WO2016071876A1 - Method for preparing type i collagen and for preparing unidirectional and multidirectional scaffolds containing same - Google Patents

Abstract

The invention hereby claimed relates to the field of tissue engineering and concerns a method for isolating type I collagen from rat tail and producing multidirectional scaffolds (Pérez et al., 2001). In recent years, the isolating method has been modified and standardised. Likewise, changes have been introduced into the method for obtaining scaffolds from this collagen and a novel method has been developed for producing unidirectional laminar scaffolds. In the optimised method, the animal collagen source is cleaned, cut and suspended in an acetic acid solution until a liquid phase pH between 2.5 and 3.2 is reached, agitating constantly at 4°C. Said suspension is centrifuged and the pH of the supernatant obtained is neutralised with NaOH in order to re-separate the solid and liquid phases. The collagen precipitated in this way is re-precipitated with salts and solubised in acetic acid, and the solution is poured into moulds, frozen at -20°C and freeze-dried. Lastly, the scaffolds obtained are cross-linked and then sterilised with ethylene oxide.

Description

COLLAGEN PREPARATION PROCEDURE             TYPE 1 AND UNIDIRECTIONAL AND MULTIDIRECTIONAL SUPPORTS             THAT CONTAIN IT

This application relates a process                 for the purification of type I collagen, as well as the                 obtaining collagen powder, collagen hydrogels and                 development of unidirectional supports and                 multidirectional from this one.

Collagen in tissue engineering:

Collagen is the main component of                 connective tissue and the most abundant protein in the                 body; It has an important role in membership,                 Chemotaxis and cell differentiation. They have                 described 28 types of collagen that are grouped into                 Subfamilies according to their structure: Colágenos                 fibrillar (types I, II, III, V, XI, XXIV and XXVII),                 fibrillar related and associated collagen                 (types IX, XII, XIV, XVI, XIX, XX, XXI and XXII),                 filamentous reticulate forming collagens (type                 VI), collagen associated to the basement membrane (types IV,                 VII, XV and XVIII), short chain collagens (types VIII                 and X), transmembrane collagens (types XIII, XVII,                 XXIII and XXV) and other collagens (types XXVI and XXVIII)                 (Ricard, 2011; Gordon and Hahn, 2010).

In the family of collagens, type I is                 the one with the greatest presence in connective tissues                 soft as the skin, oral mucosa and hard as the                 bone. This protein provides great resistance to                 the tension, promotes the adhesion and migration of                 numerous cell types including monocytes,                 neutrophils, keratinocytes and fibroblasts (Friess,                 1998). In the healing process, collagen I                 gradually replace other types of collagen when the                 tissue is remodeled and the scar matures (Gordon and                 Hahn, 2010). Once secreted by the fibroblasts of the                 wound bed is crosslinked to form fibers                 that help in the organization of cells, in the                 contractile and tissue replacement processes that                 they occur during healing (Gelse et al, 2003).

Collagen I is the most widely used protein in                 the development of supports for tissue engineering                 (Kirubanandan & Sehgal, 2010; Sauerbier et al.,                 2006; Fumiko et al., 2004; Takashi et al., 2001), since                 for its biocompatibility, biodegradability and                 bioactivity can interact with the environment in which it                 placed to induce tissue regeneration                 (Williams, 2003).

The most common form of presentation of                 collagen is in solid state, since it allows to increase its                 half-life, because the                 molecular mobility, reducing the speed of                 degradation reactions (hydrolysis, oxidation,                 deamination, racemization, etc.) (Manning, Chou et al.                 2010), in addition to having a low water activity,                 factor that decreases the likelihood of contamination                 and microbiological degradation of collagen (Miyawaki                 2009). By obtaining the collagen in powder form, the                 material acquires a combination of properties (size                 of particle, shape, surface area and morphology) that                 determine process qualities such as fluency,                 humidification, hydration and dispersion, which make it                 in a good alternative as material for                 formulation procedures and / or excipients,                 allowing an easy transformation to other forms of                 presentation: films, particles, suspensions,                 granules, matrices among others (Cape, Villa et al.                 2008). In addition, the powdered form of collagen allows a                 Easy storage process, due to the use of                 slightly bulky and light containers, in addition to not                 require special and expensive conditions of                 Storage as refrigeration.

Among the most common processes for                 Powder processing are freeze drying, the                 spray drying and the use of supercritical fluids                 (Cape, Villa et al. 2008), methodologies that require                 robust equipment, which makes these processes                 expensive. A cheaper alternative is the use of                 forced air convection drying, which allows                 control the processes of heat and mass transfer,                 without altering the quality of the product (Paul Malumba,                 2008), however, this process has not been used in the                 collagen drying

Tissue engineering supports:

One of the greatest engineering challenges                 of tissues is to reproduce the complex organization                 cell and restore functions that cells                 meet in the native tissue of the human body (Griffith                 & Swartz, 2006). The structure of a fabric and its                 Function are highly related. In the tendon the                 fibers are arranged unidirectionally, which                 provides you with high tensile strength (Kuo et                 al., 2010); the random fiber pattern of collagen in                 the skin gives it its mechanical properties (Bottcher et                 al., 2010); the cup shape of the pulmonary alveoli                 gives efficiency to the gas exchange process (Chen et                 al., 2005; Faraj et al., 2007) and the complex arrangement and                 Polygonal hepatocyte is essential for the                 appropriate liver function (Tsang & Bhatia, 2006).                 To imitate this organization the supports used in                 tissue engineering are modified in their                 microstructure, which is defined by porosity,                 interconnectivity, size and direction of the pores.

The manufacture of unidirectional supports has                 been investigated in tissue regeneration that                 they have a structure oriented like: nerves                 peripherals (Xueyu et al., 2009; Chamberlain et al.,                 1998; Stokols & Tuszynski, 2004), spinal cord                 (Spilker et al., 2001), tendon (Louie et al., 1997;                 Liu., Et al, 2008), cornea (Wray & Orwin, 2009) and                 bone (Silva et al., 2006, Yunoki, et al., 2006).                 In vivo studies in animals have shown that supports                 for nerve regeneration with microchannels                 Unidirectional oriented longitudinally allow                 achieve functional regeneration and recovery                 equivalent to self grafting, without the use of agents                 external regenerative or cells (Xueyu et al., 2009;                 Yannas, 2005). Another study showed that the                 microstructure affects the fibroblast phenotype                 corneal, since the collagen fibers aligned                 they act as a signaling mechanism to decrease                 alpha-actin expression in corneal fibroblasts of                 rabbit (Wray & Orwin, 2009).

To develop these supports have                 employed manufacturing methods with techniques of                 phase separation (Ma & Zhang, 2001), manufacturing                 of solids with free forms (Taboas et al., 2003),                 rapid prototyping (Wilson et al., 2004) and freezing                 unidirectional (Madaghiele et al., 2007), however,                 these methods produce supports of small dimensions                 (approximately 3mm x 5mm), also require                 complex and expensive equipment.

Lyophilization of collagen suspensions                 It is the most used method in the elaboration of supports                 of this biomaterial. The microstructure of the support                 after lyophilization is a replica of the                 ice crystal morphology that forms after                 be frozen, so the structure can be                 directly controlled by freezing (Schoof et                 al., 2000).

Given the importance of obtaining                 of type I collagen supports, the development of this                 optimized process claimed here, shows the                 convenience of establishing improved processes that also                 of overcoming the disadvantages present in the processes                 existing allow the obtaining of collagen supports                 I with characteristics superior to the existing ones.

The present invention relates the                 procedures for the purification of type I collagen,                 collagen hydrogels, collagen powder, supports                 multidirectional collagen and collagen supports                 Unidirectional, comprising the following stages:

1. Purification of type I collagen.

Sources of type I collagen are taken                 from animal connective tissue such as fascia,                 tendons or bone

Said source of collagen is prepared by eliminating                 adjacent tissues that are attached to it,                 it is cut into pieces that allow its treatment and                 wash with water

The material is degreased by washing                 Consecutive starting with constant low solvents                 dielectric (20) and ending with high solvents                 dielectric constant (80), for example with ethanol at                 concentrations higher than 70%, ether and / or purified water.

An acidic acid solution is performed                 acetic or hydrochloric until a pH between 2.5 and                 3.2, at temperatures below 5 ° C and stirring                 permanent

The product obtained is liquefied and centrifuged and                 then the supernatant is recovered

An alkali is added to the supernatant as                 NaOH or KOH until a pH between 4.0 and 5.0 is achieved; then I know                 centrifuge and recover the pellet obtained

A solution of a salt is added to the pellet                 that gives the solution an ionic potential                 equivalent to a 0.7M sodium chloride solution                 until reaching a conductivity of between 50 and 70 mS / cm,                 stir for 1 to 24 hours, centrifuge and recover                 again the pellet obtained

Successive water washes are performed at                 temperatures below 5 ° C and stirring,                 centrifuge and recover the pellet; all this repeats                 until a conductivity in the wash water is achieved                 between 200 and 500 µS / cm

Type I collagen is obtained.

In the present invention, it was determined that the                 conductivity is important as it allows you to control the                 ionic strength and the ratio of charges to which it is                 submitted the collagen suspension. The present                 invention allows the initial salt concentration                 (controlled by conductivity) help                 re-precipitate collagen I and solubilize proteins                 pollutants The final conductivity control                 ensures that the remaining salt content does not affect the                 cellular behavior, when cells are exposed                 to the isolated and purified material.

2. Preparation of collagen powder:

The collagen suspension is quantified                 (gravimetric method or HPLC) and adjusted with water                 deionized between 2-10% P / P.

On Teflon molds, a film is served                 homogeneous of a thick collagen suspension                 desired (between 3 and 8 mm).

Air bubbles are removed from the                 Vacuum film (6.7 to 133.3 Pa) for ten minutes.

It is dried by forced air convection at a                 constant air flow between 20m3 / hour at 40 m3 / hour and                 at a temperature of 55 ± 5 ° C for 48 hours

The film formed is removed by a                 spatula type tool and is crushed (production by                 fracture) in a mortar (application of a mechanical force).

The powder obtained is subsequently ground                 (intermediate mill type - rotary disc cutter).                 The powder mixture obtained is classified by size                 by screening technique. The dust fraction                 Majority must have a size range between 1.00 and                 0.30 mm equivalent to the fraction that passes through a sieve                 standard number 30 and retained by a standard sieve                 number 20.

3. Obtaining collagen hydrogel

Weigh between 1 and 10 g of Collagen I powder

Add between 100 and 1000 mL of acetic acid                 0.5N according to the desired collagen concentration.

Homogenize in a rotor homogenizer                 stator at a speed between 3000 and 8000 rpm.

Hydrate for a time between 12 and 18 hours

Add 0.5 N acetic acid until                 achieve the desired concentration

Homogenize in a rotor homogenizer                 stator at a speed between 3000 and 8000 rpm

Centrifuge for 15 minutes at a speed                 between 3000 and 8000 rpm.

Discard supernatant.

Quantify the pellet formed.

Adjust the suspension to the concentration                 desired (between 1 and 10% w / w collagen), with acid                 acetic 0.5 N

Keep stored at 4 ° C.

4. Obtaining collagen supports

The suspension obtained is placed in molds of                 chemically inert and nonstick material such as                 Teflon Subsequently, the following procedures must be performed:

Degassing the suspension by applying vacuum.

Freeze the suspension at a temperature between                 -20 to -40 ° C to obtain multidirectional supports.                 To make unidirectional supports one of                 the walls of the mold with a thermally conductive material                 inert and part of it is contacted with an agent                 adequate freezing ensuring a gradient of                 temperature of 20 to 60 ° C through the suspension.

Freeze dry in order to produce a                 interconnected porous structure

Dip the lyophilized material in a                 aqueous solution with a crosslinking agent (genipin,                 glutaraldehyde, dendrimers, carbodiimides or a mixture                 from them)

Degassing the system

Leave to react for 24 to 48 hours

Remove the solution containing the crosslinking agent

Wash with water until the                 removal of the crosslinking agent

Freeze crosslinked support

Freeze dry

Sterilize by ethylene oxide,                 70% gamma or ethanol radiation

If you take the option of the walls                 coated with the conductive material, a                 unidirectional support (Figure 1); if not used                 multidirectional support is achieved (Figure 2).

The figures that accompany this description                 present two photographs of microscopy images                 scanning electronics on collagen supports                 obtained by the technology claimed here.

Figure 1 shows a collagen support                 type I in whose processing has been applied, in some                 of the mold walls, a thermally conductive material                 inert with which unidirectional supports are achieved.

Figure 2 presents a collagen support                 type I in which the fibers are arranged in all                 directions during the freezing stage and it turns out                 forming a multidirectional support.

Ways to realize the invention

A preferred protocol was found for                 Type 1 collagen production with the following sequences:

It starts from fragments of fascia or tendons of                 rat, cattle or pigs

They are washed with different solvents or                 solvent mixtures that allow obtaining constants                 dielectrics in a range between 10 and 80.

The tissue is dissolved with acid                 0.5 M acetic acid and stirring at cooling temperature                 for a space of 24 hours

The product is liquefied and centrifuged

The supernatant is recovered

2M NaOH supernatant is added

It is centrifuged at 5000 rpm for 15 min and                 recover the pellet

Wash with a volume of water equal to                 volume of pellet obtained

It is centrifuged at 5000 rpm for 15 min and                 recover the pellet

NaCl pellet is added until a                 conductivity: 50-70 mS / cm

Stir at 60 rpm for 1 hour.

It is centrifuged and the pellet is recovered

Water addition repeated, centrifugation                 and recovery of the precipitate until the waters of                 washing reach a conductivity between 200-500 mS / cm

Collagen powder preparation:

The collagen suspension is quantified                 (gravimetric method or HPLC) and adjusted with water                 deionized at a percentage between 1-10% P / P.

A film is served on Teflon molds                 homogeneous of the collagen suspension.

Air bubbles are removed from the                 empty film

It dries up.

The film formed is crushed.

The powder obtained is subsequently ground                 (intermediate mill type - rotary disc cutter).                 The powder mixture obtained is classified by                 sizes by sieving technique. The fraction of                 Majority powders should have a size range                 between 1.00 and 0.30 mm.

These stages allow to obtain collagen                 Type I, with the desired characteristics. Starting from                 this collagen preferred protocols were established                 To develop collagen supports:

Multidirectional Collagen Supports

Get suspension of type I collagen in                 0.05 M acetic acid of the desired ratio

Serve in Teflon Molds

Degassing the suspension by vacuum of                 6,666 kPa until evidencing cessation of bubbling

Freeze

Freeze dry

Dip the lyophilized material in a                 aqueous glutaraldehyde solution

Degassing the system by vacuum

Let stand for 24 hours

Remove the glutaraldehyde solution

Wash with purified water

Freeze crosslinked support

Freeze dry

Sterilize

Unidirectional Collagen Supports

Prepare a suspension of type I collagen from                 the desired concentration

Serve in Teflon Molds

Degassing the suspension by vacuum of                 6,666 kPa until evidencing cessation of bubbling

Freeze the suspension by putting the wall                 coated with aluminum in contact with a bath                 liquid nitrogen ensuring a temperature gradient                 40 ° C through the suspension

Freeze dry

Remove the lid from the molds and continue the                 lyophilization

Dip the lyophilized material in a                 aqueous glutaraldehyde solution

Degassing the system by vacuum                 6,666 kPa until evidencing cessation of bubbling

Let stand for 24 hours

Remove the glutaraldehyde solution

Wash with purified water

Freeze crosslinked support

Freeze dry

Sterilize

Claims (4)

Procedure for the purification of type I collagen and unidirectional or multidirectional supports with this material, CHARACTERIZED BECAUSE it comprises the following stages: a) Obtaining the sources of collagen (Fascia, tendon); b) Clean the collagen source of adjacent tissues that are attached and cut into pieces that allow treatment; c) Wash with water; d) Degrease the material using washing with ethanol in concentrations higher than 70%; e) Dilute with acetic acid until a pH of 2.5 to 3.2 is achieved, at temperatures below 5 ° C and permanent agitation; f) Blend and centrifuge the product obtained and recover the supernatant; g) Add the NaOH supernatant until a pH between 4.0 and 4.3 is achieved, centrifuge and recover the pellet obtained; h) Add to the pellet a solution of sodium chloride until reaching a conductivity of between 50 and 60 mS / cm, stir for a space exceeding 50 minutes, centrifuge and recover the obtained pellet again; i) Perform successive water washes at temperatures below 5 ° C and stirring, centrifuge and recover the pellet; All this is repeated until a conductivity in the wash water is achieved between 200 and 500 mS / cm. Procedure for the production of type I collagen claimed in 1 CHARACTERIZED BECAUSE it comprises the following steps until it is sprayed: a) Quantify a collagen suspension (gravimetric method or HPLC); b) Serve in homogeneous Teflon molds a collagen suspension film of a thickness between 3 and 5 mm; c) Remove air bubbles from the film by vacuum (50-1000 mTorr) for ten minutes; d) Dry by forced air convection at a constant air flow between 25 and 35 m3 / hour and at a temperature of 55 ± 5 C for 24-48 hours; e) Remove and crush the film formed in a ceramic mortar (application of a mechanical force); f) Grind the powder obtained (intermediate mill type - rotary disk cutter), classify the powder mixture obtained by sizes by means of the sieving technique; the majority of the dust fraction must have a size range between 0.84 and 0.59 mm equivalent to the fraction that passes through a standard sieve number 30 and retained by a standard sieve number 20. Procedure for the production of multidirectional collagen supports claimed in 1 CHARACTERIZED BECAUSE it comprises the following steps: a) Obtain suspension of type I collagen in the desired concentration in acetic acid, having a pH between 3.0 and 3.2; b) Place in molds that, optionally, have chemically inert and non-stick walls; c) Degassing the suspension by vacuum until evidencing cessation of bubbling; d) Cover the mold ensuring that there are no free spaces inside it; e) Freeze the suspension until it reaches -40 ° C; f) Freeze-dry under vacuum between 20 and 48 hours; g) Remove the cover from the molds and continue lyophilization for another period of time between 20 and 28 hours; h) Immerse the lyophilized material in an aqueous solution with a crosslinking agent; i) Degassing the system by vacuum until evidencing cessation of bubbling; j) Leave at rest between 20 and 28 hours; k) Remove the solution with the crosslinking agent; l) Wash with water until the removal of the crosslinking agent is guaranteed; m) Freeze the crosslinked support until it reaches a temperature of -20 to -40 ° C; n) Freeze-dry for a period of time between 20 and 48 hours; o) Obtain multidirectional type I collagen supports. Procedure for the production of unidirectional collagen supports claimed in 1 CHARACTERIZED BECAUSE it comprises the following steps: a) Carry out a suspension of type I collagen in the desired proportion in acetic acid having a pH between 2.5 and 3.2; b) Place in molds that, optionally, have chemically inert and non-stick walls, one of these walls in contact with aluminum; c) Degassing the suspension by vacuum until evidencing cessation of bubbling; d) Cover the mold ensuring that there are no free spaces inside; e) Freeze the suspension by contacting the aluminum-coated wall with a nitrogen bath, ensuring a temperature gradient of 40 ° C through the suspension; f) Lyophilize between 20 and 48 hours; g) Remove the cover from the molds and continue lyophilization for another period of time between 20 and 28 hours; h) Immerse the lyophilized material in an aqueous solution with a crosslinking agent; i) Degassing the system until evidencing cessation of bubbling; j) Leave at rest between 20 and 28 hours; k) Remove the solution with the crosslinking agent; l) Wash with water until the removal of the crosslinking agent is guaranteed; m) Freeze the crosslinked support until it reaches a temperature of -20 to -40 ° C; n) Freeze-dry for a period of time between 20 and 48 hours; o) Obtain unidirectional type I collagen supports

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