ARTIFICIAL BIOMEMBRANE USING COCOON AND METHOD FOR MANUFACTURING SAME

Abstract

Disclosed herein are a cocoon-based artificial biomembrane and a method for manufacturing the same. A cocoon the shell of which has a first thickness is divided into two or more fragments in predetermined form. The cocoon fragments may be used as artificial biomembranes. They can be relatively simply manufactured in a more cost efficient manner than conventional artificial biomembranes and have excellent cell growth potential. Also contemplated are a cocoon-based artificial biomembrane having excellent tensile strength and elongation, and a method for manufacturing the same.

Claims

1. A cocoon-based artificial biomembrane, prepared by dividing a cocoon into two or more fragments in a predetermined form, the cocoon having a shell with a first thickness.

2. The cocoon-based artificial biomembrane of claim 1, wherein each of the fragments is delaminated into a lamellar fragment with a second thickness, the second thickness being smaller than the first thickness.

3. The cocoon-based artificial biomembrane of claim 2, wherein the lamellar fragment with a second thickness is an inner stratum of the cocoon.

4. The cocoon-based artificial biomembrane of claim 2, wherein the lamellar fragment with a second thickness is a mid stratum of the cocoon.

5. The cocoon-based artificial biomembrane of claim 2, wherein the lamellar fragment with a second thickness is an outer stratum of the cocoon.

6. The cocoon-based artificial biomembrane of claim 3, wherein the lamellar fragment is sterilized.

7. The cocoon-based artificial biomembrane of claim 4, wherein the lamellar fragment is sterilized.

8. The cocoon-based artificial biomembrane of claim 5, wherein the lamellar fragment is sterilized.

9. The cocoon-based artificial biomembrane of claim 6, wherein the lamellar fragment is packed.

10. A method for manufacturing a cocoon-based artificial biomembrane, comprising a first step of dividing a cocoon into two or more fragments in a predetermined form, the cocoon having a shell with a first thickness.

11. The method of claim 10, further comprising a second step of delaminating each of the fragments into a lamellar fragment with a second thickness, the second thickness being less than the first thickness.

12. The method of claim 10, further comprising a third step of packing the fragments of the second thickness prepared in the second step.

13. The method of claim 10, further comprising conducting sterilization before or after each step.

14. The method of claim 12, wherein the lamellar fragment with a second thickness is an inner stratum of the cocoon.

15. The method of claim 12, wherein the lamellar fragment with a second thickness is a mid stratum of the cocoon.

16. The method of claim 12, wherein the lamellar fragment with a second thickness is an outer stratum of the cocoon.

17. The cocoon-based artificial biomembrane of claim 7, wherein the lamellar fragment is packed.

18. The cocoon-based artificial biomembrane of claim 8, wherein the lamellar fragment is packed.

19. The method of claim 11, further comprising a third step of packing the fragments of the second thickness prepared in the second step.

20. The method of claim 11, further comprising conducting sterilization before or after each step.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 is a schematic view illustrating a manufacturing procedure of a cocoon-based artificial biomembrane according to one embodiment of the present invention;

[0032] FIG. 2 is a schematic view illustrating the manufacturing procedure of a cocoon-based artificial biomembrane according to a modified embodiment of the present invention;

[0033] FIG. 3 shows morphologies of cocoon fragments used in the artificial biomembranes of the present invention;

[0034] FIG. 4 is a graph showing a mechanical property (tensile strength) of the cocoon-based artificial biomembrane of the present invention; and

[0035] FIG. 5 is a graph showing the cell growth potentials of the cocoon-based artificial biomembranes of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0036] Unless otherwise specified, the terms and techniques used in the specification should be defined to have the same meanings as are generally accepted in the art to which the present invention pertains.

[0037] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0038] The term biomembrane or biological membrane, as used herein, refers to an enclosing or separating membrane within living things, such as cell membranes, and organelle membranes. That is, serving as a selectively permeable barrier to an external environment, a biomembrane functions to prevent the external release of nucleic acids, proteins and other biomolecules and to construct an independent space where various biological activities happen. Further, a cell membrane protects the cell from external factors, and plays as a passage for material transport between the cytoplasm and the external environment.

[0039] For use as an alternative to a biomembrane, an artificial biomembrane has to maintain the intended shape thereof for a predetermined period of time after application to the body. Importantly, the artificial biomembrane must avoid adhesion with tissues around the surgical site after surgical operation, mediation of infection, and tissue degeneration, and must promote regeneration.

[0040] Artificial biomembranes find applications in various fields including artificial neural tubes, artificial cord cervical spines, artificial esophagus, artificial bronchi, artificial vessels, artificial valves, artificial dura mater, and artificial ear drums.

[0041] For obtaining these properties, artificial biomembranes used thus far need pretreatment processes, such as physical crosslinking, etc. However, such pretreated artificial biomembranes may be toxic to the body and remain as foreign matter upon long-term use in vivo. Moreover, another disadvantage is that the pretreatment processes and chemicals used therefor increases the production cost of the artificial biomembranes.

[0042] Leading to the present invention, intensive and thorough research into an artificial biomembrane, conducted by the present inventor, resulted in the finding that a fragment prepared from a cocoon is biocompatible and useful as a biomembrane not only because it has high tensile strength and elongation but also it exhibits an excellent cell growth potential, and that the fragment can be produced at significantly lower cost, compared to conventional artificial biomembranes.

[0043] With reference to FIG. 1, a method for manufacturing a cocoon-based artificial biomembrane in accordance with the present invention is explained, below.

[0044] 1. Step 1: Preparation of Cocoon Fragment with First Thickness.

[0045] As shown in FIG. 1A, a cocoon 10, the shell of which has a first thickness, is prepared.

[0046] A cocoon is a casting spun of silk by silkworms and is used as a material of silk fibers, In the present invention, cocoons, which may be idle resources, are up-cycled into a new high added-value product, thus bringing economic benefits to silkworm fanners.

[0047] Naturally constructed by silkworms, which eat clean mulberry leaves, cocoons are free of toxicity and are suitable for use as an environment-friendly material.

[0048] Hence, the present invention takes a cocoon 10 as a material for an artificial biomembrane. The cocoon 10 is processed, as shown in FIGS. 1B to 1E, into two or more planar fragments, each having a first thickness.

[0049] In greater detail, the oval cocoon 10 is dissected along a cutting line 11 into halves, as shown in FIG. 1B. The dissected halves have semi-oval shapes, and are opened to expose the inside surface 13 of the cocoon, as shown in FIG. 1C.

[0050] Next, the cocoon halves with curved inside surfaces 13 are planarized to some degree by cutting many sites along the edge as shown in FIG. 1D, and planar regions are cut out to obtain cocoon fragments with a first thickness, as shown in FIG. IE.

[0051] The artificial biomembrane prepared in the present invention needs not have a planar surface. Because a cocoon originally has an elliptical ball shape, the curved shape of the dissected cocoon may be utilized to give carved artificial biomembranes if necessary. For use as a small artificial biomembrane, a cocoon fragment with a small area may be relatively planar. In contrast, when taken a relatively large area of the dissected cocoon halves, the artificial biomembranes may have curved surfaces.

[0052] With reference to FIG. 2, a modified method for preparing a cocoon fragment with a first thickness is described. In detail, an oval-shaped cocoon 10 is cut along cutting lines 15 and 17 to expose the inside 13 of the cocoon 10, as shown in FIGS. 2B and 2C. Then, the dissected cocoon with a curved surface is spread as shown in FIG. 2D, and then cocoon fragments 20 with a first thickness are obtained as shown in FIG. 2E,

[0053] The preparation methods of cocoon fragments described in FIGS. 1 and 2 are only illustrative, but are not intended to limit the manufacturing method of artificial biomembranes according to the present invention. A cocoon fragment with a first thickness may be prepared by cutting a cocoon in the manners shown in FIGS. 1 and 2, but other cutting methods may be used.

[0054] 2. Step 2: Preparation of Cocoon Fragment with Second Thickness (Artificial Biomembrane)

[0055] Because the cocoon fragments 20 with a first thickness, prepared in step 1, has a multilayer structure identical to that of the cocoon shell, the multilayer structure may be split into thinner layers for use as an artificial biomembrane.

[0056] Although the cocoon fragment 20 with a first thickness, prepared in step 1, is itself possible to be used as an artificial biomembrane, it is subjected to thickness splitting to give cocoon fragments 30 with a second thickness. In this regard, the second thickness is smaller than the first thickness.

[0057] As they are, the cocoon fragments 30 can be applied for any purpose of artificial biomembranes. If necessary, they may be sterilized or chemically treated.

[0058] In detail, a cocoon shell varies in thickness (first thickness) from 0.3 to 1.0 nun depending on silkworm species.

[0059] In principal, any kind of cocoons may be used in the present invention. For the purpose of the present invention, a cocoon with a shell thickness of 0.5-0.8 mm is employed. In the present invention, the cocoon shell is divided into inner, mid and outer strata. First, the outer stratum is defined as a layer that is deep from the outer surface to a point corresponding to 25% of the total shell thickness. The inner stratum is defined as a layer that is deep from the inner surface to a point corresponding to 15% of the total shell thickness. The other part corresponding to 60% of the total shell thickness, that is, the remaining middle cocoon shell except the outer and the inner layer is the mid stratum. The numerical values of the interlayer borders are determined according to characteristics of individual strata (inner, mid, and outer), as shown in FIG. 3. That is, the outer stratum that is deep from the outer surface to a point corresponding to 25% of the total shell thickness can be used as a cocoon fragment characterized by high elongation. The mid stratum that has a thickness corresponding to 60% of the total shell thickness exhibits high cell growth potential thanks to its high porosity. The inner stratum that accounts for the remaining 15% of the total shell thickness has a smooth surface and high tensile strength.

[0060] A cocoon fragment can be easily delaminated into up to 16 lamellas although the number of delaminations is dependent on the shell thickness. The thicknesses of the lamellas can be determined according to strength and elongation necessary for the kind and use of the artificial membrane. From a cocoon with a shell thickness of 0.5-0.8 mm, an artificial biomembrane 0.01 mm-0.7 mm thick can be prepared by delamination. According to the use of the artificial biomembrane, selection may be grade of the cocoon fragments 30 with various thicknesses.

[0061] As can be seen FIG. 4, the outer stratum 35 is suitable as an elastic biomembrane because it is higher in elongation rate than the inner stratum 31 and the mid stratum 33. The highest elongation rate is measured from the outer stratum, with the lowest elongation rate from the inner stratum.

[0062] The inner stratum 31 has a smooth surface so that is unlikely to adhere. In addition, the inner stratum has high tensile strength so that it is suitable for use as a biomembrane where strength is needed. The highest tensile strength is measured in the inner stratum, with the lowest tensile strength in the outer stratum.

[0063] Further, the mid stratum 33 exhibits has excellent cell growth potential because high porosity. Hence, it is suitably applied as a biomembrane to a defect lesion that needs fast healing.

MODE FOR THE INVENTION

[0064] A better understanding of the present invention may obtained through the following examples that are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1

Preparation of Artificial Biomembrane 1

[0065] A cocoon 10 was prepared, and cut at a proper site to exposure the inside thereof.

[0066] Next, the cut cocoon was further processed to make the curved inside planar.

[0067] The planarized cocoon was cut into rectangular fragments 20.

[0068] From the cocoon fragments 20, a layer containing the innermost surface 13, that is, an inner stratum was delaminated at a thickness of 0.07 mm.

[0069] The delaminated inner stratum was sterilized and used as an artificial biomembrane

EXAMPLE 2

Preparation of Artificial Biomembrane 2

[0070] From the remaining cocoon after the inner stratum was delaminated in Example 1, a layer containing the outermost surface, that is, an outer stratum opposite to the inner stratum was delaminated at a thickness of 0.07 mm.

[0071] The delaminated outer stratum was sterilized and used as an artificial biomembrane

EXAMPLE 3

Preparation of Artificial Biomembrane 3

[0072] The remainder after the inner stratum and outer stratum were delaminated into mid strata, each 0.07 mm thick, followed by sterilization to give an artificial biomembrane 3.

TEST EXAMPLE 1

Morphology of Artificial Biomembranes from Cocoon

[0073] 1. Test Method

[0074] Morphologies of the artificial biomembranes prepared in Examples 1 to 3 were observed by scanning electron microscopy (SEM) and with the naked eye. The results are shown in FIG. 3.

[0075] 2. Test Results

[0076] As can be seen in FIG. 3, the inner turn (A, Example 1), the mid stratum (B, Example 3), and the outer stratum (C, Example 2) were different from one another in terms of morphological properties, such as fiber thickness, pore form, etc. Under the naked eye, the mid stratum was observed to have a smoother surface. Thus, the morphological results indicate that the inner stratum, the mid stratum, and the outer stratum can be used where their unique characteristics are needed.

[0077] For example, as shown in FIG. 3, the inner stratum has a smooth surface and is water resistant so that it is suitably used as art artificial biomembrane where a non-sticky property is needed. Having high cell growth potential thanks to the high porosity thereof, the mid stratum can be suitably used as a biomembrane in a defect region that needs fast healing.

TEST EXAMPLE 2

Physical Properties of Cocoon-Based Artificial Biomembrane According to Cocoon Stratum

[0078] 1. Test Method

[0079] Physical properties of the cocoon-based artificial biomembranes prepared in Examples 1 to 3 by cocoon stratum were measured. In this regard, a tensile test was conducted using a universal testing machine (DAEYEONG, Korea).

[0080] Test specimens with sizes of 2.50.07 (widthlength) mm were used. The specimens were extended at a rate of 10 mm/min, with an initial gauge length set to be 10 mm.

[0081] Results are given in FIG. 4 (strain (mm) versus stress (MPa)) and Table 1 below.

[0082] 2. Test Results

[0083] Table 1

TABLE-US-00001 TABLE 1 Tensile Strength Elongation (MPa) (%) Inner Stratum (Ex. 1) 60.20 5.3 12.45 1.5 Mid Stratum (Ex. 3) 46.19 2.2 15.05 1.7 Outer Stratum (Ex. 2) 29.36 3.1 18.93 1.3

[0084] As is understood from the data of FIG. 4 and Table 1, the cocoon-based artificial biomembranes were different from one another in tensile strength and elongation by stratum. The highest tensile strength was detected in the inner stratum while the highest elongation was measured from the outer stratum.

[0085] In other words, the inner stratum is suitable for use as an artificial biomembrane in a site where strength is important whereas the outer stratum is preferably applied to a site that needs elasticity.

TEST EXAMPLE 3

Physical Properties of Mid Cocoon Stratum-Based Artificial Biomembrane According to Thickness

[0086] 1. Test Method

[0087] Physical properties of the mid stratum-based artificial biomembranes were measured according to thickness. In this regard, a tensile test was conducted using a universal testing machine (DAEYEONG, Korea). Test specimens with sizes of 202.5 (widthlength) min were used in a dry state or a wet state. In the latter case, the specimens were immersed in physiological saline for 1 hrs. For a control, a commercially available collagen membrane was employed. The specimens were extended at a rate of 10 mm/min, with an initial gauge length set to be 10 mm. Results are given in Table 2, below.

[0088] 2. Test Results

[0089] Table 2

TABLE-US-00002 TABLE 2 Max. Load Tensile Strength Elongation Thick. (N) (MPa) (%) Kind (mm) Dry Wet Dry Wet Dry Wet Mid 0.01 6 3 2 13 2 23 Stratum 0.02 9 8 2 20 1.4 29.4 0.04 17 11 4 14 1.8 23 0.06 16 16 4 13 1.6 31.8 0.08 33 15 8 9 2.4 25.2 0.1 37 20 9 10 2 30.2 0.2 88 62 22 16 2.8 26.6 0.4 113 80 28 10 2.8 31.6 Collagen 0.2 3.3 0.25 0.8 0.05 7.8 18.8

[0090] As is understood from the data of Table 2, the cocoon-based artificial biomembrane differs in tensile strength and elongation by thickness. Both the tensile strength and the elongation increased with the increase of thickness, and were better in a wet state than in a dry state. contrast, the tensile strength of the commercially available collagen membrane decreased 16 times when it was in a wet state compared to when it was in a dry state. Taken together, the data indicates that physical properties of the cocoon-based biomembranes can be maintained for a longer period of time than those of the wet collagen membrane.

TEST EXAMPLE 4

Cell Growth Potential of Artificial Biomembrane

[0091] 1. Test Method

[0092] In order to test the artificial biomembranes for cell growth potential, the mouse fibroblast cell line L929 was cultured on the artificial biomembranes at 37 C. in a 5% CO.sub.2 condition. For this, DMEM (Dulbecco's modified Eagles medium-high glucose, WelGENE, Korea) supplemented with 10% (v/v) FBS (fetal bovine serum, GIBCO), and 100 U streptomycin and 100 g/ml penicillin (GIBCO) was used. The artificial biomembrane was assayed for cell growth potential by MTT.

[0093] The results are depicted in FIG. 5 wherein cell growth potential (relative activity) is given according to cocoon stratum.

[0094] 2. Test Result

[0095] As can be seen in FIG. 5, all the cocoon-based artificial biomembranes exhibited good cell growth potential, with 4-fold higher cell growth on the mid stratum than the inner stratum.

[0096] In other words, the mid stratum is more suitable than the inner stratum for use in a site that needs cell growth. In addition, the cocoon-based artificial biomembranes of the present invention were observed to grow the cells at higher efficiency, compared to the collagen membrane.

[0097] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[0098] Description of Numerical References in Drawings

[0099] 10: Cocoon

[0100] 11: cutting line 1

[0101] 13: inside surface

[0102] 15: cutting line 2

[0103] 17: cutting line 3

[0104] 20: Cocoon fragment with a first thickness

[0105] 30: Cocoon fragment with a second thickness

[0106] 31: Inner stratum

[0107] 33: Mid stratum

[0108] 35: Outer stratum