WO2016071876A1 - Method for preparing type i collagen and for preparing unidirectional and multidirectional scaffolds containing same - Google Patents
Method for preparing type i collagen and for preparing unidirectional and multidirectional scaffolds containing same Download PDFInfo
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- WO2016071876A1 WO2016071876A1 PCT/IB2015/058583 IB2015058583W WO2016071876A1 WO 2016071876 A1 WO2016071876 A1 WO 2016071876A1 IB 2015058583 W IB2015058583 W IB 2015058583W WO 2016071876 A1 WO2016071876 A1 WO 2016071876A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Definitions
- 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 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 XVIII) (Ricard, 2011; Gordon and Hahn, 2010).
- type I 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 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).
- 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.
- unidirectional supports have 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).
- 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).
- 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:
- 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
- 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
- Type I collagen is obtained.
- 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.
- the collagen suspension is quantified (gravimetric method or HPLC) and adjusted with water deionized between 2-10% P / P.
- Air bubbles are removed from the Vacuum film (6.7 to 133.3 Pa) for ten minutes.
- 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.
- 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.
- 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
- NaCl pellet is added until a conductivity: 50-70 mS / cm
- Collagen powder preparation 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.
- 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.
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
La presente solicitud relaciona un proceso
para la purificación de colágeno tipo I, así como la
obtención de colágeno en polvo, hidrogeles de colágeno y
elaboración de soportes unidireccionales y
multidireccionales a partir de éste.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.
Colágeno en ingeniería de tejidos:Collagen in tissue engineering:
El colágeno es el principal componente del
tejido conectivo y la proteína más abundante en el
cuerpo humano; tiene un papel importante en la adhesión,
quimiotaxis y en la diferenciación celular. Se han
descrito 28 tipos de colágeno que se agrupan en
subfamilias de acuerdo a su estructura: Colágenos
fibrilares (tipos I, II, III, V, XI, XXIV y XXVII),
colágenos relacionados y asociados a los fibrilares
(tipos IX, XII, XIV, XVI, XIX, XX, XXI y XXII),
colágenos formadores de reticulados filamentosos (tipo
VI), colágenos asociados a la membrana basal (tipos IV,
VII, XV y XVIII), colágenos de cadena corta (tipos VIII
y X), colágenos transmembranales (tipos XIII, XVII,
XXIII y XXV) y otros colágenos (tipos XXVI y XXVIII)
(Ricard, 2011; Gordon and Hahn, 2010).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).
En la familia de los colágenos, el tipo I es
el que mayor presencia tiene en tejidos conectivos
blandos como la piel, la mucosa oral y en duros como el
hueso. Esta proteína proporciona una gran resistencia a
la tensión, promueve la adhesión y migración de
numerosos tipos celulares incluyendo monocitos,
neutrófilos, queratinocitos y fibroblastos (Friess,
1998). En el proceso de cicatrización, el colágeno I
reemplaza gradualmente otros tipos de colágeno cuando el
tejido es remodelado y la cicatriz madura (Gordon and
Hahn, 2010). Una vez secretado por los fibroblastos del
lecho de la herida es entrecruzado para formar fibras
que ayudan en la organización de las células, en los
procesos contráctiles y de recambio de tejido que
ocurren durante la cicatrización (Gelse et al, 2003). 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).
El colágeno I es la proteína más empleada en
la elaboración de soportes para ingeniería de tejidos
(Kirubanandan & Sehgal, 2010; Sauerbier et al.,
2006; Fumiko et al., 2004; Takashi et al., 2001), ya que
por su biocompatibilidad, biodegradabilidad y
bioactividad puede interactuar con el ambiente en que se
coloca para inducir regeneración de los tejidos
(Williams, 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).
La forma de presentación más común del
colágeno es en estado sólido, ya que permite aumentar su
tiempo de vida media, debido a que se disminuye la
movilidad molecular, reduciendo la velocidad de las
reacciones de degradación (hidrólisis, oxidación,
deaminación, racemización, etc.) (Manning, Chou et al.
2010), además de poseer una baja actividad de agua,
factor que disminuye las probabilidades de contaminación
y degradación microbiológica del colágeno (Miyawaki
2009). Al obtener el colágeno en forma de polvo, el
material adquiere una combinación de propiedades (tamaño
de partícula, forma, área superficial y morfología) que
determinan cualidades de proceso como fluidez,
humectación, hidratación y dispersión, que lo convierten
en una buena alternativa como material para
procedimientos y/o excipientes de formulación,
permitiendo una fácil transformación a otras formas de
presentación: películas, partículas, suspensiones,
gránulos, matrices entre otros (Cape, Villa et al.
2008). Además, la forma en polvo del colágeno permite un
proceso de almacenamiento fácil, debido al uso de
recipientes poco voluminosos y livianos, además de no
requerir condiciones especiales y costosas de
almacenamiento como la refrigeración.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.
Entre los procesos más comunes para la
elaboración de polvos se encuentran la liofilización, el
secado por aspersión y el uso de fluidos supercríticos
(Cape, Villa et al. 2008), metodologías que requieren
equipos robustos, lo que hace que estos procesos sean
costosos. Una alternativa más económica es el uso de
secado por convección forzada de aire, que permite
controlar los procesos de transferencia de calor y masa,
sin alterar la calidad del producto (Paul Malumba,
2008), sin embargo, este proceso no se ha empleado en el
secado de colágeno.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
Soportes en la ingeniería de tejidos:Tissue engineering supports:
Uno de los mayores desafíos de la ingeniería
de tejidos es reproducir la compleja organización
celular y restablecer las funciones que las células
cumplen en el tejido nativo del cuerpo humano (Griffith
& Swartz, 2006). La estructura de un tejido y su
función están altamente relacionadas. En el tendón las
fibras están ordenadas de manera unidireccional, lo que
le proporciona su alta resistencia a la tensión (Kuo et
al., 2010); el patrón de fibras al azar del colágeno en
la piel le da sus propiedades mecánicas (Bottcher et
al., 2010); la forma de copa de los alveolos pulmonares
da eficiencia al proceso de intercambio gaseoso (Chen et
al., 2005; Faraj et al., 2007) y el arreglo complejo y
poligonal de los hepatocitos es esencial para la
apropiada función hepática (Tsang & Bhatia, 2006).
Para imitar esta organización los soportes empleados en
ingeniería de tejidos son modificados en su
microestructura, la cual se define por la porosidad,
interconectividad, tamaño y dirección de los poros.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.
La fabricación de soportes unidireccionales ha
sido investigada en la regeneración de tejidos que
presentan una estructura orientada como: nervios
periféricos (Xueyu et al., 2009; Chamberlain et al.,
1998; Stokols & Tuszynski, 2004), medula espinal
(Spilker et al., 2001), tendón (Louie et al., 1997;
Liu., et al, 2008), cornea (Wray & Orwin, 2009) y
hueso (Silva et al., 2006, Yunoki, et al., 2006).
Estudios in vivo en animales han demostrado que soportes
para regeneración nerviosa con microcanales
unidireccionales orientados longitudinalmente permiten
alcanzar una regeneración y recuperación funcional
equivalente al auto injerto, sin el uso de agentes
regenerativos externos o células (Xueyu et al., 2009;
Yannas, 2005). Otro estudio mostró que la
microestructura afecta el fenotipo de fibroblastos
corneales, ya que las fibras de colágeno alineadas
actúan como un mecanismo de señalización para disminuir
la expresión de alfa-actina en fibroblastos corneales de
conejo (Wray & Orwin, 2009). 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).
Para desarrollar estos soportes se han
empleado métodos de fabricación con técnicas de
separación de fases (Ma & Zhang, 2001), fabricación
de sólidos con formas libres (Taboas et al., 2003),
prototipado rápido (Wilson et al., 2004) y congelamiento
unidireccional (Madaghiele et al., 2007), sin embargo,
estos métodos producen soportes de dimensiones pequeñas
(aproximadamente 3mm x 5mm), además requieren de
equipamiento complejo y costoso.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.
La liofilización de suspensiones de colágeno
es el método más empleado en la elaboración de soportes
de este biomaterial. La microestructura del soporte
después de la liofilización es una réplica de la
morfología del cristal de hielo que se forma después de
ser congelado, por lo que la estructura puede ser
controlada directamente por el congelamiento (Schoof et
al., 2000).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).
Dada la importancia que conlleva la obtención
de soportes de colágeno tipo I, el desarrollo de este
proceso optimizado aquí reivindicado, muestra la
conveniencia de establecer procesos mejorados que además
de superar las desventajas presentes en los procesos
existentes permitan la obtención de soportes de colágeno
I con características superiores a las existentes.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.
La presente invención relaciona los
procedimientos para la purificación de colágeno tipo I,
hidrogeles de colágeno, colágeno en polvo, soportes de
colágeno multidireccionales y soportes de colágeno
unidireccionales, que comprenden las siguientes etapas: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. Purificación de colágeno tipo I.1. Purification of type I collagen.
Se toman fuentes de colágeno tipo I
provenientes de tejido conectivo animal como fascia,
tendones o hueso.Sources of type I collagen are taken
from animal connective tissue such as fascia,
tendons or bone
Dicha fuente de colágeno se prepara eliminando
tejidos adyacentes que se encuentren adheridos a ella,
se corta en trozos que permitan su tratamiento y se
lavan con aguaSaid 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
El material se desengrasa mediante lavados
consecutivos empezando con solventes de baja constante
dieléctrica (20) y finalizando con solventes de alta
constante dieléctrica (80), por ejemplo con etanol a
concentraciones superiores a 70%, éter y/o agua purificada.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.
Se realiza una disolución ácida con ácido
acético o clorhídrico hasta lograr un pH entre 2,5 y
3,2, a temperaturas inferiores a 5 °C y agitación
permanente 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
Se licua y centrifuga el producto obtenido y
luego se recupera el sobrenadanteThe product obtained is liquefied and centrifuged and
then the supernatant is recovered
Se adiciona al sobrenadante un álcali como
NaOH o KOH hasta lograr un pH entre 4,0 y 5.0; luego se
centrifuga y recupera el pellet obtenidoAn 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
Se adiciona al pellet una solución de una sal
que le otorgue a la solución un potencial iónico
equivalente al de una solución de cloruro de sodio 0,7M
hasta alcanzar una conductividad de entre 50 y 70 mS/cm,
se agita entre 1 y 24 horas, se centrifuga y recupera
nuevamente el pellet obtenidoA 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
Se realizan sucesivos lavados con agua a
temperaturas inferiores a 5 °C y agitación, se
centrifuga y recupera el pellet; todo esto se repite
hasta lograr una conductividad en el agua de lavado
entre 200 y 500 µS/cmSuccessive 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
Se obtiene colágeno Tipo I.Type I collagen is obtained.
En la presente invención, se determinó que la
conductividad es importante ya que permite controlar la
fuerza iónica y la relación de cargas a la que es
sometida la suspensión de colágeno. La presente
invención permite que la concentración de sal inicial
(controlada mediante la conductividad) ayude a
re-precipitar el colágeno I y a solubilizar proteínas
contaminantes. El control de la conductividad final
asegura que el contenido de sal remanente no afecte el
comportamiento celular, cuando las células son expuestas
al material aislado y purificado.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. Preparación de colágeno en polvo:2. Preparation of collagen powder:
Se cuantifica la suspensión de colágeno
(método gravimétrico o HPLC) y se ajusta con agua
desionizada entre 2-10% P/P.The collagen suspension is quantified
(gravimetric method or HPLC) and adjusted with water
deionized between 2-10% P / P.
Sobre moldes de teflón, se sirve una película
homogénea de la suspensión de colágeno de un espesor
deseado (entre 3 y 8 mm).On Teflon molds, a film is served
homogeneous of a thick collagen suspension
desired (between 3 and 8 mm).
Se eliminan las burbujas de aire de la
película mediante vacio (6,7 a 133.3 Pa) por diez minutos.Air bubbles are removed from the
Vacuum film (6.7 to 133.3 Pa) for ten minutes.
Se seca por convección forzada de aire a un
flujo de aire constante entre 20m3/hora a 40 m3/hora y
a una temperatura de 55 ± 5 °C por 48 horasIt 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
La película formada es retirada mediante una
herramienta tipo espátula y se tritura (producción por
fractura) en un mortero (aplicación de una fuerza mecánica).The film formed is removed by a
spatula type tool and is crushed (production by
fracture) in a mortar (application of a mechanical force).
El polvo obtenido es posteriormente molido
(tipo molino intermedio - cortador de disco rotatorio).
La mezcla de polvos obtenida es clasificada por tamaños
mediante la técnica de tamizado. La fracción de polvos
mayoritaria debe tener una rango de tamaño entre 1.00 y
0,30 mm equivalente a la fracción que pasa por un tamiz
estándar número 30 y retenido por un tamiz estándar
número 20.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. Obtención de hidrogel de colágeno 3. Obtaining collagen hydrogel
Pesar entre 1 y 10 g de Colágeno I en polvoWeigh between 1 and 10 g of Collagen I powder
Adicionar entre 100 y1000 mL de acido acético
0,5N de acuerdo a la concentración de colágeno deseada.Add between 100 and 1000 mL of acetic acid
0.5N according to the desired collagen concentration.
Homogeneizar en un homogenizador de rotor
estator a una velocidad entre 3000 y 8000 rpm.Homogenize in a rotor homogenizer
stator at a speed between 3000 and 8000 rpm.
Hidratar por un tiempo entre 12 y 18 horas Hydrate for a time between 12 and 18 hours
Adicionar ácido acético 0,5 N hasta para
lograr la concentración deseadaAdd 0.5 N acetic acid until
achieve the desired concentration
Homogeneizar en un homogenizador de rotor
estator a una velocidad entre 3000 y 8000 rpmHomogenize in a rotor homogenizer
stator at a speed between 3000 and 8000 rpm
Centrifugar por 15 minutos a una velocidad
entre 3000 y 8000 rpm.Centrifuge for 15 minutes at a speed
between 3000 and 8000 rpm.
Desechar sobrenadante.Discard supernatant.
Cuantificar el pellet formado.Quantify the pellet formed.
Ajustar la suspensión a la concentración
deseada (entre 1 y 10 % p/p de colágeno), con ácido
acético 0,5 NAdjust the suspension to the concentration
desired (between 1 and 10% w / w collagen), with acid
acetic 0.5 N
Mantener almacenado a 4°C.Keep stored at 4 ° C.
4. Obtención de soportes de colágeno4. Obtaining collagen supports
La suspensión obtenida se coloca en moldes de
material químicamente inerte y antiadherente como
teflón. Posteriormente se deben realizar los siguientes procedimientos:The suspension obtained is placed in molds of
chemically inert and nonstick material such as
Teflon Subsequently, the following procedures must be performed:
Desgasificar la suspensión aplicando vacío.Degassing the suspension by applying vacuum.
Congelar la suspensión a una temperatura entre
-20 a -40°C para obtener soportes multidireccionales.
Para elaborar soportes unidireccionales se cubre una de
las paredes del molde con un material termoconductor
inerte y se pone en contacto parte de este con un agente
de congelamiento adecuado asegurando un gradiente de
temperatura de 20 a 60°C a través de la suspensión.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.
Liofilizar con el fin de producir una
estructura porosa interconectadaFreeze dry in order to produce a
interconnected porous structure
Sumergir el material liofilizado en una
solución acuosa con un agente de reticulación (genipina,
glutaraldehido, dendrímeros, carbodiimidas o una mezcla
de ellos)Dip the lyophilized material in a
aqueous solution with a crosslinking agent (genipin,
glutaraldehyde, dendrimers, carbodiimides or a mixture
from them)
Desgasificar el sistemaDegassing the system
Dejar reaccionar entre 24 y 48 horasLeave to react for 24 to 48 hours
Retirar la solución que contiene el agente de reticulaciónRemove the solution containing the crosslinking agent
Hacer lavados con agua hasta garantizar el
retiro del agente de reticulaciónWash with water until the
removal of the crosslinking agent
Congelar el soporte entrecruzadoFreeze crosslinked support
LiofilizarFreeze dry
Esterilizar mediante oxido de etileno,
radiación gamma o etanol 70%Sterilize by ethylene oxide,
70% gamma or ethanol radiation
Si se toma la opción de las paredes
recubiertas con el material conductor, se logra un
soporte unidireccional (Figura 1); en caso de no usarse
se logra un soporte multidireccional (Figura 2).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).
Las Figuras que acompañan esta descripción
presentan dos fotografías de imágenes de microscopía
electrónica de barrido sobre soportes de colágeno
obtenidos por la tecnología aquí reivindicada.The figures that accompany this description
present two photographs of microscopy images
scanning electronics on collagen supports
obtained by the technology claimed here.
La Figura 1 muestra un soporte de colágeno
tipo I en cuyo procesamiento se ha aplicado, en alguna
de las paredes del molde, un material termoconductor
inerte con lo cual se logran soportes unidireccionales.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.
La Figura 2 presenta un soporte de colágeno
tipo I en el cual las fibras se disponen en todas las
direcciones durante la etapa de congelación y resulta
conformando un soporte multidireccionales.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.
Se encontró un protocolo preferido para la
producción de colágeno tipo 1 con las siguientes secuencias:A preferred protocol was found for
Type 1 collagen production with the following sequences:
Se parte de fragmentos de fascia o tendones de
rata, bovinos o porcinosIt starts from fragments of fascia or tendons of
rat, cattle or pigs
Se hacen lavados con diferentes solventes o
mezclas de solventes que permitan obtener constantes
dieléctricas en un intervalo entre 10 y 80.They are washed with different solvents or
solvent mixtures that allow obtaining constants
dielectrics in a range between 10 and 80.
Se hace la disolución del tejido con ácido
acético 0,5 M y agitación a temperatura de refrigeración
por un espacio de 24 horasThe tissue is dissolved with acid
0.5 M acetic acid and stirring at cooling temperature
for a space of 24 hours
Se licua y centrifuga el producto The product is liquefied and centrifuged
Se recupera el sobrenadanteThe supernatant is recovered
Se adiciona al sobrenadante NaOH 2M2M NaOH supernatant is added
Se centrifuga a 5000 rpm por 15 min y se
recupera el pelletIt is centrifuged at 5000 rpm for 15 min and
recover the pellet
Se lava con un volumen de agua igual al
volumen de pellet obtenidoWash with a volume of water equal to
volume of pellet obtained
Se centrifuga a 5000 rpm por 15 min y se
recupera el pelletIt is centrifuged at 5000 rpm for 15 min and
recover the pellet
Se adiciona al pellet NaCl hasta lograr una
conductividad: 50-70 mS/cmNaCl pellet is added until a
conductivity: 50-70 mS / cm
Se agita a 60 rpm por 1 horaStir at 60 rpm for 1 hour.
Se centrifuga y se recupera el pelletIt is centrifuged and the pellet is recovered
Se repiten la adición de agua, centrifugación
y recuperación del precipitado hasta que las aguas de
lavado alcancen una conductividad entre 200-500 mS/cmWater addition repeated, centrifugation
and recovery of the precipitate until the waters of
washing reach a conductivity between 200-500 mS / cm
Preparación de colágeno en polvo:Collagen powder preparation:
Se cuantifica la suspensión de colágeno
(método gravimétrico o HPLC) y se ajusta con agua
desionizada a un porcentaje entre 1-10% P/P.The collagen suspension is quantified
(gravimetric method or HPLC) and adjusted with water
deionized at a percentage between 1-10% P / P.
Se sirve sobre moldes de teflón una película
homogénea de la suspensión de colágeno.A film is served on Teflon molds
homogeneous of the collagen suspension.
Se eliminan las burbujas de aire de la
película mediante vacioAir bubbles are removed from the
empty film
Se seca.It dries up.
La película formada se tritura.The film formed is crushed.
El polvo obtenido es posteriormente molido
(tipo molino intermedio - cortador de disco rotatorio).
La mezcla de polvos obtenida es clasificada por
tamaños mediante la técnica de tamizado. La fracción de
polvos mayoritaria debe tener un rango de tamaño
entre1.00 y 0,30 mm.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.
Estas etapas permiten la obtención de colágeno
tipo I, con las características deseadas. Partiendo de
este colágeno se establecieron los protocolos preferidos
para desarrollar los soportes de colágeno:These stages allow to obtain collagen
Type I, with the desired characteristics. Starting from
this collagen preferred protocols were established
To develop collagen supports:
Soportes multidireccionales de colágenoMultidirectional Collagen Supports
Obtener suspensión del colágeno tipo I en
ácido acético 0,05 M de la proporción deseadaGet suspension of type I collagen in
0.05 M acetic acid of the desired ratio
Servir en moldes de teflónServe in Teflon Molds
Desgasificar la suspensión mediante vacío de
6,666 kPa hasta evidenciar cese de burbujeoDegassing the suspension by vacuum of
6,666 kPa until evidencing cessation of bubbling
CongelarFreeze
LiofilizarFreeze dry
Sumergir el material liofilizado en una
solución acuosa de glutaraldehidoDip the lyophilized material in a
aqueous glutaraldehyde solution
Desgasificar el sistema mediante vacíoDegassing the system by vacuum
Dejar en reposo por 24horasLet stand for 24 hours
Retirar la solución de glutaraldehidoRemove the glutaraldehyde solution
Lavar con agua purificadaWash with purified water
Congelar el soporte entrecruzadoFreeze crosslinked support
LiofilizarFreeze dry
EsterilizarSterilize
Soportes unidireccionales de colágenoUnidirectional Collagen Supports
Preparar una suspensión del colágeno tipo I de
la concentración deseada.Prepare a suspension of type I collagen from
the desired concentration
Servir en moldes de teflónServe in Teflon Molds
Desgasificar la suspensión mediante vacío de
6,666 kPa hasta evidenciar cese de burbujeoDegassing the suspension by vacuum of
6,666 kPa until evidencing cessation of bubbling
Congelar la suspensión poniendo la pared
recubierta con aluminio en contacto con un baño de
nitrógeno líquido asegurando un gradiente de temperatura
de 40°C a través de la suspensiónFreeze 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
LiofilizarFreeze dry
Retirar la tapa de los moldes y se continúa la
liofilización Remove the lid from the molds and continue the
lyophilization
Sumergir el material liofilizado en una
solución acuosa de glutaraldehidoDip the lyophilized material in a
aqueous glutaraldehyde solution
Desgasificar el sistema mediante vacío de
6,666 kPa hasta evidenciar cese de burbujeoDegassing the system by vacuum
6,666 kPa until evidencing cessation of bubbling
Dejar en reposo por 24horasLet stand for 24 hours
Retirar la solución de glutaraldehidoRemove the glutaraldehyde solution
Hacer lavados con agua purificadaWash with purified water
Congelar el soporte entrecruzadoFreeze crosslinked support
LiofilizarFreeze dry
EsterilizarSterilize
Claims (4)
- Procedimiento para la purificación de colágeno tipo I y soportes unidireccionales o multidireccionales con este material, CARACTERIZADO PORQUE comprende las siguientes etapas: a) Obtención de las fuentes de colágeno (Fascia, tendón); b) Limpiar la fuente de colágeno de tejidos adyacentes que se encuentren adheridos y cortar en trozos que permitan su tratamiento; c) Lavar con agua; d) Desengrasar el material empleando lavando con etanol en concentraciones superiores al 70%; e) Diluir con ácido acético hasta lograr un pH de 2,5 a 3,2, a temperaturas inferiores a 5 °C y agitación permanente; f) Licuar y centrifugar el producto obtenido y recuperar el sobrenadante; g) Adicionar al sobrenadante NaOH hasta lograr un pH entre 4,0 y 4,3 centrifugar y recuperar el pellet obtenido; h) Adicionar al pellet una solución de cloruro de sodio hasta alcanzar una conductividad de entre 50 y 60 mS/cm, agitar por un espacio superior a 50 minutos, centrifugar y recuperar nuevamente el pellet obtenido; i) Realizar sucesivos lavados con agua a temperaturas inferiores a 5 °C y agitación, centrifugar y recuperar el pellet; todo esto se repite hasta lograr una conductividad en el agua de lavado entre 200y 500 mS/cm.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.
- Procedimiento para la producción del colágeno tipo I reivindicado en 1 CARACTERIZADO PORQUE comprende las siguientes etapas hasta su pulverizado: a) Cuantificar una suspensión de colágeno (método gravimétrico o HPLC); b) Servir en moldes de teflón una película homogénea de la suspensión de colágeno de un espesor entre 3 y 5 mm; c) Eliminar las burbujas de aire de la película mediante vacio (50-1000 mTorr) por diez minutos; d) Secar por convección forzada de aire a un flujo de aire constante entre 25 y 35 m3/hora y a una temperatura de 55±5 C por 24- 48 horas; e) Retirar y triturar la película formada en un mortero de cerámica (aplicación de una fuerza mecánica); f) Moler el polvo obtenido (tipo molino intermedio - cortador de disco rotatorio), clasificar la mezcla de polvos obtenida por tamaños mediante la técnica de tamizado; la fracción de polvos mayoritaria debe tener una rango de tamaño entre 0,84 y 0,59 mm equivalente a la fracción que pasa por un tamiz estándar número 30 y retenido por un tamiz estándar número 20.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.
- Procedimiento para la producción de soportes multidireccionales de colágeno reivindicado en 1 CARACTERIZADO PORQUE comprende las siguientes etapas: a) Obtener suspensión de colágeno tipo I en la concentración deseada en ácido acético, que tengan un pH entre 3,0 y 3,2;b) Colocar en moldes que, opcionalmente, cuenten con paredes químicamente inertes y antiadherentes;c) Desgasificar la suspensión mediante vacío hasta evidenciar cese de burbujeo; d) Cubrir el molde asegurando que en su interior no existan espacios libres;e) Congelar la suspensión hasta que alcance -40°C; f) Liofilizar bajo vacío entre 20 y 48horas; g) Retirar la cubierta de los moldes y continuar la liofilización por otro período de tiempo entre 20 y 28 horas; h) Sumergir el material liofilizado en una solución acuosa con un agente de reticulación; i) Desgasificar el sistema mediante vacío hasta evidenciar cese de burbujeo; j) Dejar en reposo entre 20 y 28 horas; k) Retirar la solución con el agente de reticulación; l) Hacer lavados con agua hasta garantizar el retiro del agente de reticulación; m) Congelar el soporte entrecruzado hasta alcanzar temperatura de -20 a -40°C; n) Liofilizar por un espacio de tiempo entre 20 y 48 horas; o) Obtener soportes de colágeno tipo I multidireccionales.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.
- Procedimiento para la producción de soportes unidireccionales de colágeno reivindicado en 1 CARACTERIZADO PORQUE comprende las siguientes etapas: a) Realizar una suspensión de colágeno tipo I en la proporción deseada en ácido acético que tengan un pH entre 2,5 y 3,2; b) Colocar en moldes que, opcionalmente, cuenten con paredes químicamente inertes y antiadherentes, una de estas paredes en contacto con aluminio; c) Desgasificar la suspensión mediante vacío hasta evidenciar cese de burbujeo; d) Cubrir el molde asegurando que en su interior no existan espacios libres; e) Congelar la suspensión poniendo en contacto la pared recubierta en aluminio con un baño de nitrógeno, asegurando un gradiente de temperatura de 40°C a través de la suspensión; f) Liofilizar entre 20 y 48 horas; g) Retirar la cubierta de los moldes y continuar la liofilización por otro período de tiempo entre 20 y 28 horas; h) Sumergir el material liofilizado en una solución acuosa con un agente de reticulación; i) Desgasificar el sistema hasta evidenciar cese de burbujeo; j) Dejar en reposo entre 20 y 28 horas; k) Retirar la solución con el agente de reticulación; l) Hacer lavados con agua hasta garantizar el retiro del agente de reticulación; m) Congelar el soporte entrecruzado hasta alcanzar temperatura de -20 a -40°C; n) Liofilizar por un espacio de tiempo entre 20 y 48 horas; o) Obtener soportes de colágeno tipo I unidireccionalesProcedure 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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| Application Number | Priority Date | Filing Date | Title |
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| CO14-245779 | 2014-11-06 | ||
| CO14245779 | 2014-11-06 |
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Cited By (1)
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| WO2022131896A1 (en) | 2020-12-18 | 2022-06-23 | Top Health, S.A.P.I. De C.V. | Method for producing human collagen structures with controlled characteristics |
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