KR100427557B1 - Bone collagen scaffold - Google Patents

Abstract

The present invention relates to a collagen support for bone, and provides a bone collagen support comprising a porous body of 5 to 500 ㎛ size prepared by decal cification of bone. The bone collagen support of the present invention can be used as a bioactive factor transporter as well as a dermal substitute for fibroblasts to proliferate in molding, artificial skin culture, or dermal defect filling.

Description

Bone COLLAGEN SCAFFOLD}

The present invention relates to a collagen support of bone, and more particularly, to a method for using the bone collagen support as a material for biotissue engineering and dermal material in the preparation of artificial skin.

Biotissue engineering is a new field that has emerged with the development of science. By integrating and applying the basic concepts and technologies of bioscience and engineering, it is possible to understand the correlation between the structure and function of biological tissues, and to create and transplant surrogate tissues. This is an applied research aimed at maintaining, improving or restoring the function of our body. The theoretical background of biotissue engineering can be summarized into two categories: selective cell transplantation and artificial substrates. Selective cell transplantation refers to the selective harvesting and transplantation of only the tissues or tissues that are most needed among organs. The artificial substrate is an artificially synthesized substrate based on the fact that the extracellular matrix secreted by cells plays an important role in tissue formation. Means to transplant using. Based on this theoretical background, experiments have been made to form tissues or organs by attaching and transplanting only cells necessary for various kinds of natural or artificial substrates.

The most important in biotissue engineering techniques is the scaffold for three-dimensional cell culture, which provides a foundation for cells to grow three-dimensionally. However, if cells are cultured without these three-dimensional supports, the cells will only grow on the bottom of the plate and grow or float in culture. In both cases, the cell loses its three-dimensional shape and cannot develop into tissue.

However, with the development of materials science, biodegradable polymers have been incorporated into biotissue engineering, and biodegradable polymers have the property of self-decomposing and disappearing after a certain time in the human body. It is widely used as a biomaterial suitable for the purpose of biotissue engineering.

Initially, cell cultures using biodegradable polymers were attempted by culturing cells on polymer plates, but they did not yield satisfactory results. After that, the polymer was made into a thin thread-like form and then woven together to form a mesh-like shape, and the cells were planted and cultured thereon. In the above method, sufficient space is secured between cells to facilitate the supply of oxygen and nutrients due to the diffusion of body fluids, and the formation of neovascularization is facilitated, resulting in successful cell growth, differentiation, and tissue formation. In addition, since the polymer used is artificially synthesized, the composition and shape of the polymer can be adjusted at will according to the preparation. It can be prepared by calculating the tension, decomposition rate, and cell adhesion ability of the material in advance. Also, a technique that contains the factors involved in cell growth in the polymer can cause such factors to flow out gradually as the polymer is degraded. It became possible.

To summarize the basic biotissue technique, we first collect a small amount of tissue from the patient's body, separate the cells from the tissues, and then proliferate the separated cells in the required amount through the culture, and incubate them in a biodegradable polymer for a period of time. This cell-polymer construct is then implanted back into the body. After transplantation, blood vessels grow in the human body and blood is supplied with angiogenesis. Cells proliferate and differentiate to form new tissues and organs, and polymers are broken down.

On the other hand, collagen has been widely used as an adjuvant for biodegradable polymer and artificial skin regeneration due to its excellent cell compatibility. Collagen is the most fibrous protein contained in the animal's body. It is the main component of the dermis and connective tissue of the skin, and 20% of the bone weight is collagen.

In general, collagen is used for medical or cosmetics, collagen is known to act on the cell adhesion function, maintaining the structure of the body and organs, activation of cell function, cell proliferation, hemostatic action, immune strengthening in vivo.

Also, a biotissue equivalent containing collagen of Korean Patent Publication No. 90-14547, a method for producing a chitin-based interpenetrating structure film for artificial skin of Korean Patent Publication No. 96-6788, and a biocompatible perforated membrane in Korean Patent Publication No. 91-8999 And a method of manufacturing the same, artificial skin tissue such as three-dimensional regenerated artificial skin tissue and a method of manufacturing the same in Korean Patent Publication No. 97-60646, but using collagen or a biodegradable polymer, but only artificial skin It has not been able to provide dermal substitutes that can reproduce the appearance of human skin, confined to the three-dimensional culture.

Therefore, it is required to develop dermal substitutes that are suitable for academic research or medical treatment by maintaining artificial skin cultured in vitro.

Accordingly, an object of the present invention is to provide a bone collagen support that can be used as a dermal filler material.

1 is a photograph taken by scanning electron microscopy at 270-fold 3 days after sprinkling fibroblasts to bone collagen,

Figure 2 is a photograph observed with a scanning electron microscope at 250 times 10 days after sprinkling fibroblasts to bone collagen,

3 is a photograph observed with a scanning electron microscope at a magnification of 1000 times 3 days after sprinkling fibroblasts to bone collagen,

4 is a photograph taken with a scanning electron microscope at a magnification of 700 times after 10 days after sprinkling fibroblasts to bone collagen,

FIG. 5 shows the amount of bone collagen scaffold comprising antibiotics eluting antibiotics over time.

In order to achieve the above object, the present invention provides a bone collagen support for dermal filler material.

In another aspect, the present invention provides a bone as a physiologically active factor carrier comprising a collagen support.

Hereinafter, the present invention will be described in detail.

The bone collagen support of the present invention is characterized in that it is obtained by decalcification of bone of an animal. The decalcification method is preferably carried out in HCl bone, more preferably 12 hours to 24 hours after immersing the bone in 0.5 N HCl, the reaction is washed with PBS (Phosphate buffered saline) buffer Titration to pH 7.4.

The bone collagen support of the present invention is characterized by including a porous body. The porous body formed in the support provides sufficient space between the cells to facilitate the supply of oxygen and nutrients by the diffusion of body fluids and help to form new blood vessels, resulting in the successful growth and differentiation of cells and the formation of tissues. . Therefore porosity is also as important as the biocompatibility of the support. As for the porous body of the bone collagen sponge of this invention, 5-500 micrometers is preferable, More preferably, it is 10-100 micrometers. When the porous body is less than 5 ㎛, sufficient space between cells is not secured, and cell growth can be suppressed.In the case of more than 500 ㎛, the spacing between cells is too wide, which makes it difficult to develop normal skin tissue and supports the dermis. There is also a problem with tensile strength. The porous body in the support of the present invention is preferably a support having an excellent biocompatibility as 10 to 100 m.

The bone collagen support of the present invention is suitable for proliferation of cells, can be stored for a long time, and can be commercialized. In addition, the bone collagen scaffold is a pore-type sponge having various shapes and sizes well developed to the center, and has a form in which cells can be well introduced into the cell when inoculating the scaffold, and it is a substance directly obtained from an organism. The biocompatibility is also very good.

The present invention also provides a bone collagen support as a dermal filler. The dermal filler may be applied to the treatment of skin scars or to areas where there is a defect in the dermis to regenerate the skin, and may be used when shaping organs of the body such as penis enlargement.

In addition, the bone collagen support of the present invention may provide an artificial skin prepared by culturing fibroblasts on the bone collagen support.

When culturing fibroblasts on the bone collagen support, fibroblasts are inoculated at 100 to 1000 / mg and preferably cultured for 5 days to 7 days, and after about 10 days, the fibroblasts form a layer and proliferate.

In addition, the present invention can utilize the bone collagen support as a physiologically active factor carrier. More preferably, the bone collagen support of the present invention may be used as a growth factor encapsulation, growth factor release, vascular growth factor encapsulation, vascular growth factor release, antibiotic encapsulation, antibiotic release, drug delivery agent, etc. in vivo or in vitro. Can be. In other words, the bone collagen support may contain the above factors (growth factor, drug, etc.) to serve as a drug carrier to release the drug over a period of time according to the encapsulation method.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

Fibroblast Culture

The subcutaneous fat layer of the skin obtained from the foreskin was completely removed, the dermis was separated by treatment with colnase, and then fibroblasts were separated from the dermis by treatment with trypsin-EDTA. The isolated fibroblasts were cultured in 5% CO ₂ , 37 ℃ incubator using Dulbeccos modified eagles medium (DMEM) medium containing 10% FBS (fetal bovine serum) and antibiotics.

Preparation of Bone Collagen

100 g of bone obtained from the animal was placed in 0.5 N HCl and decalcified. After deliming it was washed with PBS (phosphate buffered saline) and titrated to pH 7.4. The demineralized collagen sponge was dehydrated by a general alcohol concentration gradient, dried in air, stored in an airtight container, and taken out if necessary. The dried collagen scaffold is a porous sponge with various shapes and sizes that are well developed to the center, and when the cells are inoculated into the scaffold, the cells can be well introduced into the scaffold. The biocompatibility was also very good.

Example 2

The bone collagen support prepared in Example 1 was sterilized with low temperature ethylene oxide gas, washed several times with PBS, and sprinkled with the same number of fibroblasts (100-1000 / mg). Incubated for 3 or 10 days in DMEM medium containing 10% FBS and antibiotics.

Experimental Example 1

The fibroblasts cultured in Example 2 were observed with a scanning electron microscope.

First, fibroblasts were seeded and cultured for 3 or 10 days, bone collagen was fixed in para-glutaraldehyde solution at 4 ° C. for 2 hours, and then washed several times with distilled water. In order to remove moisture in the sample, dehydrated with acetone for each concentration, and dried on a sample bed with double-sided tape. The dried samples were gold-coated with an ion coater (EiKO, 1B-3) and observed with a scanning electron microscope (AKAShi, ISI-SS40).

1 is a photograph observed by scanning electron microscopy at 270 times 3 days after sprinkling fibroblasts to bone collagen, Figure 2 is a picture observed at 250 times after 10 days. In addition, Figure 3 is a photograph observed with a scanning electron microscope at a magnification of 1000 times 3 days after sprinkling fibroblasts to bone collagen, Figure 4 is a photograph observed at a magnification of 700 times 10 days. As shown in Figures 1 and 3, on the third day of culture, fibroblasts were implanted in bone collagen and blocked pores formed in the collagen sponge, and as shown in FIGS. 2 and 4, the culture On day 10, fibroblasts were further proliferated and observed at high magnification, indicating that the cells were layered.

Example 3

The release experiment of antibiotics was performed using the chitosan sponge and the bone collagen support of Example 1. The elution of the antibiotic was determined by dipping the sponge or bone collagen scaffold containing the antibiotic in an aqueous solution and taking a sample at a predetermined time to determine the concentration in the solution, and then converted to the total amount of the solution. The results are shown in Table 1 and FIG. 5, and the bone collagen support showed a lower dissolution rate compared to the antibiotic dissolution rate by the chitosan sponge, and it was thought that the therapeutic effect by the antibiotic for a long time could be maintained.

% Of antibiotic released 0 min 1 minute 5 minutes 10 minutes 30 minutes Chitosan Sponge 10.8% 13.1% 35.3% 55.2% 102.1% Example 1 4.8% 7.5% 28.4% 44.8% 70.9%

As mentioned above, the bone collagen support of the present invention can be easily prepared, as well as excellent cell suitability, including porosity, to facilitate cell culture. It can also be used as a skin defect, cosmetic, bioactive factor transporter.

Claims (6)

Bone collagen support for dermal filler material prepared by deliming bone to form a porous body of 5 to 500 ㎛ size. delete delete The bone collagen support according to claim 1, wherein the bone collagen support is used as a dermal substitute during proliferation, molding, artificial skin culture, or filling of dermal defects by proliferating fibroblasts. The bone collagen support according to claim 1, wherein the bone collagen support further carries a bioactive factor and is used as a bioactive factor carrier. 6. The bone collagen support according to claim 5, wherein the bioactive factor is a growth factor, an antibiotic or a medicament.