TWI482781B - Method for preparing porous collagen matrices - Google Patents

Description

Method for preparing porous collagen matrix

The present invention relates to a method of preparing a porous collagen matrix from connective tissue.

Collagen matrices can be used to make, in particular, artificial tissues, artificial organs and drug delivery vehicles. Collagen matrix is generally reconstituted from collagen fibers, which are extracted from colloid-rich connective tissue. Conventional methods for extracting collagen fibers require harsh physical or chemical treatments such as grinding, homogenization or acid/base degradation, which can damage the network structure of the collagen fibers.

The present invention is based on the unpredictable finding that when connective tissue is treated with a weak acid solution that does not substantially contain a salt, the connective tissue can be expanded to a great extent without destroying the network structure of the collagen.

Accordingly, a feature of the present invention is a method of preparing a porous collagen matrix directly from connective tissue. The method comprises (i) providing a connective tissue having a surface area of from 20 square centimeters to 2 square meters (preferably from 25 square centimeters to 900 square centimeters); (ii) volume of the connective tissue with an acid solution Expanding at least 50% (preferably 100% to 500%) to form an expanded connective tissue; and (iii) washing the expanded connective tissue to remove non-collagen material thereby forming a porous collagen matrix. The above connective tissue is derived from dermal tissue or tendons. The above-mentioned acid solution for swelling has a pH value (pH value) of 1 to 6 (preferably 2 to 4), and the acid liquid does not substantially contain a salt, that is, there is no salt component in the solution, or The salt concentration is very low, so that the ionic strength of the solution is not more than 0.005M. In some embodiments, the acid system described above may be prepared from formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, or mixtures thereof. Preferably, the acid solution is a 0.1-6 M acetic acid solution.

Prior to the expanding step, the connective tissue described above may be rinsed with a solvent comprising an organic solvent and optionally water. When the solvent contains an organic solvent and water, the volume ratio of the solvent to water is from 1:4 to 9:1. In some embodiments, the organic solvent includes, but is not limited to, an alcohol, a ketone, acetone, acetonitrile, hexane, chloroform, N,N-dimethylformamide, dimethyl hydrazine, and mixtures thereof. When the connective tissue is derived from the epidermis and contains hair or hair roots, it is preferred to pretreat the epidermis containing hair or hair roots with a microalkaline solution or proteolytic enzyme to remove hair or hair roots, and the microalkali solution may be soda or small. For soda, calcium chloride, or low concentration hydrogen peroxide, the proteolytic enzyme may be, for example, a neutral protease (dispase), trypsin, papain, pepsin, chymotrypsin. (chymotrypsin), bromelain, figin or a mixture thereof.

The above expansion step can be achieved by immersing the connective tissue in an acid solution. Alternatively, the soaking step described above is accomplished by simultaneously treating the connective tissue with an ultrasonic wave or simultaneously ejecting a liquid to the connective tissue.

After the expansion step described above, the expanded connective tissue can be washed to remove non-collagen material, thereby preparing a porous collagen matrix. The above washing step can be achieved by immersing the expanded connective tissue in a cleaning solution, which may further comprise a detergent, a protease or a mixture thereof. Alternatively, the soaking step may be accomplished by injecting a liquid together to the expanded connective tissue to achieve or together with an ultrasonic treatment of the connective tissue, or in combination with two physical forces.

A porous collagen matrix prepared by the foregoing method is also within the scope of the present invention.

The above and other objects, features, and advantages of the present invention will become more apparent from the scope of the appended claims. as follows.

The present invention provides a method for preparing a porous collagen matrix directly from connective tissue, wherein collagen is the major protein in connective tissue. Collagen is a triple-helix rod-shaped molecule having a length of about 300 nanometers (nm) and a diameter of about 1.5 nanometers (nm). A large amount of collagen molecules form collagen filaments, and a bundle of collagen filaments form a collagen fiber. Covalent cross-linking exists between the collagen molecules and within the collagen molecules, thereby forming a fibrous network within the connective tissue.

In the method of the present invention, the starting material is connective tissue and may be taken from animals such as cattle, pigs, horses, sheep, chickens, ducks, turkeys, geese, whales or sharks. Connective tissue suitable for use in the methods of the invention includes, but is not limited to, dermal tissue, subcutaneous tissue, ligaments, tendons, aponeurosis, cartilage, bone tissue. If necessary, the connective tissue is first manually removed (for example by gross dissection) or mechanically to remove unwanted materials such as fats and lipids. In one embodiment, the dermal tissue is taken from fresh animal skin, the animal skin is removed from the lipid, and the skin is washed several times with saline, and then the surface layer of the animal skin is removed by a dermatome to obtain the dermis. organization. The resulting dermal tissue can be further washed with a phosphate buffer solution.

If necessary, the connective tissue can be treated with a suitable organic solvent or a mixture of an organic solvent and water so that the organic solvent can penetrate into the connective tissue. The organic solvent includes, but is not limited to, an alcohol, a ketone, acetone, acetonitrile, hexane, chloroform, N,N-dimethylformamide, dimethyl hydrazine or a mixture thereof. When a mixture of an organic solvent and water is used, the ratio of the organic solvent to water is higher than 1:4 (for example, 1:1, 2:1 or 4:1).

When the connective tissue contains hair or hair roots, it can be treated with a microalkaline solution or proteolytic enzyme to remove hair or hair roots. The microalkaline solution can be soda, baking soda, calcium chloride, or low concentration hydrogen peroxide, etc. Such as, for example, a neutral protease (dispase), trypsin, papain, pepsin, chymotrypsin, bromelain, ficin or its mixture.

Next, soak any of the above connective tissues in an appropriate amount of acid solution, and after a suitable period of time, expand the connective tissue to a desired standard, that is, a thickness that is at least 50% greater than the original thickness (preferably the original thickness of 2) -10 times). The above acid solution may be prepared as an organic acid such as formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid or a mixture thereof. In one embodiment, the acid solution may be acetic acid having a concentration of 0.1 to 6 M (preferably 0.1 to 2 M or 0.5 to 1.25 M). In order to obtain a better expansion effect, the acid used in the present invention does not substantially contain a salt.

In the expansion step described above, the connective tissue is suspended in the acid solution described above. If desired, one or more fluids can be used to treat connective tissue, causing the acid to penetrate into the connective tissue and shorten the time required to swell the connective tissue. The liquid can be ejected from a nozzle or orifice of a container in which the acid and connective tissue are placed.

Alternatively, or in addition, an ultrasonic vibration device can generate an ultrasonic wave or a high frequency water wave generated by an electromagnetic field or a cam to apply to the connective tissue soaked in the acid solution to promote the penetration of the acid solution. Connective tissue.

The expanded connective tissue prepared in the above expansion step is washed with a washing solution to substantially remove the non-collagen material in the expanded connective tissue, thereby preparing a porous collagen matrix. The cleaning solution may contain a cleaning agent, a chelating agent, a protease, or a mixture thereof.

Exemplary cleaners for preparing cleaning solutions include, but are not limited to, sodium dodecyl sulphate (SDS), Tego compounds (eg, surfactant Tween-80, nonionic detergent TWR (Triton WR 1339), P-isooctylpolyoxy-ethylene phenol polymer and tetrabutyl aldehyde (Triton A20) surfactant, cetylpyridinium chloride, brominated ten Cetyltrimethyl ammonium bromide, dioctyl sodium sulphosuccinate, polyoxyethylene sorbitol oil available from Japan Kao Soap Co., Ltd. under the name "Emasol 4130" Polyoxyethylene sorbitan monoleate surfactant, non-ionic surfactant available from Imperial Chemical Industries Ltd. under the name "Lubrol W", and non-ionic surfactant available from European Sigma under the name "Nonidet P40") . In one embodiment, the expanded connective tissue is treated for 1 hour to 150 hours at a temperature of 4 ° C to 45 ° C using a cleaning solution containing 0.01% to 10% SDS concentration.

The chelating agent contained in the cleaning liquid includes, but is not limited to, ethylene diamine tetra-acetic acid (EDTA), 1,4,7,10-tetraazacyclododecyl-1,4 , 7,10'-tetraacetic acid (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA), 1,4,7,10-tetraazacyclododecyl- 1,4,7,10'-tetramethylene phosphate (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene phosphonic acid), DOTP), cyclohexanediaminetetraacetic acid ( Trans-1,2-diaminocyclohexantetra-acetic acid (CDTA), 4,5-dihydroxybenzene-1,3-disulphonic acid (Tiron), thiourea ), 8-hydroxyquinoline-5-sulphonic acid, 3,6-disulfo-1,8-dihydroxynaphthalene (3,6-disulpho-1,8-dihydroxy) -naphthalene), Eriochromeschwarz T(1-(1-hydroxy-2-naphthylazo)-2-hydroxy-5-nitro-4-naphthalenesulfonic acid) (Eriochromeschwarz T(1-(1-hydroxy-2-) Naphthylazo)-2-hydroxy-5--nitro-4-naphthalene sulphonic acid)), ammonium purpurate, and the like. A preferred chelating agent is from 0.01 mM to 100 mM EDTA.

Alternatively, or in addition, the cleaning solution may comprise one or more proteolytic enzymes, such as ficin, pepsin, trypsin, papain, chymotrypsin, pineapple Protease (bromelain), hemolysin or a mixture thereof to remove proteins linked to the extracellular matrix, other non-collagen proteins and telopeptides of collagen molecules. In this field of technology, the conditions for limited enzyme cleavage (and even the maintenance of collagen fibers without the degradation of protein by non-collagen proteins) are well known.

In the washing step, the expanded connective tissue may be immersed in any of the aforementioned acid solutions for a suitable period of time. In one embodiment, one or more liquids are sprayed from a nozzle or orifice to the connective tissue to facilitate removal of non-collagen material. The liquid may be water, a solution containing a detergent or a solution containing an enzyme. In another embodiment, ultrasonic cleaning of the expanded connective tissue soaked in the cleaning fluid can improve cleaning efficiency.

The porous collagen matrix that has been subjected to the above washing step can be frozen and the freezing rate controlled so that the matrix contains the desired ice crystal size. It can then be stored by freeze drying. Alternatively, the porous collagen matrix can be immersed in a phosphate buffer solution and stored at a temperature of 4 °C. When necessary, the above porous collagen matrix can be crosslinked by a general chemical or physical method. The crosslinking agent for crosslinking the above porous collagen matrix comprises glutaraldehyde, formaldehyde, carbodiimide or other polyepoxy compound, such as B. Glycol diglycidyl ether, polyol polyglycidyl ether, dicarboxylic acid diglycidyl ester.

The aforementioned method for preparing a porous collagen matrix differs from conventional methods in at least two aspects. First, the method of the present invention does not require harsh physical or chemical treatments (e.g., grinding, homogenization, or acid/base degradation) which destroy the network structure of collagen fibers in connective tissue. Second, the acid used to swell the connective tissue does not substantially contain salts, and conventional methods use salts to stabilize the collagen fibers.

Also disclosed herein is a porous collagen matrix prepared by any of the foregoing methods of preparation. The porous collagen matrix can be used to make artificial tissues, artificial organs, implants, and drug delivery carrier systems, or as a scaffold for cell growth. The present invention will be fully utilized by those skilled in the art based on the foregoing description without further detailed description. The following examples are, by way of example only, and are not intended to limit the invention in any respect. All documents cited herein are incorporated herein by reference.

Example 1 : Preparation of porous collagen matrix from pig skin

The skin tissue was taken from the pig, and after removing the lipid, the skin was washed several times with saline, and the epidermal layer of the skin was removed with a dermatome to obtain a dermal tissue having a thickness of 0.3 mm. The dermal tissue is further washed with a phosphate buffer solution. After washing, all buffer solution residues were completely removed from the surface of the dermal tissue.

The dermal tissue was placed in a container containing a 0.5 M acetic acid solution and allowed to stand at 37 ° C for a different time to expand the dermal tissue. During constant temperature placement, the container is placed on a vibrator to suspend the dermal tissue. The thickness of the dermal tissue after expansion through different times is listed in Table 1 below:

The expanded dermal tissue was then immersed in a solution containing 0.5% SDS and 0.5 mM EDTA for two hours at room temperature to remove non-collagen material to produce a porous collagen matrix.

The porous collagen matrix was washed with a sterilized phosphate buffer solution to remove residual SDS and EDTA. Next, after freezing at a temperature of -20 ° C, the above porous collagen matrix was stored by freeze-drying. The lyophilized porous collagen matrix was vacuum-sputtered and photographed using a scanning electron microscope (SEM). As shown in Fig. 1 and Fig. 2, the porous collagen matrix does not contain cells and cell remnants, and has a matrix structure formed of collagen fibers.

Example 2 : Preparation of porous collagen matrix by liquid jet method

A porcine dermal tissue was prepared by the procedure described in Example 1 and immersed in a 0.5 M acetic acid solution. In particular, the container used to soak the dermal tissue in the acetic acid solution has a plurality of nozzles from which a plurality of acetic acids are sprayed onto the dermal tissue. This injection step accelerates the expansion process as shown in Table 2 below:

Next, the soaked and sprayed expanded dermal tissue was washed with the SDS/EDTA solution described in Example 1 to prepare a porous collagen matrix.

Example 3 : Preparation of porous collagen matrix by ultrasonic treatment

A porous collagen matrix was prepared by the procedure described in Example 2, except that ultrasonic treatment was performed using an ultrasonic oscillating device instead of the blasting treatment.

The results obtained indicate that, like the blasting process, the ultrasonic treatment also significantly shortens the time to expand the dermal tissue to an appropriate standard.

Example 4 : Pretreatment of porcine dermal tissue with ethanol to prepare a porous collagen matrix

Porcine dermal tissue was prepared by the procedure described in Example 1, and the porcine dermal tissue was placed in a container containing 30% ethanol (ethanol: water = 30:70 (volume ratio)). The container is placed on a vibrator so that the dermal tissue can be suspended in the ethanol solution. The above dermal tissue was immersed in 30% ethanol for 14 hours.

After pouring 30% ethanol, a 0.5 M acetic acid solution was poured into the container, and placed on a vibrator to expand the dermal tissue soaked in the acetic acid solution. After one day, the dermal tissue expanded to a thickness of 0.45 millimeters (mm) (1.5 times the original thickness). This means that pretreatment with ethanol also shortens the time during which the dermal tissue expands to a thickness of 0.45 mm.

After removing the 0.5 M acetic acid solution, the expanded dermal tissue was immersed in 20% ethanol (ethanol: water = 20:80 (volume ratio)) for 12 hours at room temperature. The resulting porous collagen matrix was washed with deionized water, frozen at a temperature of -20 ° C, and finally stored by freeze drying.

Example 5 : Preparation of a porous collagen matrix using a proteolytic enzyme treatment

A pig dermal tissue was washed and placed in a solution containing protease (15 U/ml) for one day. The dermal tissue was then expanded and washed by the procedure described in Example 2. The results showed that it took only six hours for the protease-treated dermal tissue to expand to a thickness of 0.45 millimeters (mm).

Comparative Example 1 : Preparation of a porous collagen matrix by a conventional method

The dermis of the pig was prepared by the procedure of Example 1, and it was placed in an acetic acid solution at a temperature of 37 ° C and 0.5 M for 12 hours. The treated dermis was then placed in a solution containing 0.2 M acetic acid and 0.5 M sodium chloride for 72 hours. The dermis thus treated had a thickness of 0.36 millimeters (mm) (1.2 times the original thickness of the dermis). Thereafter, the treated dermis was placed in a solution containing 1% (w/v) hydrogen peroxide and allowed to stand for 24 hours, and then placed in a solution containing SDS/EDTA at a temperature of 37 ° C for further tempering. Set for 24 hours. Finally, it was washed with a sterilized phosphate buffer solution, and frozen at a temperature of -20 ° C, followed by freeze drying.

As described above, when it is expanded by a solution containing acetic acid and a salt (e.g., sodium chloride), it takes more than seventy-two hours to obtain a thickness of 0.36 mm in a thickness of 0.3 mm (mm). The difference is that, as shown in Example 1, when the solution containing acetic acid is expanded for three days, the thickness of the dermis having a thickness of 0.3 mm can be made 0.9 mm (refer to Table 1). This means that a solution containing no acetic acid in the salt can achieve a higher degree of swelling of the dermis compared to a solution containing acetic acid and a salt such as sodium chloride. This may be due to the stabilizing effect of the salt. In particular, salts stabilize collagen fibers so that collagen can bind tightly, rather than swell.

Other embodiments

All of the features disclosed in the present invention should be implemented in any combination. Each feature disclosed in the present invention should be substituted with a substitute of the same, equal or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is only one embodiment of the one of the

From the above, it will be apparent to those skilled in the art that the present invention can be readily understood from the scope of the present invention without departing from the spirit and scope of the invention as defined by the appended claims. , when various changes, substitutions, and corrections can be made here. Therefore, other embodiments are also within the scope of the appended claims.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 1 is a scanning electron micrograph (magnification: 100) of a porous collagen matrix prepared according to the method described in Example 1.

Figure 2 is a scanning electron micrograph (magnification: 400) of a porous collagen matrix prepared according to the method described in Example 1.

Claims (19)

A method for preparing a porous collagen matrix from connective tissue, the method comprising: providing a connective tissue having a surface area of 20 square centimeters to 2 square meters; and an expanding step of: causing the connective tissue with an acid solution Forming at least 50% of the volume to form an expanded connective tissue, wherein the acid solution has a pH (pH) of 1 to 6 and the acid solution does not substantially contain a salt; and a washing step: washing the The connective tissue is expanded to remove non-collagen material thereby forming a porous collagen matrix. The method of claim 1, wherein the connective tissue has a size of from 25 square centimeters to 900 square centimeters. The method of claim 1, wherein the acid is a weak acid. The method of claim 3, wherein the weak acid solution comprises an acid selected from the group consisting of formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, and mixtures thereof. The method of claim 3, wherein the acid solution has a pH (pH) of from 2 to 4. The method of claim 5, wherein the acid solution comprises a concentration 0.1 to 6 M acetic acid. The method of claim 1, wherein in the expanding step, the connective tissue has a volume after expansion of 100% to 1000%. The method of claim 1, wherein the expanding step comprises soaking the connective tissue in the acid solution and simultaneously spraying a liquid to the connective tissue. The method of claim 1, wherein the washing step comprises immersing the expanded connective tissue in a cleaning solution comprising a detergent, a protease or a mixture thereof. The method of claim 9, wherein the washing step comprises immersing the expanded connective tissue in the cleaning solution while simultaneously injecting a liquid into the expanded connective tissue. The method of claim 1, wherein the expanding step comprises soaking the connective tissue in the acid solution while simultaneously treating the connective tissue with ultrasound. The method of claim 9, wherein the washing step comprises immersing the expanded connective tissue in the cleaning solution while simultaneously treating the connective tissue with ultrasound. The method of claim 1, further comprising treating the connective tissue with a solvent prior to the expanding step, the solvent comprising an organic solvent and optionally comprising water, wherein the organic solvent is selected from the group consisting of an alcohol, a ketone, and an acetone a group consisting of acetonitrile, hexane, chloroform, N,N-dimethylformamide, dimethyl hydrazine, and mixtures thereof. The method of claim 13, wherein the solvent comprises an organic solvent and water and the volume ratio between the organic solvent and water is from 1:4 to 9:1. The method of claim 1, wherein the connective tissue comprises a plurality of hairs or hair roots, and the method further comprises, prior to the expanding step, treating the connective tissue with a slight alkaline solution or proteolytic enzyme to relax or Remove the hair or hair roots. The method of claim 15, wherein the proteolytic enzyme is selected from the group consisting of a dispase, a trypsin, a papain, a pepsin, a chymotrypsin, A group consisting of bromelain, ficin, and mixtures thereof. The method of claim 1, wherein the connective tissue is derived from dermal tissue or tendons. The method of claim 1, further comprising drying the porous collagen matrix after the washing step. The method of claim 1, wherein the cleaning step comprises treating the expanded connective tissue with a first cleaning solution and a second cleaning solution, the first cleaning solution comprising a cleaning agent, a chelating agent or a mixture thereof The second cleaning solution comprises a protease.