WO2003080143A1

WO2003080143A1 - Base material for reconstructing tissue or organ containing cell-free tissue matrix and cell growth factor

Info

Publication number: WO2003080143A1
Application number: PCT/JP2003/003581
Authority: WO
Inventors: Yasuhiko Tabata; Osamu Ogawa
Original assignee: Kaken Pharmaceutical Co., Ltd.
Priority date: 2002-03-25
Filing date: 2003-03-25
Publication date: 2003-10-02
Also published as: AU2003221081A1; JPWO2003080143A1; JP4431399B2

Abstract

It is intended to provide a base material for reconstructing a tissue or an organ which is highly biocompatible and shows regulated graft contraction. Namely, a base material for reconstructing a tissue or an organ which contains a cell-free tissue matrix and a cell growth factor.

Description

Description Base material for tissue or organ reconstruction containing decellularized tissue matrix and cell growth factor

The present invention relates to a substrate for reconstructing a biological tissue or organ, that is, a substrate for regeneration that induces invasion of cells from surrounding tissues and regenerates and repairs the biological tissue or organ. A substrate for reconstructing a tissue or organ, comprising a decellularized tissue matrix and a cell growth factor. Background art

Biological tissue engineering is an attempt to regenerate and repair damaged or defective parts of living tissues or organs. In this attempt, scaffold materials for proliferation and differentiation of various cells have been researched and developed. To date, synthetic scaffolds have been created, but there are many issues that need to be improved. Therefore, the production of a scaffold material from a biological material has been considered and attempted. To date, attempts to use various biological tissues or organs, perform decellularization to suppress immune rejection of tissues or organs, collect only extracellular matrix, and use it as a scaffold There is. These biological scaffolds have better mechanical properties and body fluid storage properties than synthetic scaffolds. However, when used in vivo, the scaffold shrinks. One way to solve this is to insert cells. However, in this method, isolation and seeding of cells are complicated, and another method is desired.

Therefore, we considered adding a cell growth factor as a method to suppress the contraction of the scaffold by promoting the invasion of the cell into the scaffold material instead of the cell in the body. This is the point of the present invention. Of course, in some cases, various cells and cell growth factors can be combined and put into a decellularized tissue matrix and used.

One cause of graft contraction is poor regeneration of tissues or organs, such as smooth muscle, which contains cell growth factors and releases them slowly from the decellularized tissue matrix. It is thought that the regeneration of the above-mentioned tissue or organ is promoted by the conversion.

For example, considering bladder reconstruction surgery, bladder dysfunction due to a congenital disease or bladder enlargement surgery is necessary when the bladder is removed due to cancer. Patch repair is usually performed on the gastrointestinal tract, but there are many problems such as reabsorption of urine from the intestinal tract, rupture and carcinogenesis, and the regeneration of the bladder itself is ideal.

The history of bladder reconstruction by patch repair using various artificial materials dates back to the 1960s, and a wide variety of materials have been used from animal experiments to clinical trials. However, non-absorbable materials are not suitable for use in the urinary tract due to foreign body reactions, stone formation, etc. On the other hand, absorbent polymers and various biological materials show good regeneration on grafts On the other hand, the contraction of the graft area itself could result in insufficient function addition. After that, progress was made in bladder reconstruction using the gastrointestinal tract, but as the long-term problems mentioned above became apparent, bladder reconstruction is gaining attention again.

In the 1990s, animal experiments were conducted on two biological materials, small intestine submucosa (SIS) and decellularized bladder matrix (bl adder acel lul ar matrix). Repair was reported to be a new material for bladder reconstruction, but none of the major defects were repaired due to the above-mentioned graft contraction, which did not provide sufficient function. did. However, these two biological materials are already routinely used in the United States for urethral reconstruction, etc., and in particular, SIS is distributed as a product of COOK.

On the other hand, another group has reported that a large amount of autologous bladder cells cultured outside the body can be seeded on a graft and bladder repair performed to suppress graft contraction, and are now in clinical trials. Although the usefulness of autologous cell transplantation has been proved by this study, it is clear that such complicated operations and medical care requiring large medical costs will not be widely accepted in the world. There are many problems to be solved.

If the bladder itself can be fully rehabilitated in some way, it may be possible to regenerate the bladder without taking complicated procedures such as cell transplantation. Development is the current challenge. Disclosure of the invention

The present invention has solved these problems, that is, it can be used not only for bladder but also for reconstruction of various other living tissues or organs, is excellent in biocompatibility, does not cause graft shrinkage, and is easy to operate. It is an object of the present invention to provide a simple and economically inexpensive base material for reconstructing a tissue or organ that can bring out tissue repair ability.

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, have made the present invention.

The present invention provides a living tissue or a tissue having a function of inducing regeneration of a living tissue or organ and regenerating and repairing the living tissue or organ, comprising a decellularized tissue matrix and a cell growth factor. It is a substrate for organ reconstruction.

The decellularized tissue matrix in the present invention refers to a matrix in which most of the cellular components of a living tissue or organ have been removed and a porous extracellular matrix remains, and decellularized from any living tissue or organ. Includes all products that have been processed.

The tissue or organ from which the decellularized tissue matrix can be obtained is not particularly limited as long as it is a living tissue or organ.For example, esophagus, bladder, urethra, small intestine, liver, lung, lung, skeletal muscle, smooth muscle , Myocardium, kidney, bone, cartilage, skin, hair, brain, nerve, muscle, blood vessels, kidney, retina, cornea, diaphragm, pericardium, serosa, amnion, tendon, ligament, large intestine, duodenum, trachea, Examples include vas deferens, fallopian tubes, ureters and lymph vessels. Preferred are the bladder, the small intestine and the esophagus, and particularly preferred is the bladder.

Cell growth factors in the present invention include cell growth factors (cell growth factors) such as bFGF (basic fibroblast growth factor), aFGF PDGF TGF-β1 VEGF HGF HB_EGF CTG FI GF-I and IGF-II. It includes what is called or interleukins, cytokins, bioactive peptides and chemokines, but is preferably bFGF HGF CTGF and PDGF, and particularly preferably bFGF. These growth factors can be used alone or in combination of two or more. Specifically, bFGF that can be used in the present invention is an organ such as the pituitary gland, brain, retina, corpus luteum, adrenal gland, etc. Extracted products, those produced by genetic engineering techniques such as recombinant DNA technology, and modified products thereof that can act as fibroblast growth factors. Examples of the modified form of bFGF include those obtained by adding, substituting or deleting an amino acid in the amino acid sequence of bFGF obtained by the above-described extraction or genetic engineering technique. The bFGF that can be used in the present invention preferably includes, for example, those described in WO 87/01728 and WO 89704832, particularly those described in the former.

The amount of cell growth factor in the tissue or organ reconstruction substrate of the present invention may be determined by the following: decellularized tissue matrix, type of cell growth factor, type of tissue or organ to be reconstructed, lesion site, extent of lesion, patient condition, etc. For example, in the decellularized tissue matrix lg, 1,000 to 100,000 g, preferably 5,000 to 50,000 g, more preferably 2,000 to 30,000 / g g.

The tissue or organ to be reconstructed in the present invention is not particularly limited as long as it is a living tissue or organ, and for example, esophagus, bladder, urethra, small intestine, liver, lung, skeletal muscle, smooth muscle, cardiac muscle, kidney, bone, Cartilage, skin, hair, brain, nerve, muscle, blood vessels, kidney, retina, cornea, diaphragm, pericardium, serosa, amniotic membrane, tendon, ligament, large intestine, duodenum, trachea, intubation, fallopian tubes, ureters and Examples include lymphatic vessels and the like. Preferred are the bladder, small intestine, and urethra, and particularly preferred is the bladder.

In the tissue or organ reconstruction substrate of the present invention, the tissue or organ for obtaining the decellularized tissue matrix and the tissue or organ to be reconstructed may be the same or different, but both tissues or organs are the same. In some cases, it is preferable. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram (A) of the decellularized tissue matrix from rat bladder and the HE staining result (B).

Figure 2 shows the preparation of decellularized tissue matrix from rat bladder and the inclusion of cell growth factor in rat bladder before decellularization (1) and decellularized tissue matrix from rat bladder ( 2) Decellularized tissue mat after lyophilization Lix (3) and a decellularized tissue matrix (4) after impregnation with an aqueous cell growth factor solution.

FIG. 3 is a graph showing the release of cell growth factor from a decellularized tissue matrix containing cell growth factor in an aqueous solution (PBS).

FIG. 4 is a graph showing degradation of a decellularized tissue matrix and attenuation of a cell growth factor contained in the decellularized tissue matrix in a subcutaneous mouse. Figure 5 is a graph (top) and a macroscopic image showing the granulation-forming ability of the decellularized tissue matrix containing cell growth factors embedded in the living body for a certain period of time, 7 days after subcutaneous implantation in mice. (Bottom).

Figure 6 shows HE staining results of bladder tissue on grafts of rats in groups A, B, C and D 4 weeks after surgery ((A), (B), (C) and ( D)).

FIG. 7 shows the bladder ((A), (B), (C) and (D)) of the rats in groups A, B, C and D 4 weeks after the operation. The region surrounded by the arrow is the graft site.

FIG. 8 is a graph showing the graft area of rats in groups A, B, C, and D four weeks after surgery.

Figure 9 shows HE staining results of bladder tissue on grafts of rats in groups A, B, C and D 12 weeks after surgery ((A), (B), (C) and (D )). BEST MODE FOR CARRYING OUT THE INVENTION

The substrate for tissue or organ reconstruction of the present invention can be produced by including a cell growth factor in a decellularized tissue matrix.

The decellularized tissue matrix can be obtained by subjecting the cells or tissues to the above-described tissues or organs by a conventional method, for example, treatment with a surfactant such as Triton X-100 or other known treatments, for example, repeated freeze-thawing, osmotic pressure change, etc. Can be prepared according to the destruction method. The decellularized tissue matrix can be lyophilized and stored for later use.

The method for incorporating the cell growth factor into the decellularized tissue matrix may be any method that allows the cell growth factor to be uniformly distributed in the decellularized tissue matrix. The method is not particularly limited, and examples thereof include a method of impregnating a cell growth factor solution into a decellularized tissue matrix, a method of freeze-drying a decellularized tissue matrix, and impregnating or adding a cell growth factor solution thereto.

The tissue or organ reconstruction base material of the present invention may be used, for example, when there is a tissue dysfunction due to a congenital disease, when the tissue or organ is excised due to cancer, or when the wound healing power is poor due to complications such as diabetes. Or, when wound healing power is poor due to infection of surrounding tissues, etc., it can be used effectively for reconstruction of the tissues or organs.

The reconstruction of a tissue or an organ using the reconstruction substrate of the present invention can be performed, for example, by using the reconstruction substrate as a graph and patch-repairing the tissue or organ to be reconstructed. Example

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.

Example 1: Preparation of decellularized tissue matrix from rat bladder and its morphological evaluation

(Method)

The bladder of a rat weighing 250 to 300 g was excised and the surrounding tissues were excised and decellularized by the following method.

1) 1 OmM PBS + 0.1% sodium azide solution was added, and shaken at 37 ° C for 6 hours.

2) 0.1M PBS was added and washed at room temperature for several minutes.

3) 1M NaC1 + 200 OKunitz units DNase I was added and shaken at 37 ° C for 6 hours.

4) 4% Triton-X aqueous solution was added and shaken at 37 ° C for 6 hours.

5) 70% ethanol was added and washed for 1 hour. This was repeated three times.

6) Distilled water was added and washed for 1 hour. This was repeated three times.

After fixing the obtained decellularized tissue matrix with 10% formalin, HE staining Specimens were prepared and the morphology was recorded with a light microscope. Another specimen was fixed with 2.5% aqueous dartaldehyde solution, dehydrated, replaced with t-butyl alcohol, freeze-dried, coated with platinum vapor deposition, and observed and recorded with a scanning electron microscope.

(Results) As shown in Fig. 1 (A) and (B), in the obtained decellularized tissue matrix, most of the cell components of the bladder were removed, and the porosity of the extracellular matrix was reduced. The tissue remained. Example 2: Cell growth factor binding method by impregnation of lyophilized decellularized tissue matrix in aqueous solution and evaluation of binding state in aqueous solution

(Method)

The matrix prepared by the above method was frozen in distilled water at 180 ° C. for 12 hours, and then lyophilized for 48 hours using a vacuum pump. This was impregnated with an aqueous bFGF solution and allowed to stand at 37 ° C for 1 hour to prepare a decellularized tissue matrix containing cell growth factors. Separately, the matrix before lyophilization was impregnated with an aqueous bFGF solution.

^An aqueous solution of 100 to 200 basic fibroblast growth factors (hereinafter abbreviated as FGF) radiolabeled with ¹²⁵ I was impregnated with 20 ^ 1 per rat matrix (dry weight 10 to 12 mg) at a rate of 20 ^ 1. A decellularized tissue matrix containing cell growth factors was obtained (Fig. 2). This was shaken in 1 ml of PBS or urine at 37 ° C, and the radioactivity in the solution after a certain time was measured by gamma. A fixed amount of the counter was used to evaluate the interaction between bFGF and the matrix.

(Result)

A certain amount of bFGF impregnated in the decellularized tissue matrix was detached in the first 24 hours, before and after freeze-drying, and reached a plateau thereafter (Fig. 3). Freeze-dried matrices had a lower release than the undried matrices. Also, release in urine was even lower than in PBS.

(Discussion)

By this method, it is possible to attach a certain amount of bFGF to the matrix. Also, when this matrix is used for urethral reconstruction, it is lost in large quantities in the urine. It was not thought to be. Example 3: Evaluation of correlation between in vivo attenuation of cell growth factor and matrix degradation (method)

bFGF in vivo release experiments: A matrix containing radiolabeled cell growth factor, prepared in a similar manner as described above, was implanted subcutaneously in the back of 6-week-old female d dY mice. Thereafter, this was sacrificed over time and the remaining radioactivity of the matrix and the surrounding tissue was measured to examine the remaining bFGF.

Matrix degradation experiments: Bolton-Hunter reagent in ¹²⁵ 1 with the decellularized tissue matrix was radiolabeled in the same manner as described above, were implanted subcutaneously into mice, was examined the degradation rate in vivo.

(Result)

bFGF gradually attenuated over 6 weeks after implantation. The matrix decomposition followed the same process (Fig. 4).

It is considered that bFGF bound to the decellularized tissue matrix disappears from the living body as the matrix is degraded. Example 4: Evaluation of residual cell growth factor activity in vivo

(Method)

A 5 mg decellularized tissue matrix from rat bladder is impregnated with an aqueous solution containing 5 g of bFGF or PBS lOl in the same manner as above, and one group is directly implanted under the mouse subcutaneously in the same manner as above. The remaining two groups were sealed in a diffusion chamber and implanted subcutaneously in a mouse in the same manner as described above. After this was removed 7 or 14 days later, the matrix alone was reimplanted subcutaneously in another mouse. Seven days after matrix implantation (or re-implantation) in each group, the mice were sacrificed and subcutaneous tissues 2 cm square around the matrix were collected to evaluate the tissue weight and the like.

(Result)

In the matrix impregnated with bFGF, granulation around the matrix was observed for more than 2 weeks (Fig. 5). It was considered that the activity of bFGF bound to the matrix was maintained even when implanted in vivo. Example 5: Bladder Patch Repair Surgery Using Decellularized Tissue Matrix from Rat Bladder Containing FGF, Use b Examination of relationship between FGF concentration and bladder reconstructed image on matrix graft, patch size.

(Method)

The rat bladder was decellularized, and the portion corresponding to the bladder apex 2Z3 was removed therefrom to prepare a decellularized tissue matrix containing various concentrations of bFGF. Another 10-week-old female Wistar rat was anesthetized with xylazine-ketamine, a midline incision was made in the lower abdomen to expose the bladder, the apical 2/3 of the bladder was excised, and the decellularized tissue matrix was used. This defect was patch repaired as a graft.

The suture between the bladder and the graft was continuously sutured using 8-0 absorbent thread, and the four corners were marked with 7-0 nylon thread. After the restoration was completed, a tube was inserted through the urethra and physiological saline was injected to confirm that there was no leak. At the same time, the graft size at the time of filling was recorded as the major and minor diameters of the marking thread.

For each decellularized tissue matrix containing 0, 5, 25, and 100 bFGF, 9 duplicates were prepared, and 4 were evaluated 4 weeks after operation and 5 were evaluated at 12 weeks. did. Each was designated as A, B, C, D groups. In addition, as a control, 6 groups of sutures were closed after resection of the bladder 2Z3, and 3 groups were evaluated 4 and 12 weeks later.

Under anesthesia with urethane (900 mg / kg, sc), the lower abdomen was incised to expose the bladder, and a 3 Fr polyethylene tube was placed from the urethra. A T-tube was connected to the end of the tube, one to the pressure transducer and the other to the infusion pump. After evacuating the bladder from the wound to make it empty, measure the bladder pressure by injecting physiological saline at a rate of 6.Oml / hr from the infusion route, and measure the maximum by leaking the infusate around the urethral indwelling tube. Bladder capacity. The test was performed five times in a row for each individual, and the average of the three median values was taken as the bladder capacity of that individual. After the test is completed, the rats are sacrificed and the bladder is emptied.After injecting 10% formalin or Krebs solution equivalent to the bladder capacity, the urethral opening is clamped, and the distance between the marking threads is reduced. It was measured. The graft area was defined as major axis X minor axis Z2. Tissues were fixed in formalin, photographed, and dehydrated, paraffin-embedded, and HE-stained in all groups at 4 weeks and 3 animals at 12 weeks in each group.

(Result)

At week 4, epithelial regeneration was complete in all groups. In the submucosa, the higher the concentration of bFGF, the greater the amount of mesenchymal cells infiltrating into the graft (Figures 6-7). At this time, the contraction of the graft area was suppressed depending on the bFGF concentration (FIG. 8).

At the 12th week, smooth muscle layer formation was observed in each group (FIG. 9). The invention's effect

The substrate for tissue or organ reconstruction of the present invention can be used in tissue or organ reconstruction such as patch repair for bladder reconstruction, is excellent in biocompatibility, does not bring about graft repair, is a simple and economical method. Can bring out the tissue repair ability.

The substrate for tissue or organ reconstruction according to the present invention suppresses contraction of a graft, which cannot be obtained with a conventional biologically-derived scaffold, by gradually releasing cell growth factors and promoting cell invasion from the tissue to the substrate. Has an effect. Moreover, the method of the present invention is more convenient than the conventional method of inserting cells into a biologically-derived scaffold.

Claims

The scope of the claims

1. A substrate for reconstructing a tissue or organ, comprising a decellularized tissue matrix and a cell growth factor.

2. The reconstruction substrate according to claim 1, wherein the cell growth factor is bFGF.

3. The reconstruction substrate according to claim 1 or 2, wherein the decellularized tissue matrix is derived from the bladder.

4. The method for producing a substrate for reconstruction according to any one of claims 1 to 3, which comprises impregnating or adding a cell growth factor solution to the decellularized tissue matrix.