KR20180078625A - Collagen Membrane and Method for Fabricating the Same - Google Patents

Abstract

Disclosed is a collagen membrane capable of being applied to various medical purposes including dental barriers. Moreover, disclosed is a production method thereof. To this end, the collagen membrane includes a laminated structure of a plurality of plate-like collagen layers.

Description

[0001] The present invention relates to a collagen membrane and a method for manufacturing the same,

The present disclosure relates to collagen membranes and methods for their manufacture. More particularly, this disclosure relates to collagen membranes applicable to a variety of medical applications, including dental shields, and methods of making the same.

When tissue, organs, etc. are damaged, the body initiates self-healing, and in the process, scar tissue is formed, which is a factor that hinders normal tissue regeneration. Particularly, in the regeneration of the bony tissue, a phenomenon occurs in which the surrounding tissue penetrates and hinders normal bone formation. To overcome this problem, new healing methods such as guided bone regeneration or guided tissue regeneration (GTR) have been developed.

In the above-mentioned method, a bioactive scaffold for regenerating bone tissue is used, which supports the bone defect site through implantation and filling of the damaged or missing bone site, and at the same time, Refers to a biomaterial that induces or regenerates tissue. Generally. The bioactive scaffold for bone tissue regeneration can be divided into (i) an absorbent material filled in a bone defect portion and (ii) a bone tissue regeneration member for regenerating a bone tissue at a bone loss site.

In the early days, biomaterials for bone tissue regeneration were dependent on the inactivation characteristics in vivo, and there were many restrictions on the application due to infection and inflammatory reaction of surrounding tissues after the procedure. Recently, with the rapid development of biomaterial technology using metals, ceramics, polymers, etc., materials having biocompatible properties rather than bioinert have been developed and various types of bioactive materials for bone tissue regeneration Supports have been introduced. These bioactive scaffolds for regenerating bone tissue may have different physical properties to be used depending on the position of implantation, have no toxicity to surrounding tissues, and may require higher mechanical properties than other body parts. The above-mentioned bioactive scaffold for bone tissue regeneration is commercially available as various biomaterials depending on the characteristics and uses of the raw materials.

The materials for human body implantation, particularly bone tissue regeneration material, are excellent in processability and moldability, provide an environment suitable for cell adhesion and growth, and cell differentiation, and further, adducts formed by degradation thereof are also biocompatible .

In recent years, the demand for the development of implantable materials in the oral cavity has been rapidly increasing due to an increase in the number of implant treatment patients due to the improvement of living standards. The dental implant procedure is generally accomplished by cutting the patient's gums, inserting the implant post into the alveolar bone therein, and attaching an artificial tooth to the implant post. In order to perform the implant procedure, it is required that the alveolar bone on which the fixture is placed has a sufficient length and width. However, bones are often absorbed or lost in other tissues. The bone regeneration procedure using bone graft material and shielding membrane is mainly used for the regeneration and treatment of the dislocated bone during implantation. After the bone graft is placed on the bone defect site requiring implant placement, the shielding film is covered It is a factor that determines bone implant success, especially implant success rate. As such, the biological requirements of bone induction regeneration include supply of blood flow, stabilization, osteoblasts, limited space, and space maintenance, and various surgical procedures can be performed for this purpose.

As mentioned above, membranes that induce tissue regeneration should be biocompatible, not infectious of surgical wound infection or tissue degeneration, and made of materials with good cell growth characteristics. These membranes are divided into absorbent membranes and non-absorbable membranes, depending on the nature of the degradation.

The absorptive membrane is naturally biodegraded after implantation in the bone defect site, so that there is no need for a secondary operation for removal thereof and the operation is comparatively easy. On the other hand, since the durability is lower than that of a nonabsorbable membrane, The amount of tissue regeneration may be limited if the negative external shape is not good and the bone graft material does not help. Also, after transplantation in the human body (e.g. oral), rapid biodegradation is likely to be difficult to complete bone recovery and intermediate byproducts may cause local tissue reactions.

On the other hand, non-absorbable membranes induce sufficient tissue regeneration but require secondary surgery to remove the membrane.

As described above, since the existing absorbent and non-absorbable membranes have disadvantages in addition to the advantages, there is a continuing need for a membrane for bone tissue regeneration having improved characteristics.

According to a first aspect of the present disclosure,

A laminate structure of a plurality of collagen layers having a plate-like shape,

There is provided a collagen membrane which meets the following properties:

(i) a density of at least 0.4 g / cm < 3 &

(ii) an absorbance of 100 to 2,000%, measured according to ISO 13726-1,

(iii) an enzymatic degradation resistance (8h) measured according to ASTM F2212 of 40 to 80%, and

(iv) tensile strength and tensile elongation, measured according to ASTM D638, of at least 0.5 MPa and at least 5%, respectively.

According to one embodiment, the multiple collagen layer may have a cross-linked collagen structure.

According to a second aspect of the present disclosure,

a) pulverizing the collagen-containing dermis layer derived from the swine dermis together with water;

b) shaping the crushed dermal layer into a membrane by vacuum filtration; And

c) performing a double bridge comprising physical crosslinking and chemical crosslinking on the membrane-shaped molding;

/ RTI >

There is provided a method for producing a collagen membrane, wherein the crosslinked membrane-shaped molding has a laminated structure of a plurality of plate-like collagen layers.

According to one embodiment, the method may further comprise lyophilizing the membrane-shaped molding between steps b) and c).

According to one embodiment, prior to said step a)

Deglossing the pork to obtain a pig dermis; And

Enucleation of the pig dermis;

As shown in FIG.

According to one embodiment, step a) may be carried out after lyophilization of the enucleation-oxidized porcine dermis.

The collagen membrane according to the embodiment of the present disclosure is produced by treating the porcine dermis by a specific method, thereby being harmless to the human body and preventing cellular inflow of surrounding tissues and promoting osteogenesis by proliferation of cells derived from bone tissue . In particular, it has an excellent biochemical durability and mechanical properties capable of maintaining a constant shape for a period of time required for bone regeneration, and also has an advantage that affinity to peripheral tissues is good and side effects such as inflammation are not caused. Therefore, wide commercialization is expected in the future.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process flow diagram exemplarily showing a series of steps for producing a collagen membrane according to another embodiment of the present disclosure; FIG.
FIG. 2 is a photograph showing the results of observation of an H & E stained tissue slice and optical microscopic observation (40-fold and 100-fold) for evaluating the content of nucleic acid components before and after treating the porcine dermal layer with the nucleic acid solution in Example 1 ;
3 is a scanning electron microscope (SEM) image of a cross-section of a membrane-shaped molding which has undergone reduced pressure filtration and freeze-drying in Example 1;
FIG. 4 is an optical microscope photograph showing the cytotoxicity of the collagen membrane prepared according to Example 1; FIG.
FIG. 5 is a graph showing the result of comparing absorbency between the collagen membrane manufactured according to Example 1 and a commercial membrane product; FIG. And
6A and 6B are graphs showing the results of comparing mechanical properties (tensile strength and tensile elongation) between the membrane member and the commercial membrane product of the shielding membrane produced according to Example 1. FIG.

The present invention can be all accomplished by the following description. The following description should be understood to describe preferred embodiments of the present invention, but the present invention is not necessarily limited thereto. It is to be understood that the accompanying drawings are included to provide a further understanding of the invention and are not to be construed as limiting the present invention. The details of the individual components may be properly understood by reference to the following detailed description of the related description.

In this specification, when an element is referred to as "comprising ", it means that it may further include other elements unless otherwise stated.

It will be understood that terms such as " on "or" above ", and "below" or "below " may be understood to describe relative positional relationships between components or members, Quot; position "can be understood as expressing a relative positional relationship not only in a state of being in contact with a specific object but also in a non-contact state.

"Collagen" is a natural biodegradable polymer, which means a protein that is a major constituent of connective tissue (skin, bone, tendon, etc.) of an animal, and may be understood to mean a protein family rather than a specific single protein. Specifically, collagen is a structural support of the body constituting the flesh and connective tissues of mammals. Its basic constituent unit is a tropocollagen protein (composed of three polypeptide chains of the same size) They are wrapped around each other to form a superhelical cable or a triple-helix structure. Collagen can be largely classified into Type I, Type II, Type III, and Type V, and this type of collagen forms a thin and long fibrous structure and is dense.

One of the greatest features of the collagen membrane according to the embodiment of the present disclosure is that it has a laminated structure of a plate-like multiple collagen layer. By having a laminate structure of a plurality of thin collagen layers as described above, it is possible to maintain the external shape or structure required for bone regeneration in the environment (particularly, the oral environment) of the human body, and furthermore, When used as a shielding film made of a metal material (for example, titanium or its alloy), it is possible to suppress the phenomenon that the membrane is torn or damaged by the frame member even when tensile force is applied, such as bending of the shielding film Mechanical properties (or durability) can be ensured.

In an exemplary embodiment, a plate-like multiple collagen layer constituting a laminate structure in a collagen membrane may have a crosslinked collagen structure. By thus introducing a crosslinking (or crosslinking network) structure into the collagen, It is possible to suppress the phenomenon that the member absorbs the body fluid in an excessive amount and can not maintain the mechanical properties. In addition, the collagen degradation in the membrane due to the enzyme contained in the saliva when contacted with the saliva can be suppressed.

Particularly, by introducing a laminated structure of a plurality of collagen layers, the shielding effect against external factors can be enhanced, and the decomposition resistance in the oral environment can be improved by enhancing the collagen layer mutual adhesion property. In this connection, when the amount of the hydroxyl group (-OH) in the hydroxyproline (OH-Pro) among the total amino acid composition of the collagen component in the membrane member into which the crosslinked structure is introduced is not crosslinked (or before the crosslinking structure is introduced) For example, about 90% or less, specifically about 50 to 80%, and more specifically about 60 to 70%.

At this time, the thickness of the individual collagen layer constituting the laminated structure may be, for example, in the range of about 1 to 50 mu m, specifically about 10 to 45 mu m, more specifically about 15 to 35 mu m. However, in the laminate structure, most of the collagen layer may exist in intimate contact with other collagen layers adjacent to the upper surface and / or lower surface, but may be slightly spaced at some surface portions in some cases.

In an exemplary embodiment, the thickness of the collagen membrane can range from about 0.1 to 1 mm, specifically from about 0.2 to 0.6 mm, more specifically from about 0.3 to 0.5 mm, the bone regeneration period, the required mechanical and biochemical Physical properties and the like.

According to one embodiment, the improved collagen membrane in terms of mechanical and biochemical properties meets a plurality of physical properties requirements.

Specifically, the density of the collagen membrane may be at least about 0.4 g / cm3, specifically about 0.48 to 0.7 g / cm3, more specifically about 0.5 to 0.65 g / cm3. The density of the membrane is a factor that affects the shape and mechanical properties, and below a certain level, the collagen has a spongy-like property rather than a lamellar structure. On the other hand, if the density is excessively high, the physical strength is excellent but the usability is lowered and the flexibility in hydration may be deteriorated. Therefore, it may be advantageous to have the characteristics within the above-mentioned density range.

On the other hand, the absorption of the membrane may range from about 100 to 2,000%, specifically about 500 to 1,000%, more specifically about 100 to 200%. At this time, the absorption rate of the membrane can be measured by ISO 13726-1 (Methods for the direct testing of wounds. Part 1: Aspects concerning absorption). The reason why it is preferable to maintain the membrane absorption rate within a specific range is to secure a proper absorption rate for the liquid component (moisture) contained in the saliva, blood, etc. in the defect space in the human body, And to obtain a rapid re-vascularization effect using these factors in the blood. However, when the water absorption rate is excessively low or high, problems may arise in terms of the utilization of the growth factors contained in the blood and the function of the membrane. Therefore, it is advantageous to appropriately control the water content within the above-mentioned range as much as possible.

In addition, the membrane must have a resistance to enzymatic degradation of a certain level or higher so that it can not be degraded by enzymes contained in saliva or body fluids during the bone induction regeneration period. Enzymolysis resistance (8 hours) can be measured according to ASTM F2212 to quantify these properties, and in this particular example about 40 to 80%, specifically about 50 to 70%, more specifically about 55 to 65% Lt; / RTI > If the resistance to degradation of the enzyme is too low, the inflammation due to the infection may be induced in the periphery due to exposure to the membrane degradation in the state where the bone regeneration is not sufficient, whereas when the degradation resistance of the enzyme is excessively high, Secondary elimination is needed to remove. Therefore, it is preferable to have a value within the above-mentioned range.

In an embodiment according to the present disclosure, the mechanical properties of the collagen membrane are determined by a support function for maintaining the bone regeneration space and an external force (for example, tensile force) It provides an important function for suppressing the membrane tearing or damage. In view of the above, it may be required that the collagen membrane has a tensile strength and a tensile elongation higher than a certain level in this specific example, and such mechanical properties can be measured according to ASTM D638.

In one embodiment, the tensile strength of the membrane may be in the range of at least about 0.5 MPa, specifically about 0.5 to 10 MPa, more specifically about 1 to 5 MPa. In addition, the tensile elongation percentage may be in the range of at least about 5%, specifically about 10 to 40%, more specifically about 15 to 30%. Thus, the tensile strength and the tensile elongation must be kept within an appropriate range, since they must have appropriate strength and flexibility.

The above-mentioned biochemical and mechanical properties are not only complementary properties but also conflicting properties, so it is required to satisfy them at the same time. In view of the above, another embodiment of the present disclosure provides a method for producing a membrane structure (molded product) through a specific procedure using collagen derived from a porcine bark.

FIG. 1 shows an example of a series of processes for producing collagen membranes, which can largely include a preprocessing step of porcine dermis, a membrane structure forming step, and a two-step crosslinking step.

Pretreatment step (optional)

In the pork loin, various kinds of foreign substances such as blood are contained or attached, and various fat components and nucleic acid components are contained. Thus, one or more pigs pretreatment steps are performed prior to the preparation of the membrane using the porcine dermis-derived collagen. Hereinafter, an example of a pretreatment process of the poultry obtained and at least one of the illustrated pretreatment steps can be performed.

(i) washing / removing the fat layer

According to one embodiment, it is possible to carry out a step of washing the poultry obtained as one of the pretreatment processes of the poultry and removing the fat layer. In order to wash the ponzu, it may be firstly immersed in an aqueous solution to which sodium chloride or the like is added, and subjected to shaking culture with or without stirring. At this time, the concentration of the additive component may be in the range of, for example, about 0.5 to 2 M, specifically about 1 to 1.7 M, and for example, about 30 to 45 캜, specifically about 35 to 40 캜, Lt; / RTI > In addition, the shaking culture time can be adjusted within the range of, for example, about 50 to 100 hours, specifically about 65 to 80 hours.

After the shaking culture process, it is washed once more with water (specifically, deionized water), and in some cases, the fat portion existing around the pork skin is washed using a cutting means (for example, scissors, etc.) Can be removed. As such, when the washing is completed, it may be required to remove the fat layer (subcutaneous fat layer) present under the pork pie, so that the pork fat can be frozen and then the fat layer can be removed.

(ii) degreasing treatment step

The pre-wash / fat removal stage involves mainly physical treatments and still retains the fat (lipid) components contained in the pork. Therefore, it may be preferable to perform degreasing treatment so as to be in a state containing only the collagen component as much as possible. For this purpose, a degreasing solution is used to chemically remove the remaining lipids, and further, the external virus (or primary virus) remaining in the pork can be deactivated.

Illustratively, a mixed solution of a base component (for example, sodium hydroxide) and acetone is prepared for the degreasing treatment, and then the solution is heated at a temperature of about 1 to 10 DEG C, specifically about 2 to 5 DEG C for about 1 to 5 hours, For about 2 to 4 hours, and in some cases, stirring may be carried out. Subsequently, further treatment in ethanol (e.g., at a concentration of about 50% or more, specifically about 70% or more, more specifically about 90% or more) for about 10 to 20 hours, specifically about 15 to 18 hours Residual lipids can be removed. Then, the external virus can be inactivated by treatment with a germicidal component, such as hydrogen peroxide.

The lipid content (crude fat content) in the swine dermis obtained through the above-described treatment process is reduced to, for example, about 0.005% or less, specifically about 0.003% or less, more specifically about 0.002% or less.

 (iii)

Even after the de-lipidization step is completed, the pig's dermis contains nucleic acid components, and if contacted with the oral mucosa of the human body, it may cause immune reaction and rejection of transplantation. Therefore, it is possible to carry out the nucleic acid treatment for removing the nucleic acid component (for example, DNA, RNA, etc.) in the swine dermis. The treatment solution that can be used for the nucleic acid treatment may be a buffer solution, for example, a buffer solution containing Triton-X, Trizma base (or Tris base), SDS (sodium dodecyl sulfate) and the like. After the enucleation oxidation treatment is completed in this manner, neutralization washing (treatment) can be performed using disinfection water.

(iv) lyophilization step

After the above-mentioned nucleic acid treatment, the lyophilization layer of the porcine dermis layer can be subjected to a lyophilization step so as to easily form a pulverized product of the pig dermis necessary for the process of forming the membrane-like complex structure. At this time, the freeze-drying is carried out under a temperature condition of, for example, about -90 to -50 ° C, specifically about -85 to -60 ° C, more specifically about -80 to -70 ° C, , Specifically about 15 to 40 hours, more particularly about 20 to 30 hours.

Molding step of membrane shape

According to one embodiment, a slurry (or suspension) of porcine dermis powder is prepared by physically breaking the porcine dermis layer (specifically, pretreated porcine dermis layer) with water (e.g. deionized water). Here, in the pulverization process, a pulverizing means known in the art, for example, a blender, a homogenizer, a freezing pulverizer, an ultrasonic pulverizer, a hand blender, a plunger mill or the like may be used, but the present invention is not limited thereto.

The size of the pulverized product of the porcine dermal layer can be adjusted in consideration of the size of the filter net used in the vacuum filtration process to be described later, the required physical properties of the membrane member, and the like, and is about 0.7 to 2 탆, Mu] m, more specifically in the range of about 0.9 to 1.2 [mu] m, but this is for illustrative purposes and may be adjusted to an appropriate range in consideration of the size of the filter net.

The amount of water to be added in the pulverization process of the duck bark is not limited to a specific amount as long as it can secure the viscosity, the supporting force, and the like necessary for shaping into a membrane shape. Illustratively, about 500 to 1000 parts by weight, specifically about 550 to 800 parts by weight, more specifically about 600 to 700 parts by weight, can be selected for 100 parts by weight of the dermal layer. However, when the addition amount of water is too small or too large, it is difficult to form a membrane structure, so that it may be advantageous to control the addition amount of water within the above-mentioned range.

According to an exemplary embodiment, the slurry of the prepared porcine bark crushed material is formed into a membrane shape through reduced pressure filtration. The membrane forming process through the reduced pressure filtering is performed by pouring a predetermined amount of the slurry of the dermis crushed material onto the filter net and applying a reduced pressure to the bottom of the filter net so that the crushed dermis layer is closely adhered to the filter net and moisture is removed from the filter net, A membrane-like structure made of water can be formed (reduced pressure filtering step). At this time, the filter network may be formed of a stainless steel (for example, SUS series), a cellulose (for example, cellulose acetate), a fluorine (e.g., PTFE, PVDF, Glass), a sulfone system (e.g., polyethersulfone), a polyaramid system (e.g., par a-aramid, Nylon 6, Nylon 6,6).

On the other hand, the pressure applied in the above-described vacuum filtration process may be, for example, about 600 Torr or less, more specifically about 10 to 500 Torr, and more specifically about 200 to 300 Torr. Further, the size of the filter net may be in the range of, for example, about 0.2 to 1 mu m, specifically about 0.4 to 0.8 mu m, more specifically about 0.5 to 0.7 mu m. However, the size of the filter net can be set to be smaller than the size of the crushed dermal layer so that the crushed dough layer of pigs does not exit the filter net.

The thus-formed membrane structure still contains a certain amount of water and is in a deformable state while exhibiting a viscosity, so that it may undergo a drying process. According to an exemplary embodiment, the membrane structure may be dried in a lyophilized manner. In lyophilization, the temperature may be set, for example, in the range of about -90 to -50 ° C, specifically about -85 to -60 ° C, more specifically about -80 to -70 ° C. At this time, drying (or lyophilization) of the membrane structure can be performed under reduced pressure, and the pressure can be about 600 Torr or less, more specifically about 1 to 100 Torr, more specifically about 2 to 10 Torr.

Optionally, the membrane structure may be pre-frozen prior to drying (or lyophilization), for example, at a temperature of about -70 to -50 캜, more specifically about -65 to -55 캜 for at least about 1 hour , Specifically about 1 to 3 hours.

After the above-described molding step by the reduced pressure filtration and the drying (freeze-drying) step, the membrane structure (molded product) has a laminated form of a plate-shaped collagen layer.

2 step crosslinking step

According to one embodiment, the dried membrane structure is formed by introducing a cross-linking structure (cross-linking network) to the collagen layer through a two-step sequential cross-linking step to provide the necessary bone regeneration period (e.g., about 4-8 months, ≪ / RTI > to 7 months). Specifically, the amount of the hydroxyl group (-OH) in the collagen decreases due to the crosslinking reaction. For example, in the total amino acid composition of the collagen component in the membrane member into which the crosslinking structure is introduced, the hydroxyl group in the hydroxyproline (OH-Pro) For example, about 90% or less, specifically about 50 to 80%, more particularly about 60 to 70%, as compared to when the amount of (-OH) is not crosslinked (or before the crosslinking structure is introduced) .

In this embodiment, one of the important features is that a plurality of crosslinking reactions, specifically, a heat-treating crosslinking reaction and a chemical crosslinking reaction are sequentially carried out. The reason for progressing in the order of physical crosslinking-chemical crosslinking is as follows. In the case of a membrane structure freeze-dried after reduced pressure filtration, the binding force between pulverized tissues is weak, so that pulverized tissues can easily dissociate during hydration. Therefore, heat treatment crosslinking reaction and chemical crosslinking reaction are sequentially performed to maintain the physical strength of the membrane structure and the shape of the membrane structure during hydration through heat treatment crosslinking. Further, there is an advantage that the structure of the multilayer film of the membrane structure becomes more dense through heat treatment crosslinking. Chemical cross-linking has a higher degree of cross-linking than thermal cross-linking, which is a physical cross-linking method, so that it has a high resistance to decomposition and can serve as a stable membrane during the bone formation period after transplantation.

Details of the exemplary two-step crosslinking reaction are as follows.

(i) Heat treatment Crosslinking reaction (dehydration heat treatment)

Heat treatment of collagen The cross-linking reaction is a type of physical cross-linking reaction, which removes moisture in the collagen to increase the shrinkage temperature of the collagen. Collagen is formed by removing water from the collagen, and the collagen is decomposed at a temperature of, for example, about 80 to 130 DEG C (specifically about 90 to 125 DEG C, more specifically about 100 to 120 DEG C) , About 750 Torr or less), for example, for about 12 to 30 hours (specifically about 15 to 25 hours, more specifically about 18 to 22 hours). The heat-treated cross-linking reaction mechanism is shown in the following Reaction Scheme 1.

[Reaction Scheme 1]

In an exemplary embodiment, the cross-linking degree is increased, for example, by about 5 to 30%, specifically about 10 to 25%, more specifically about 15 to 20%, by heat-treated crosslinking reaction.

(ii) chemical cross-linking reaction

The chemical cross-linking reaction of collagen may involve the use of a cross-linking agent, typically by reacting a bifunctional compound with an amine group of a lysine or hydrolysin moiety on a different polypeptide chain, or by activating the carboxyl group of glutamic acid and aspartic acid moiety And then reacted with an amine group of another polypeptide chain to form an amide bond, whereby a chemical cross-linking reaction can be performed. In an exemplary embodiment, glutaraldehyde, an epoxy compound, a carbodiimide compound, an acyl azide, and the like can be used. According to certain embodiments, carbodiimide compounds can be used as chemical cross-linking agents. The water-soluble carbodiimide compound can activate the free carboxyl group of glutamic acid and aspartic acid moiety in collagen, and it is also possible to use a carbodiimide (for example, 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxyl group to form the O-acylisourea group. The free amine group of the (hydroxy) lysine residue with urea as the leaving group forms an amide cross-link through a condensation reaction by nucleophilic attack. The mechanism of this chemical cross-linking reaction is shown in the following reaction formula (2).

[Reaction Scheme 2]

According to an exemplary embodiment, the EDC crosslinking agent can be used in the form of a solution, for example, a solution using water and ethanol as a solvent. The concentration of the EDC crosslinking agent in the solution may be, for example, in the range of about 1 to 5 mM, specifically about 2 to 4 mM, more specifically about 2.5 to 3.5 mM. The reaction using the EDC solution is carried out in a shaking culture manner, for example, at about 1 to 10 DEG C, specifically about 3 to 6 DEG C, for example, for about 15 to 30 hours, specifically about 20 to 25 hours . At this time, it may be advantageous to carry out agitation for promoting the reaction. In the case of the chemical crosslinking reaction, a high degree of crosslinking (for example, up to about 90%, specifically about 60 to 80%) can be obtained, as compared with the above-mentioned heat-treated crosslinking reaction.

When the above-described two-step crosslinking reaction is completed, a step of washing the crosslinking agent remaining on the crosslinked membrane structure may be selectively performed. Illustratively, ultrasonic cleaning can be used. Specifically, ultrasonic cleaning may be performed at least once, and shaking culture may be performed to remove the crosslinking agent component remaining in the composite structure.

Subsequently, neutralization can be performed using a phosphate buffered saline (PBS), in order to control the osmotic pressure and the pH in a condition similar to body fluids. At this time, the pH of the PBS can be adjusted to, for example, about 7 to 8, specifically about 7.3 to 7.5. At this time, the neutralization treatment can be performed at least once using ultrasonic waves in a manner similar to the cleaning step described above.

Subsequently, the composite structure that has undergone the cleaning and / or neutralization treatment is again freeze-dried to finally produce a collagen membrane for bone tissue regeneration. At this time, the drying conditions may be set within the range described above, and a preliminary freezing process may be followed before drying (or lyophilization).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the present invention is not limited thereto.

Example 1

Purification of Ponzu (Pretreatment)

The pork purchased in the market was immersed in a 1.5 M aqueous NaCl solution and shaken for 72 hours under stirring conditions of 37 ° C and 150 rpm. Thereafter, the peripheral fat portion of the pork loin was removed, cut to a predetermined size, and washed three times with deionized water. The washed pork was frozen at -60 ° C and the fat layer was removed using a mortar.

Then, a defatted lipid-treated solution prepared by mixing 1.5M NaOH and acetone at a volume ratio of 6: 4 (volume basis) was prepared, and the mixture was incubated at 4 ° C and 150 rpm for 2 hours with shaking for 2 hours. Ethanol for 16 hours to remove residual lipids, and the duck skin layer of the pig was separated. Then, 12% concentration of hydrogen peroxide was added to the obtained duck skin layer to remove discoloration and foreign matter, and to deactivate the primary virus, followed by shaking culture at 4 ° C and 150 rpm for 2 hours.

According to ISO 1444 (1996, Meat and meat products-Determination of free fat contents) and Soxhlet extraction method (sample of more than 0.2 g, 100 mL ether, 8 hour extraction (80 캜)), Evaluation was carried out. The results are shown in Table 1 below.

Sample number Sample weight (g) Sample weight
Total (g) flask
Weight (g) Weight of flask after measurement (g) Crude fat
content (%) One 0.0905 0.2465 3 times measurement
Average 135.8132 g 4 measurements
135.8167g 0.002% 2 0.0729 3 0.0831 4 0.0623 0.2662 3 times measurement
Average 129.4189g 4 measurements
Average 129.4209g 0.001% 5 0.0916 6 0.1123

As shown in the above table, it can be confirmed that the lipid component in the pork dermis layer was substantially removed through the degreasing treatment.

To remove the residual nucleic acid in the dregs-treated swine duck layer, a nucleic acid solution containing 2.4 g of Tris, 4 g of EDTA, 2.34 mL of Triton X100 and 2.5 g of SDS was prepared in 2000 mL of distilled water, and the prepared nucleic acid solution 250 mL of porcine dermis and incubated for 24 hours under agitation at 150 rpm.

H & E staining tissue slices were prepared and the pig dermal layer before and after enucleation was observed with an optical microscope (40-fold and 100-fold) in order to evaluate the degree of reduction of nucleic acid components by enucleation oxidation treatment. 2. As shown in the figure, it can be confirmed that the nucleic acid component in the pig dermal layer was significantly reduced through the nucleic acid treatment.

The duck skin layer of the enucleation-oxidized pig was pre-frozen at -84 DEG C and then lyophilized for 24 hours.

Manufacture of Membrane Structures

20 g of the lyophilized swine duck layer was placed in a beaker, and 130 mL of distilled water was added thereto. The duck skin layer was pulverized for 2 minutes using a hand mixer to prepare a slurry of the dyneposite ground product. A Buchner 2000 mL filtration flask and a vacuum pump were connected. A 0.7 μm disc filter was placed in Buchner and distilled water was flowed to make the Buchner and the filter closely contact each other, and a vacuum pump was operated.

100 mL of the pulverized slurry of porcine dermal layer prepared prior to Buchner was added and filtered under reduced pressure for 24 hours (460 Torr). The membrane structure obtained after the completion of the reduced pressure filtering was separated into tweezers, put in a freeze dryer, and preliminarily frozen at -60 ° C for 1 hour. The pre-frozen membrane structure was then placed on the top shelf and the vacuum valve connected to the top shelf was opened to ensure that the vacuum was maintained and then lyophilized for 24 hours at -86 < 0 > C and 5 Torr.

A scanning electron microscope (SEM) photograph of the section of the lyophilized membrane structure through the above-described reduced pressure filtration is shown in Fig. From the figure, it was confirmed that the lamellar structure was formed while the plate-like multiple collagen layers were in intimate contact with each other under reduced pressure filtration and freeze-drying.

Step 2 (physical crosslinking - chemical crosslinking) step

After removing the lyophilized membrane structure and moving it into a vacuum oven using tweezers, the vacuum valve was opened and the external air inlet was closed. After 18 hours and 30 minutes, heat treatment was carried out in a vacuum oven (110 ° C) ) Crosslinking. After completion of heat treatment crosslinking, the temperature was lowered and the vacuum was released.

Then, the heat-treated crosslinked membrane structure was added to 600 mL of a 2.5 mM EDC crosslinking solution (water: ethanol (99%) = 1: 1) and incubated at 4 ° C and 150 rpm for 24 hours with shaking. After completion of the chemical crosslinking reaction by shaking culture, the crosslinking solution was removed, 2000 mL of distilled water was added, and ultrasonic washing was performed three times at intervals of 5 minutes.

The washed composite structure was then neutralized in PBS (pH 7.4) for 24 hours under agitation conditions. The neutralization treatment solution was discarded and the membrane structure bridged with distilled water was washed and pre-frozen at -60 ° C for 1 hour. Then, the collagen membrane was obtained by freeze-drying at -86 캜 and 5 Torr for 24 hours.

Characterization of collagen membrane

The obtained collagen membrane was subjected to a characterization test as described below.

(1) Cytotoxicity evaluation test

end. Materials Used

- Cell line: L929 cell (CCL-1, ATCC, USA)

- Positive Control: A polyurethane film (RM-A, Hatano Research Institute, Japan) containing 0.1% zinc diethyldithiocarbamate (ZDEC)

- Negative Control: High density polyethylene film (RM-C, Hatano Research Institute, Japan)

I. step

(i) Preparation of test solution: 2 ml per sample was added to an alpha-MEM culture medium to which no 10% FBS (fetal bovine serum) was added and eluted for 48 hours.

(ii) Preparation of culture control solution: The test substance was not added and the cells were added to an alpha-MEM culture medium to which 10% FBS was not added, followed by elution for 48 hours

(iii) Preparation of negative control solution: A high-density polyethylene film as a negative control component was added to a culture medium of alpha-MEM containing 10% FBS at a rate of 6 cm2 / mL and eluted for 48 hours.

(iv) Preparation of positive control solution: ZDEC polyurethane film 0.1 g / mL was added to a 15 mL tube containing a 10% FBS-free alpha-MEM culture medium and eluted at 37 DEG C for 48 hours.

All. Evaluation Method and Criteria

(i) Testing and evaluation were conducted in accordance with ISO 10993-5 and the Common Criteria for Biological Safety of Medical Devices (Korea Food and Drug Administration Notice 2006-3).

(ii) Evaluation method: Cells were observed under a microscope, and the living cells were attached to the bottom in a long branch shape, whereas the dead cells were round in shape, floated in the culture medium, Could not be distinguished.

(iii) On the next day, when the cells proliferated to about 80% of the plate bottom area, the culture broth was removed from the plate, and the test solution and the reference solution were added and cultured in a 5% CO2 incubator for 24 hours.

(iv) After 24 hours, when the positive and negative controls were correct, the cells were observed under a microscope and determined to be cytotoxic if the response to the test was less than grade 1. The detailed criteria are shown in Table 2 below.

The above cytotoxicity evaluation results (culture control, negative control, positive control and sample) are shown in FIG.

According to the figure, in the case of the collagen membrane obtained in this example, a large number of living cells in the shape of long branches were attached to the bottom. This means that the membrane is not substantially cytotoxic. On the other hand, in the culture control and negative control groups, the ratio of dead cells to living cells was high, especially in the positive control group.

(2) Biochemical and mechanical properties testing

Comparative experiments were carried out on collagen membranes obtained in Example 1 on the basis of commercially available collagen membranes (Comparative Example) and biochemical and mechanical properties. The results are shown in Table 3, and in FIGS. 5 and 6.

Test Methods Example 1 Comparative Examples 1 to 5
Psalm Information Crosslinking method 2 step bridge
(DHT / EDC) Chemical bridge
(EDC / NHS) Heat treatment bridging Non-bridge Non-bridge Decomposition period more than 6 month more than 6 month 3 to 5 months 3 to 4 months 3 to 5 months No. Test Evaluation Items raw materials Nongpie Cow tendon Nongpie Small sacrum One Thickness (mm) - 0.50 0.45 0.24 0.48 - 0.45 2 Density (g / cm3) - 0.51 0.34 0.66 0.08 - 0.26 3 Absorption (%) ISO 13726-1 165.1 169 567 1689.04 - - 4 Enzyme degradation resistance (%)
(Collagenase 50U, 8h) ASTM F2212 82.73 (10h)

67.79 (8H) 43.05 (10h) 8.00 (8H) - 0.00 (8h) 6.00 (8H)
5 Tensile Strength (MPa) ASTM D638 5.63 3.27 4.69 0.74 - - 6 Tensile elongation (%) 33.12 14.4 14.8 14.1 - -

According to the above tables and figures, the collagen membrane produced according to Example 1 has good properties all over a plurality of evaluation indexes.

In the case of Comparative Example 1 in which only chemical crosslinking was carried out, the absorbency showed values similar to those in Example 1, but the tensile strength and the tensile elongation were significantly lower than those in Example 1. In addition, Comparative Examples 2 and 3 which had undergone the heat treatment crosslinking exhibited lower physical properties than those of Example 1 in absorbency, enzyme decomposition resistance, tensile strength and tensile elongation.

In addition, Comparative Examples 4 and 5, which were not accompanied by a crosslinking reaction, exhibited poor physical properties in comparison with Example 1 on all evaluation surface planes.

(3) Evaluation of amino acid composition in collagen membrane

The amino acid composition was measured in Example 1 in which the two-step crosslinking step was performed and in Comparative Example 4 in which no crosslinking step was performed. Specifically, the PITC-labeled sample was dissolved in 400 μL of buffer solution and diluted 10-fold, and 10 μL of the sample was taken and loaded on HPLC. The analytical instrument used is as follows.

- System: Two Waters 510 HPLC pumps, a Waters Gradient controller and a Waters 717 automatic sampler

- Column: Waters Pico-tag column (3.9 x 300 mm, 4 mm)

Detector: Waters 2487 UV detector (254 nm)

- Data Analysis: empower 2 software

As a result of analysis, the OH-Pro content of Example 1 was 1054.276 pmol while the OH-Pro content of Comparative Example 4 was 1186.949 pmol. As described above, in Example 1, the content of -OH group in the collagen protein constituting the collagen membrane is lower than that in Comparative Example 4, indicating that the decomposition resistance is high in the oral environment.

(4) Evaluation of crosslinking density

Crosslinking of cross-linked collagen was confirmed by nynhydrin analysis to confirm cross-link density after heat-treated cross-linking and chemical cross-linking. First, a ninhydrin solution was prepared as follows:

Potassium iodate solution was prepared by adding ninhydrin (0.5 g) and proctose (0.3 g) to 200 ml of 95% (v / v) ethanol. . 1 mL of the sample and about 1 mL of the ninhydrin solution in 4 mL of phosphate-buffered PBS, pH = 6.8) was added to a 25 mL Colorimetric Tube and heated in a hot water bath for 20 minutes, It was cooled in a water bath. Then 5 mL of potassium iodate solution was added to the standard colorimetric tube and 25 mL of the solution was maintained by addition of deionized water. The absorbance of the solution was measured by a spectrophotometer (Mecasys, Optizen-a, UV / vis Spectrophotometer) at 568 nm. Glycine (0, 0.01, 0.05, 0.1, 0.5, 2, 4 and 6 mg / L) was used as a reference.

The crosslink density was determined by the following equation:

[Equation 1]

(Crosslinking density (%) = (NH ₂ (total) -NH ₂ (after) / NH ₂ (total) × 100).

As a result, the degree of crosslinking increased to about 17% by the heat - treatment crosslinking reaction, and the degree of crosslinking increased to about 80% by chemical crosslinking.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (18)

A laminate structure of a plurality of collagen layers having a plate-like shape,
Collagen membranes meeting the following properties:
(i) a density of at least 0.4 g / cm < 3 &
(ii) an absorbance of 100 to 2,000%, measured according to ISO 13726-1,
(iii) an enzymatic degradation resistance (8h) measured according to ASTM F2212 of 40 to 80%, and
(iv) tensile strength and tensile elongation, measured according to ASTM D638, of at least 0.5 MPa and at least 5%, respectively. The collagen membrane of claim 1, wherein the multiple collagen layer has a crosslinked collagen structure. The collagen membrane according to claim 1, wherein the thickness of each of the plurality of plate-like multiple collagen layers ranges from 1 to 50 mu m. The collagen membrane according to claim 2, wherein the amount of hydroxyl group (-OH) in hydroxyproline in the total amino acid composition of the multiple collagen layer having the crosslinked structure is 90% or less as compared with the case where crosslinked. The collagen membrane according to claim 1 or 2, wherein the thickness of the collagen membrane ranges from 0.2 to 0.6 mm. a) pulverizing the collagen-containing dermis layer derived from the swine dermis together with water;
b) shaping the crushed dermal layer into a membrane by vacuum filtration; And
c) performing a double bridge comprising physical crosslinking and chemical crosslinking on the membrane-shaped molding;
/ RTI >
Wherein the cross-linked membrane-shaped molding has a laminated structure of a plurality of plate-shaped collagen layers. The collagen membrane according to claim 6, wherein the collagen membrane has the following physical properties:
(i) a density of at least 0.4 g / cm < 3 &
(ii) an absorbance of 100 to 2,000%, measured according to ISO 13726-1,
(iii) an enzymatic degradation resistance (8h) measured according to ASTM F2212 of 40 to 80%, and
(iv) tensile strength and tensile elongation, measured according to ASTM D638, of at least 0.5 MPa and at least 5%, respectively. [7] The method of claim 6, further comprising lyophilizing the membrane-shaped molding between steps b) and c). 7. The method of claim 6, further comprising: deglossing the pork to obtain a pig dermis prior to step a); And
Enucleation of the pig dermis;
≪ / RTI > 10. The method of claim 9, wherein the step (a) is performed after lyophilization of the enucleated oxidized pig's dermis. [7] The method of claim 6, wherein the particle size of the crushed dermal layer is in the range of 0.7 to 2 [mu] m. [7] The method of claim 6, wherein the amount of water in step a) ranges from 500 to 1000 parts by weight based on 100 parts by weight of the dermal layer. 7. The method of claim 6, wherein the pressure applied for the reduced pressure filtration is 600 Torr or less. 9. The method of claim 8, wherein the step of lyophilizing the membrane-shaped molding is performed at a temperature ranging from -90 to -50 < 0 > C and a pressure of 600 Torr or less. 7. The method of claim 6, wherein the physical crosslinking is carried out at 80 to 130 DEG C under reduced pressure for 12 to 30 hours as heat treatment crosslinking. 16. The method of claim 15, wherein the cross-linking degree is increased to 5 to 30% by the heat-treatment crosslinking reaction. 7. The method of claim 6, wherein the chemical crosslinking is carried out using a carbodiimide compound. 18. The method of claim 17, wherein the cross-linking degree is increased up to 90% by the cross-linking reaction.

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