KR100744833B1 - Artificial intervertebral disc comprising disc cell and the method of tissue engineered disc regeneration using the said disc cell - Google Patents

Abstract

An artificial intervertebral disc comprising a disc cell and a method of regenerating a tissue engineered disc using the same disc cell are provided to regenerate a disc using an artificial disc comprising a scaffold having the inside and outside comprising different component. An artificial intervertebral disc comprising a disc cell includes: an inside disc including a disc central shaft in a disc shape and comprising biodegradable porous scaffold including nucleus pulposus cells; and an outside disc surrounding the inside disc in a doughnut shape, and comprising biodegradable porous scaffold including annulus fibrous cells. The artificial intervertebral disc further includes a natural or synthetic biomaterial added to the biodegradable porous scaffold of the inside or the outside of the disc.

Description

Artificial intervertebral disc comprising disc cell and the method of tissue engineered disc regeneration using the said disc cell}

1 is a schematic diagram of a composite support for a disk made of a different component inside and outside the disk. (a) is the disc which comprises an artificial disk, (b) is the outside (b).

Figure 2 shows the lumbar disc tissue of an 8-week-old rabbit.

Figure 3 is a single layer culture of the disk cells cultured separated from the rabbit disk.

Figure 4 is a view before implantation of the artificial disk prepared in Example 1.

5 is a view before implantation of the artificial disk prepared in Example 2.

Figure 6 is a state before implantation of the artificial disk prepared in the comparative example.

Figure 7 is a state after two weeks (top), three weeks (bottom) of the artificial disk prepared in Example 1.

FIG. 8 is a view after 2 weeks (top) and 3 weeks (bottom) of the artificial disc manufactured in Example 2. FIG.

FIG. 9 is an X-ray photograph of 6 weeks after implanting the artificial disk prepared in Example 1 and Comparative Example into the lumbar spine of a rabbit. FIG.

The present invention relates to a method for tissue engineering disk regeneration using disc cells, and more particularly, to separate and culture the annulus fibrosus-derived fibrosus cells and nucleus pulposus-derived nucleus cells from the disc tissue. The internal and external parts of the artificial discs are seeded on a complex support composed of different components and inserted into the damaged discs to regenerate the tissues by histological engineering. [Qi-Bin Bao, et al. , Biomaterials 17, 1157 (1996), US Patent 2,677,369 (1954), US Patent 4,759,769 (1998), US Patent 4,772,287 (1988), US Patent 4,874,389 (1989), US Patent 4,904,260 (1990), US Patent 4,917,704 (1990) , US Patent 4,946,378 (1990), US Patent 5,035,716 (1991), US Patent 5,047,055 (1991), US Patent 5,192,326 (1993), US Patent 5,320,644 (1994), US Patent 5,489,308 (1996), US Patent 5,534,028 (1996), US P Atent 6,264,695B1 (2001), US Pat. No. 6,280,475B1 (2001)] relates to a method for regenerating a tissue engineering disc using disc cells having superior biological activity and advantageous advantages for tissue regeneration.

Treatment for spinal cord injuries is currently progressing almost similarly to treatments for knee and hip defects. Degeneration of the vertebral joints, such as degeneration of the knee and hip joints, causes pain first, and then deforms, and further progresses, eventually failing. Treatment in early onset includes methods for improving spinal activity, distributing the load directly to the spine by a cane and crutches, or stabilizing externally in the form of clasps and splints. Immobilized fusion of the affected vertebral joint reduces the inflammatory response caused by abnormal joint movement. Such spinal fusion has the effect of providing a sense of safety and reducing pain, but has a disadvantage in that the load on the neighboring spinal plate is suddenly concentrated and the weight is increased biomechanically again.

Spinal pain may be caused by (1) pronounced disc symptoms by traditional diagnostic methods, (2) neural compression at radiographic findings, and (3) unstable movement of the patient and physiological loads of daily exercise. If the damage is the same or if the case is rich, the operation will proceed. Surgical techniques for degenerated vertebrae include discs that compress the nerves, decompression of protruding bones or ligaments, and fusion or coalescence of two adjacent vertebrae. In this way, the lesion is removed, and then an artificial disc is inserted for physiological motility or biomechanical function. (1) A total disc substitute [US Patent 2,677,369 (1954), US Patent 4,759,769 (1998), US Patent 4,772,287 ( 1988), US Patent 4,874,389 (1989), US Patent 4,904,260 (1990), US Patent 4,917,704 (1990), US Patent 4,946,378 (1990), US Patent 5,035,716 (1991), US Patent 5,320,644 (1994)] and (2) nucleus nuclei (nucleus pulposus) [US Patent 5,047,055 (1991), US Patent 5,192,326 (1993), US Patent 5,489,308 (1996), US Patent 5,534,028 (1996), US Patent 6,264,695B1 (2001), US Patent 6,280,475B1 (2001). Two alternative types of implants will be inserted.

Current materials using artificial disc substitutes include metal biocompatible alloys such as stainless steel, titanium alloys, and cobalt-chromium (Co-Cr) alloys. In addition, polymers such as polyurethane and silicone elastomers are also used because of their similar physical properties to those of natural disk structures. In addition, polymers such as polyethylene and epoxy resins and materials such as calcium nitride, ceramics, or a combination thereof are used. . In recent years, partial transplants have been proposed to replace the nucleus pulposus, which is less invasive surgically than artificial discs implanted whole and has the advantage of using the physiological properties of the natural fibers of the annulus itself. The methods and conditions have difficult drawbacks. The most popular artificial discs currently used in the clinic are Steffie disc, Link SB Carte, and Hou model. Eventually, this implant material is showing a limitation in application because of its lack of bioactivity. Therefore, in recent years, interest in the development of histological biodisks has been increasing.

On the other hand, the present inventors have proposed a porous biodegradable artificial organs and a method for producing the same by the emulsion freeze drying method consisting of a polymer solution and water to solve the existing disadvantages (Korean Patent No. 201,874). However, the manufacturing method according to Korean Patent No. 201,874 has a disadvantage in that the surface properties of the synthetic biodegradable polymer used are hydrophobic so that the culture solution and cells do not penetrate and do not grow into the biodegradable polymer carrier. In addition, when the biodegradable polymer carrier contains cytokines of various growth factors, various properties such as angiogenesis and neuronal cell formation can be remarkably increased, but bioactive substances are denatured and water is produced during the manufacturing process. And other solvents are pre-flowed out and the original function is lost. Moreover, the growth factor or cytokines are very expensive, so if you use them, you have to have a lot of economic burden.

On the other hand, Korean Patent No. 482,651 of the present inventors is an economical and bioactive natural / synthetic hybrid carrier for tissue engineering and a method of manufacturing the hydrophobicity of the synthetic polymer material not only solved by using the small intestinal submucosa and bone powder as a natural material It is suitable for the manufacture of artificial organs because it contains excessive amounts of physiologically active substances and is easy for tissue regeneration. However, in spite of the advantages of the existing carrier, the disc tissue has different characteristics of the nucleus pulposus and the annulus of the annulus.

The present invention is characterized in that the inside of the disc including the disc central axis is composed of a biodegradable porous support including nucleus cells, and the donut shape surrounding the inside of the disc is composed of a biodegradable porous support comprising a fibrous ring cell. Provide a disk.

In another aspect, the present invention provides a method for producing an artificial disk comprising the step of separately preparing the biodegradable porous support of the inside and outside of the artificial disk, and including the nucleus cells and the annulus fibroblasts in each.

The present invention also provides a method for regenerating a disc by histological engineering by inserting an artificial disc of the present invention or an artificial disc made by the manufacturing method into a damaged disc.

The present invention is an artificial disk to replace the entire disk, the disk including the central axis of the disk is composed of a biodegradable porous support including a nucleated nucleus cells, the outside of the doughnut shape surrounding the disk is a biodegradable porous including a fibrous ring cells It is characterized by consisting of a support.

In another aspect, the present invention comprises the step of mixing the biodegradable polymer solution containing natural or synthetic biomaterials with salts for the manufacture of artificial disks, filling them into a doughnut-shaped mold, and drying to prepare an external porous support of the artificial disks ; Inoculating fibrosus cells to the donut-shaped porous support; And filling the suspension of the biodegradable polymer solution and nucleus cells containing the natural or synthetic biomaterial into the donut using a donut-shaped porous support inoculated with the annulus fibrosus cells as a mold.

In addition, the present invention is mixed with a salt biodegradable polymer solution containing natural or synthetic biomaterials for the manufacture of artificial disks, and filled into a doughnut-shaped mold, and dried to produce an external porous support of the artificial disk step; A biodegradable polymer solution comprising natural or synthetic biomaterials is mixed with a salt and filled into a disc-shaped mold having a diameter equal to the inner diameter of the donut-shaped porous support, and dried to prepare an internal porous support of the artificial disc. Doing; Inoculating the annulus fibrosus cells to the outer porous support, and inoculating the nucleus cells into the inner porous support; And inserting and attaching the inner porous support including the nucleated cells to the outer porous support including the annulus fibrosus.

Referring to the present invention in more detail as follows.

The present invention is an artificial disk made of a separate biodegradable porous support containing a component suitable for each of the disks constituting the artificial disk as shown in Figure 1 divided into (a) and (b).

Biodegradable polymers for preparing a biodegradable porous support inside or outside the disc are well known in the art and may use different ones or the same inside and outside the disc. Such biodegradable polymers include, for example, polylactide, polyglycolide, poly (lactide-glycolide) copolymers, polycaprolactones, polyisoesters, polyiminocarbonates, polyanhydrides (poly (SA-HDA) ) anhydride), polyethyl glutamate, polydioxanone, and the like can be used.

In addition, it is preferable to additionally use natural or synthetic biomaterials in addition to the biodegradable polymer to prepare the biodegradable porous support. The natural or synthetic biomaterials include alginic acid, chitosan, chitin, collagen, laminine, heparin, fibrin sulfate chondroitin, hyaluronic acid, methylene polyethylene glycol / polycaprolactone (MPEG / PCL), poloxamer Agarose, gelatin, small intestinal submucosa, demineralized bone meal and the like.

In addition, growth factors such as transforming growth factor (TGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and osteoinductive factor (BMP) for the production of the biodegradable porous scaffold and / or Or it is preferable to further use at least one drug such as dexamethasone, hydrocortisone, ascorbic acid, vitamin D ₃ and the like.

The support constituting the inner disk should include nucleated cells, and the support constituting the outer disk should contain fibroblast cells. The nucleated nucleus cells and annulus fibroblasts are separated from the nucleus nucleus and fibrous annulus parts of the disc tissue of the mammal, or the nucleus nucleus cells and fibrillar cells are purified from the disc tissue, respectively. The mammalian disc tissue can be used as a disc tissue of a human or a mammal except a human, for example, a disc tissue such as a cow, a pig, a rabbit and a mouse.

Referring to the artificial disk manufacturing method of the present invention in more detail as follows.

1) A biodegradable polymer solution (1.5 ~ 30 (w / v)% as a solvent) containing natural or synthetic biomaterials is mixed in a weight ratio of 1: 4 ~ 1: 16 with a salt and filled into a donut-shaped mold Molding and drying to prepare an outer porous support of the artificial disc;

2) inoculating fibrosus cells to the donut-shaped porous support; And

3) filling a donut-shaped porous support inoculated with the annulus fibrosus cells into a mold to fill a suspension of a biodegradable polymer solution and nucleus cells containing a natural or synthetic biomaterial inside the donut.

Dispersing the growth factor or drug optionally in the solution of 1) may be included.

In addition, instead of the step 3), the biodegradable polymer solution containing natural or synthetic biomaterials is mixed with a salt, and filled into a disc-shaped mold having the same diameter as the inner diameter of the donut-shaped porous support, and dried to artificially Preparing an inner porous support of the disk; Inoculating a nucleated nucleus cell in such an internal porous support; And attaching the inner porous support including the nucleated cells to the outer porous support including the annulus fibrosus cell 2).

Natural or synthetic biomaterials are first prepared in fine powder form so as to be uniformly mixed with the biodegradable polymer solution. The size of the salt for forming the pores when preparing the support is preferably 100 ~ 600 ㎛ diameter.

In addition, when the mixing ratio of the polymer solution and the salt is less than 1: 4 mass ratio, the porosity is less than 40%, making it difficult for cells to penetrate. The salts that can be used for preparing the porous support of the present invention are harmless to the human body, are easily soluble in water, and have a certain size, and are effervescent salts of sodium bicarbonate, sodium carbonate, ammonium bicarbonate, ammonium carbonate, potassium bicarbonate and carbonate. Calcium and mixtures thereof and non-foamable salts such as sodium chloride, barium chloride and the like can all be used.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are intended to illustrate the present invention and do not limit the present invention.

Example  One

4 parts by weight of dichloromethane is mixed with 1 part by weight of a biodegradable polymer (poly (lactide-co-glyclide), PLGA) to form a polymer solution, and the powder of the submucosal tissue 20 (w / v)% of the mixture was put into a donut-shaped mold and pressed to prepare a disc outer support. The support was dried at room temperature, followed by salt extraction using tertiary ultrapure distilled water, followed by lyophilization and sterilization with ethylene oxide gas. FIG inoculated in sterilized supports a Annulus cells (AP, concentration of 4 × 10 ³ gae / ㎕ in Fig. 3) 50 ㎕ derived from tissues such as the disk 2, and incubated in 37 ℃, CO ₂ incubator. Prepare MPEG / PCL in 20 (w / v)% solution in phosphate buffered saline (PBS) and suspend 20 μl of 2 × 10 ³ cells / μl of nucleated cells (NP in FIG. Filling the inside of the donut-shaped support inoculated with was prepared a tissue engineering disk as shown in the schematic diagram of Figure 1 and the photograph of FIG.

Example  2

A polymer solution was prepared by mixing 4 parts by weight of dichloromethane with 1 part by weight of PLGA, and the powder of the small intestinal submucosa was mixed at 20 (w / v)%, and put into a donut-shaped mold to press to prepare an outer disk support. The disc inner support is mixed with demineralized bone powder at 20 (w / v)% in a polymer solution in which 4 parts by weight of dichloromethane is mixed with 1 part by weight of PLGA and placed in a disc shaped mold having a size suitable for an inner diameter of a donut shape. It was prepared by pressing. The prepared disc inner and outer supports were dried at room temperature, and subjected to salt extraction using tertiary ultrapure distilled water, followed by lyophilization and sterilization with ethylene oxide gas. The sterile disk outer support was inoculated with 50 μl of fibroblasts (AP in FIG. 3, concentration 4 × 10 ³ / μl), and the inner support with 2 × 10 ³ / μl nucleated cells (NP of FIG. 3) 20 Inoculated with μl and incubated in 37 ℃, CO ₂ incubator, respectively. A tissue engineering disk as shown in the schematic diagram of FIG. 1 and the photo of FIG. At this time, the inner diameter of the outer support was slightly larger than the diameter of the inner support, but there were some pores between the two supports, but the pores were adhered with fibrin glue.

Comparative example

1 part of PLGA was mixed with 4 parts by weight of dichloromethane to make a polymer solution. Then, the submucosa powder was mixed at 20 (w / v)%, and put into a disc mold to pressurize the disk support having a single structure. Prepared. The prepared disk support was dried at room temperature, subjected to salt extraction using tertiary ultrapure distilled water, and then lyophilized, sterilized with ethylene oxide gas, and then 2 × 10 at the center (inside) of the disk. ³ / ㎕ of the recipient cells 20 ㎕ (NP in Figure 3), the peripheral (outer) has evenly along the periphery Annulus cells (Fig. 3 AP, concentration of 4 × 10 ³ / ㎕) was inoculated 50 ㎕ 37 ℃ , And cultured in a CO ₂ incubator to prepare a tissue engineering disk as shown in Figure 6.

Test Example  One: Morphology  observe

4, 5 and 6 show the visual observation of the tissue prior to implanting the tissue engineering disks prepared in Examples 1 and 2 and Comparative Examples, and the tissue engineering disks prepared in Examples 1 and 2 were nude. Artificial disks inoculated with cells 2 and 3 weeks after implantation in the dorsal subcutaneous of mice were extracted from nude mice and shown in FIGS. 7 and 8.

Artificial disk of the present invention forms a tissue very similar to the tissue of the human body compared to the conventional artificial disk consisting of only metal and synthetic materials, has excellent biological activity, and has the advantage of very good fusion with the human body is advantageous in tissue regeneration And it was found.

Test Example  2: X-ray observation

9 shows X-ray images of the tissue engineering discs prepared in Example 1 and Comparative Example in the rabbit lumbar spine after 6 weeks. In the picture, Sham is the same procedure as the surgery on the disc but does not remove the disc. Blank is the case in which the support is not removed after the disc is removed. It can be seen that the gaps between the two sites are different. In the case of implantation of the comparative example, the effect is also considered to be insignificant since the formation of the gap similar to that of the blank, while the implantation of Example 1 maintains the gap similar to that of Sham or normal disk. By verifying that the artificial disc manufactured in the present invention proves to be effective for disc reproduction.

As described above, in the case of replacing the damaged disk tissue with the artificial disk of the present invention, compared to the conventional artificial disk substitutes have higher bioactivity and biomimetics, which was much more helpful for tissue regeneration and visual observation and X-ray observation Through the composite support according to the invention it can be seen that forms a structure similar to the actual disk tissue, has the advantage of showing excellent characteristics as a support for tissue engineering disk.

Claims (7)

An internal disk comprising a disc central axis in the form of a disc made of a biodegradable porous support including nucleated cells, and A donut-shaped outer disk surrounding the inner disk made of a biodegradable porous support including fibroblast cells Artificial disk consisting of. The method according to claim 1, An artificial disc further comprising a natural or synthetic biomaterial on a biodegradable porous support inside or outside the disc. The method according to claim 2, The natural or synthetic biomaterials include alginic acid, chitosan, chitin, collagen, laminine, heparin, fibrin sulfate chondroitin, hyaluronic acid, methylene polyethylene glycol / polycaprolactone (MPEG / PCL), poloxamer , Agarose, gelatin, small intestinal submucosa, demineralized bone meal any one or more materials selected from. The method according to any one of claims 1 to 3, An artificial disc further comprising any one or more components selected from growth factors, dexamethasone, hydrocortisone, ascorbic acid and vitamin D ₃ in the biodegradable porous support inside or outside the disc. The method according to claim 1, The biodegradable nucleated pulmonary cells and annulus fibroblasts are artificial disks, characterized in that separated from the disk of the mammal. Mixing a biodegradable polymer solution comprising natural or synthetic biomaterials with a salt, filling it into a doughnut-shaped mold, and drying to prepare an external porous support of the artificial disc; Inoculating fibrosus cells to the donut-shaped porous support; And And a step of filling a donut-shaped porous support inoculated with the annulus fibrosus cells into a mold to fill a biodegradable polymer solution containing a natural or synthetic biomaterial and a suspension of nucleated cells in the donut. Mixing a biodegradable polymer solution comprising natural or synthetic biomaterials with a salt, filling it into a doughnut-shaped mold, and drying to prepare an external porous support of the artificial disc; A biodegradable polymer solution comprising natural or synthetic biomaterials is mixed with a salt and filled into a disc-shaped mold having a diameter equal to the inner diameter of the donut-shaped porous support, and dried to prepare an internal porous support of the artificial disc. Doing; Inoculating the annulus fibrosus cells to the outer porous support, and inoculating the nucleus cells into the inner porous support; And And inserting and bonding the inner porous support including the nucleated cells to the outer porous support including the annulus fibrosus cells.

Images