MG of hyaluronate 1,400 to 2,400 kDa) in a mass ratio of 1: 1 (collagen solution to hyaluronate solution) mixed. The mixture is then stirred until a coprecipitate is formed. This is separated from the liquid phase by centrifuging or decanting. The coprecipitate is highly adhesive and constitutes the adhesive. The coprecipitate contains 5.0 to 15.0% (w / w) collagen, 3.0 to 10.0% (w / w) hyaluronic acid and 75.0 to 92.0 % Water. The shows the IR spectrum of a coprecipitate of atelocollagen and Na-hyaluronate of biotechnological origin.

For the measurement of the adhesiveness of the adhesives prepared according to Examples 3 and 4 in the biological tissue, vital bone and cartilage tissue from the pig was used. The reference adhesive used was fibrin glue from Baxter (TISSUCOL Duo S).

In preparation for the measurement, cylinders of 10 mm in diameter and 20 mm in length were produced from the medial and lateral condyle of a femur bone using a round punch. This raw punch contains both bone and cartilage tissue. To prepare a cartilage punch used for the measurement, the surface was cut flat using a microtome. For a bone punch used in the measurements, the cartilage layer was completely removed.

For the measurement of adhesiveness, two cylinders (cartilage or bone) were vertically opposed, centered and placed at a distance of 5 mm in a Zwick force measuring stand (Z005) by means of a round sample holder. The course of the measurement was as follows.

40 μl of the adhesive to be tested were first distributed on the surface of the lower tissue cylinder. At a speed of 10 mm per minute, the opposing tissue surfaces were then moved against each other until a force of 1 Newton [N] acted on the measuring surface (A = 0.875 cm 2). This position was held for 30 seconds before the two measuring surfaces were pulled apart at a rate of 10 mm per minute. The course of the occurring force over time was recorded, which counteracts the vertical movement. The maximum value was corrected by the force that occurs with the same procedure without the use of an adhesive. The force thus determined is a measure of the adhesiveness.

In the measuring arrangement described, an adhesivity of on average 15.2 kN / m ² with a standard deviation of 1.0 kN / m ² could be measured for the adhesive produced according to the invention compared with cartilage tissue. For fibrin glue, this value in the same tissue was only 5.2 kN / m ² , with a standard deviation of 2.5 kN / m ² ( ). In the bone tissue, an adhesivity of 11.8 kN / m ² was determined for the collagen-hyaluronic acid adhesive, with a standard deviation of 1.9 kN / m ² . For fibrin glue it was significantly lower at 2.6 kN / m ² and the standard deviation was 1.9 kN / m ² ( ).

The adhesive effect of the adhesives prepared according to Examples 3 and 4 does not differ, but the adhesive prepared on the basis of atelocollagen has a reduced allergy potential.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 5209776 [0002]
• US 2008/0208114 A1 [0002]
• US 4280954 [0002]

Claims (15)

A method for producing an adhesive, characterized in that an aqueous collagen solution having a concentration of dissolved collagen of 0.01 to 5% (w / v) and a pH of 2 to 5 with an aqueous hyaluronate solution having a hyaluronate or hyaluronic acid concentration from 0.02 to 5% (w / v) and a pH of 6 to 7, and the resulting precipitate is separated from the liquid phase. The method of claim 1, wherein the aqueous collagen solution has a dissolved collagen concentration of 0.1 to 1% (w / v). Method according to one of the preceding claims, wherein the aqueous collagen solution has a pH of 2.3 to 4.5. The method of any one of the preceding claims, wherein the solubilized collagen in the aqueous collagen solution is substantially in native triple helical structure. A method according to any one of the preceding claims, wherein the aqueous hyaluronate solution has a hyaluronate or hyaluronic acid concentration of 0.2 to 1% (w / v). A method according to any one of the preceding claims wherein the aqueous hyaluronate solution has a pH of from 6.3 to 6.5. Method according to one of the preceding claims, wherein the hyaluronate or the hyaluronic acid of the aqueous hyaluronate solution is of bacterial origin and is preferably substantially peptide-free. Method according to one of the preceding claims, wherein the dissolved collagen in the aqueous collagen solution contains or consists of atelocollagen and / or tropocollagen. Method according to one of the preceding claims, wherein the hyaluronate or the hyaluronic acid of the aqueous hyaluronate solution has an average molecular weight of 1,400 to 2,400 kDa. Method according to one of the preceding claims, wherein the aqueous collagen solution and the aqueous hyaluronate solution are mixed in a mass ratio of 1: 0.5 to 1: 1.5, preferably from 1: 0.7 to 1: 1.2. Method according to one of the preceding claims, wherein the aqueous collagen solution and / or the aqueous hyaluronate solution may contain further constituents, for example ions, salts, organic acids, sugars, proteins, telopeptides and / or gelatin. Adhesive containing or consisting of: 1 to 20% by weight of collagen, 0.5 to 15% by weight of hyaluronic acid or hyaluronate and 65 to 98.5% by weight of water; the data in% by weight relate to the total weight of the adhesive. Adhesive according to claim 12, containing or consisting of: 5 to 15% by weight of collagen, 3 to 10 wt .-% hyaluronic acid or hyaluronate and 75 to 92% by weight of water; the data in% by weight relate to the total weight of the adhesive. Adhesive according to one of claims 12 or 13, wherein in the adhesive collagen and hyaluronic acid or hyaluronate in a weight ratio of 0.5: 1 to 5: 1 are present. An adhesive according to any one of claims 12 to 14, wherein the adhesive is made by a process according to any one of claims 1 to 11.

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