KR101269618B1 - Graft materials derived from mammalian cartilage - Google Patents

Abstract

The present invention relates to a living graft material derived from mammalian cartilage tissue. The biotransplant material according to the present invention has a significantly lower risk of adverse effects when transplanting antigenicity, pathogens and cytotoxicity through a multi-step treatment process, and thus obtains and supplies the cartilage tissue of non-human animals (mammals). This is a smooth and inexpensive and safe when applied to biotransplantation.

Description

Graft materials derived from mammalian cartilage

The present invention relates to a living graft material derived from mammalian cartilage tissue.

Cartilage transplantation has been widely used in the treatment of damaged cartilage or plastic surgery. In particular, as the rhinoplasty is activated day by day, the number of cartilage is increasing. It has been used for decades, including autologous cartilage and various types of implants. Among them, xenografts such as Gore-tex and silicone are easy to handle, save time for surgery, and eliminate donor morbidity. Prior art in related fields using heterogeneous materials is disclosed in Korean Patent Laid-Open Publication Nos. 2011-0012807 (artificial cartilage substitutes) and 2011-0097662 (support for tubular bone regeneration and methods for preparing the same).

However, long-term follow-up has reported side effects such as foreign body sensations, infections, pain, escape and mobility of the implant. In addition, autologous implants have been noted as limiting donor morbidity, limiting patient selection, prolonging operation time, and collecting a large amount, despite satisfactory surgical outcomes due to rare side effects according to complete biocompatibility.

As an alternative to overcome the problem of autologous cartilage as described above, there is a method in which cartilage tissue is supplied from an allogeneic or xenograft. In this case, there is the greatest risk of transmission of the virus along with the implant, but the Tissue Bank is currently screening the donor virus for infection and processing only selected uninfected tissues. This is solved through. This kind of allograft has been used in orthopedics, plastic surgery, ophthalmology, gynecology and urology since 1972, and unlike the xenograft, the most important advantage is that it does not require a separate autologous cartilage collection for non-adjuvant surgery. to be.

However, the use of cartilage tissue of the human body is also difficult to supply a large amount, and because it is a homogeneous transplant, there is always a risk due to the transfer of pathogens, and the price of the final product manufactured for transplantation is very expensive. It is becoming.

Under such technical background, the present inventors have completed the present invention as a result of diligent efforts to develop a biograft which is easy to obtain and low in cost while having a significantly low risk of side effects in transplantation.

After all, the technical problem to be achieved by the present invention is to provide a method for producing a biological transplant material derived from a mammal.

Another technical problem to be achieved by the present invention is to provide a biograft prepared by the above method.

According to one aspect of the invention, preparing a cartilage tissue of mammal; Treating the prepared cartilage tissue with sodium chloride hypertonic solution; Treating cartilage tissue treated with the hypertonic solution with a virus inactivation solution; Treating cartilage tissue treated with the virus inactivation solution with an alkaline solution; Dewatering or freezing the cartilage tissue treated with the alkaline solution; And it can be provided a method for producing cartilage transplant material comprising the step of irradiating the dehydrated or frozen cartilage tissue with gamma rays.

According to one embodiment, the mammal may be one or more selected from the group consisting of pigs, humans, horses, cattle, dogs, mice.

According to one embodiment, the cartilage tissue may be at least one selected from the group consisting of free cartilage, elastic cartilage and fibrocartilage.

According to one embodiment, the step of preparing the cartilage tissue of the mammal by immersing the collected cartilage tissue in a solution of [acetone or methyl alcohol]: chloroform = 1: 1 (v / v) for 48 hours Defatting may be further included.

According to one embodiment, the cartilage tissue of the mammal may be cartilage tissue of pigs.

According to one embodiment, the cartilage tissue of the pig may be an ear ear cartilage (Elastic ear cartilage) or rib cartilage (rip cartilage).

According to one embodiment, the step of treating the hypertonic solution may be to immerse in 6 ~ 12% sodium chloride solution for 4-6 hours while applying ultrasonic waves to the cartilage tissue.

According to one embodiment, the step of treating the virus inactivation solution is 0.5 to 1.5% hydrogen peroxide, 0.1 to 1.0% peracetic acid (Peracetic Acid), 70 to 99% alcohol, and 0.001 to 3% sodium hypochlorite It may be immersed in a virus inactivation solution containing (NaOCl) for 30 minutes to 1 hour.

According to one embodiment, the treatment of the alkaline solution may be immersed in 0.25 ~ 0.75M sodium hydroxide (NaOH) for 15 to 45 minutes.

According to one embodiment, the dehydration is sequentially immersed in acetone of 50%, 80% and 100% concentration, immersed in anhydrous alcohol (absolute alcohol), or sequentially immersed in 50%, 80% acetone concentration Then immersed in carbohydrate material in a solution containing 0.1-1% of antibiotic and 90-99.9% glycerol, followed by primary immersion in 100% glycerol solution and second immersion in anhydrous alcohol. There may be more than one.

According to one embodiment, the freezing treatment may be to freeze-dried for 48 to 72 hours after freezing the cartilage tissue treated in the alkaline solution for 1-2 hours at -70 ℃.

According to another aspect of the present invention can be provided cartilage graft material prepared according to the above method.

The biograft according to the present invention is prepared with a multi-step treatment process to remove antigenicity, pathogens and cytotoxicity, and to inactivate all kinds of pathogenicity without affecting biomechanics and tissue reconstitution ability. . Using cartilage tissue of non-human animals (mammals) has the advantage that the acquisition and supply is smooth and the cost is low when applied as a living transplant material.

1 is a photograph of the pig ear cartilage tissue before the process treatment according to the present invention stained with various dyes and observed the cut surface under a microscope.
Figure 2 is a photograph of the cut surface of the pork rib cartilage tissue before treatment process according to the present invention by staining with various dyes.
Figure 3 is a photograph of the pig ear cartilage tissue finally prepared after the process treatment according to the present invention stained with various dyes to observe the cut surface under a microscope.
Figure 4 is a photograph of the final section of the pork cartilage tissue prepared after the process according to the present invention stained with various dyes to observe the cut surface under a microscope.
Figure 5 is a photograph showing the collagen, keratan sulfate in the cartilage tissue of the pig ears before and after the process according to the present invention.
Figure 6 is a photograph showing fibronectin, chondroitin sulfate present in the cartilage tissue of pig ears before and after the process treatment according to the present invention.
7 is a photograph of cartilage tissue using a scanning electron microscope (SEM) of the cartilage tissue of the pig ears before and after the process treatment according to the present invention.
8 is a graph of HPLC results showing the chemical composition of cartilage tissue of pig ears before and after the process treatment according to the present invention.
9 is data comparing the thermal characteristics of acetone or glycerol treatment in accordance with the present invention.
Figure 10 is a graph confirming the presence or absence of cytotoxicity and the final cartilage of the cartilage graft according to the difference between the dehydration method acetone or glycerol dehydration method according to the present invention.
Figure 11 is a photograph showing the process of transplant testing through animal experiments to evaluate the biological safety of cartilage transplants prepared according to the present invention.
12 is a result of tissue extraction and tissue examination after performing a transplant test through animal experiments to evaluate the biological safety of cartilage transplants prepared according to the present invention.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the invention, preparing a cartilage tissue of mammal; Treating the prepared cartilage tissue with sodium chloride hypertonic solution; Treating cartilage tissue treated with the hypertonic solution with a virus inactivation solution; Treating cartilage tissue treated with the virus inactivation solution with an alkaline solution; Dewatering or freezing the cartilage tissue treated with the alkaline solution; And it provides a method for producing cartilage graft material comprising the step of irradiating the dehydrated or frozen cartilage tissue with gamma rays.

At this time, the cartilage tissue of the mammal may be cartilage tissue indicating the free cartilage, elastic cartilage, fibrocartilage of the mammal including all pigs, humans, horses, cattle, dogs, mice, etc., preferably pig cartilage It may be an organization. More preferably, it may be elastic ear cartilage or rip cartilage of pigs.

In addition, preparing the cartilage tissue of the mammal is 24 to 72 hours, more preferably in a solution in which the collected cartilage tissue [acetone or methyl alcohol]: chloroform = 1: 1 (v / v) in a mixed solution It may further comprise the step of defatting by soaking for 48 hours.

In the case of further comprising the degreasing step, when the crude fat content was measured by the chloroform-methanol extraction method, the crude fat content of the pork rib cartilage tissue was measured as follows.

Item Analysis Weight of sample taken Test Methods
Before degreasing process After degreasing process Crude fat 3.4 g 0.0g 100g Chloroform-Methanol Extraction

According to one embodiment, in the step of treating the sodium chloride hypertonic solution may be soaked cartilage tissue for 4-6 hours in 6 ~ 12% sodium chloride (NaCl) solution while applying ultrasonic waves. If the concentration of sodium chloride is less than 6%, the osmotic pressure on the cells in the cartilage tissue is insignificant, and the osmotic destruction effect on the cells may be weak.If the concentration of the sodium chloride exceeds 12%, undissolved salts are precipitated in the cartilage tissue or the container. It may be undesirable. At this time, the sonication may help penetration of the solution into the cartilage tissue and may promote osmotic destruction of the cells.

According to one embodiment, in the step of treating the virus inactivation solution virus inactivation comprising 0.5-1.5% hydrogen peroxide, 0.1-1.0% peracetic acid, 70-99% alcohol, and 0.001-3% sodium hypochlorite Immersion solution may be soaked for 30 minutes to 1 hour. Through treatment of the virus inactivation solution, pathogens such as various virus particles and prion particles can be inactivated. More preferably the peracetic acid may be 0.1 to 0.5%, most preferably 0.15 to 0.25%. When the hydrogen peroxide of the virus inactivation solution is less than 0.5% is not preferable because the inactivation effect of the pathogen is inadequate, and when it exceeds 1.5% may damage the cartilage tissue is not preferable. Here, when the alcohol is used in a composition ratio close to 100% in excess of 95.5% of the above range, it is not preferable because it may cause degeneration of cartilage and side effects of cartilage dehydration.

According to one embodiment, the treatment of the alkaline solution may be immersed in 0.25 ~ 0.75M sodium hydroxide (NaOH) for 15 to 45 minutes. Such alkaline solution treatment can inactivate viruses that are present in or are likely to be introduced into cartilage tissue and allow the removal of cartilage cells in cartilage tissue. When the sodium hydroxide is treated at less than 0.25M, the chemical reaction necessary for inactivation of such viruses and removal of cells does not occur sufficiently, and when it exceeds 0.75M, it is not preferable because it may damage cartilage tissue.

According to one embodiment, the cartilage tissue treated with an alkaline solution is subjected to a dehydration step, which is sequentially immersed in acetone at 50%, 80% and 100% concentrations, or immersed in anhydrous alcohol (absolute alcohol). Or immersed in acetone at 50% and 80% concentrations sequentially, followed by anhydrous alcohol, or primary immersion of cartilage graft in a solution containing 0.1 to 1% antibiotic and 90 to 99.9% glycerol, and then 100 It may be at least one selected from the group consisting of a secondary immersion method by moving to a% glycerol solution. When the intracellular water of the cartilage transplant material is substituted with glycerol, the preservation property is improved, and therefore, two or more of the above methods may be used in combination.

Without passing through the dehydration treatment, the cartilage tissue treated with the alkaline solution may be stored in a frozen state of -20 ° C. or lower through a washing step.

According to one embodiment, the freezing treatment may be to freeze-dried for 48 to 72 hours after freezing the cartilage tissue treated in the alkaline solution for 1-2 hours at -70 ℃.

Processed cartilage can be individually packaged and then sterilized with ethylene oxide (EO) gas. Alternatively, the cartilage graft material may be sterilized by 10 to 60 kGy, more preferably 10 to 30 kGy, gamma sterilization, to the cartilage after the process is completed. In particular, when performing high-level gamma sterilization of 30 kGy or more, the cartilage graft material may be prepared by immersing the cartilage graft material in sterile physiological saline and performing only a single treatment step by gamma sterilization.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Establishment of Optimal Conditions by Process

Example  1. Optimal Gamma dose  decision

Fresh cartilage extracted from pig ears was immersed in 9% NaCl solution for 5 hours and irradiated with ultrasound. This allows chondrocytes to be removed. The tissue was then immersed in 1% hydrogen peroxide as a virus inactivation solution and stirred for 45 minutes. During this process, viruses and other pathogens were inactivated. Furthermore, it was immersed in 0.5M sodium hydroxide (NaOH) for 30 minutes as alkaline solution. Before treating each step solution, the wash solution was used to thoroughly wash the remaining solution in the previous step. Thereafter, in order to minimize the damage to the tissue, the immersion was sequentially dehydrated in 50%, 80% and 100% acetone, and then sealed in a packaging container and irradiated with gamma rays. In order to determine the optimal audit dose, gamma rays were irradiated in 5kGy units in the range of 5 ~ 30kGy for the cartilage transplants manufactured through the above steps. The optimal gamma-ray dose was selected by quantifying the degree of active chondrocytes and the amount of substances constituting the cartilage according to gamma-irradiation, and the results are shown in Table 1 below.

After gamma irradiation, chondrocytes remaining activated through H & E staining were compared and observed. Since live chondrocytes can act as antigens after transplantation in the body, it is important to inactivate them. Accordingly, the degree of morphology of chondrocytes was maintained for each sample according to the radiation dose, and the cases where the morphology of chondrocytes were most maintained were scored out of 5 points, and the degree of morphology maintenance of chondrocytes was recorded as a score. As a result, in the 25kGy and 30kGy conditions, the chondrocytes maintaining the original shape were hardly observed.

In addition, the results of the MA (Masson's Trichrome) staining and the Verhoeff-Van Gieson (VVG) staining showed that the collagen fibers and the elastic fibers (elastic fibers) are maintained in large part. Safranine O staining confirmed the presence of proteoglycans and Alcian blue staining revealed that the presence of viscous polysaccharides (GAGs).

As gamma dose increased, the degree of removal and inactivation of chondrocytes in the matrix was increased. Referring to Table 2, when the gamma dose was 25 kGy, the removal rate of chondrocytes was the highest, and the preservation ratio for collagen, proteoglycan GAGs, and elastic fibers was also the highest, indicating the optimal dose. 5 and 6 show the results of the dye when the gamma dose is 25 kGy.

Evaluation item 5kGy 15kGy 25kGy 30kGy Morphology of chondrocytes 5 4 One One Collagen fiber 4 4 4 3 Proteoglycan 5 5 4 3 Point Polysaccharides (GAGs) 5 5 4 4 Elastic fiber 5 5 5 4

Example  2. Comparison of dehydration methods

The prepared cartilage tissue can be dehydrated using acetone treatment or glycerol. Glycerol treatment is a method of replacing the water in the tissue with glycerol by immersing the tissue in glycerol containing about 0.11% of the antibiotic, and then immersed it in 99.9% or more glycerol.

Referring to FIG. 9, data comparing thermal properties of the final product after dehydration using acetone and glycerol are shown. As a result of confirming the thermal characteristics change according to the dehydration method of Figure 9, when comparing the acetone and glycerol thermal properties, it was confirmed that the glass transition temperature of acetone is about 99 degrees, glycerol is about 96 degrees, there is no significant difference, Based on the results, it was confirmed that the thermal properties of acetone and glycerol did not affect the state of the sample. In addition, Figure 10 shows the results of observing the cytotoxicity according to the dehydration method, it was confirmed that both the dehydration method by acetone or glycerol is not toxic to the cells.

Therefore, the cartilage graft material according to the present invention was dehydrated with acetone, and in addition to the method, a method of dehydration and preservation by treating glycerol was also possible.

Validation of the selected process

Example  3. Process ECM  Argument Check

5 and 6, the contents of keratan sulfate, collagen II, fibronectin, and chondroitin sulfate were examined for fresh porcine ear cartilage and finally prepared cartilage grafts. . As a result, keratan sulfate was observed throughout the substrate, and chondroitin sulfate was observed mainly near the cartilage. In particular, the tendency of chondroitin sulfate to appear biased around one cartilage was observed. Keratan Sulfate, Collagen II, Fibronectin and Chondroitin Sulfate tended to decrease somewhat in the final cartilage grafts, but were still maintained and preserved.

Example  4. Histological examination of the final product-inactivation of chondrocytes and Extracellular  temperament( ECM ) Maintenance of arguments

The final cartilage graft treated through the above treatment was subjected to a biopsy with stains specific to each element to verify whether there are chondrocytes capable of causing antigenicity and how much extracellular matrix is maintained.

Referring to FIGS. 3 and 4, when observed through hematoxylin & eosin (H & E) staining, it was confirmed that live chondrocytes were not observed in the final cartilage graft and all chondrocytes in the matrix were inactivated. . This is in contrast to the viable chondrocytes observed in FIGS. 1 and 2. 3 and 4, the results of the Masson's Trichrome (MT) and Verhoeff-Van Gieson (VVG) staining showed that the collagen fibers and the elastic fibers were substantially maintained. Safranine O staining results confirmed the presence of proteoglycans and the presence of point polysaccharides (GAGs, Glycosaminoglycans) through Alcian blue staining.

Example  7. Confirmation of 3D porous structure

The three-dimensional structure of the final product was observed by electron microscopy (SEM). As can be seen in Figure 8, it can be seen that the porous structure and the three-dimensional structure is well preserved without damage compared to the structure of the original cartilage tissue.

Example  8. Chemical Characterization

The chemical contents of cartilage tissue (red line) not added to the process, and cartilage tissue (blue line) and final product (purple line) during the processing were examined. As can be seen in Figure 9, the same peak appears in each sample, indicating that the characteristics of the raw material do not change by processing and sterilization.

Example  9. Confirmation of biological safety

Seven-week-old Sprague Dawley rats (SD-rat, male) were obtained and bred for a week before the test to observe and purify the general health status of the animals. After that, the individuals who reached the average body weight were selected and tested. Anesthesia was induced by intramuscular injection of Zoletile (20mg / kg) and Xylazine (5mg / kg), and 2cm horizontally incisions were made on the left and right sides of the spine. In order to implant the test substance, the fascia and dermis were separated and pockets were prepared. After transplantation, 4-0 nylon (nylon) was used to suture the defect site with simple nodule closure. Three and four months after the transplantation test, the subject was sacrificed and histological examinations were performed, including the normal tissues.

At 3 months, the result of histological examination showed a weak inflammatory response that was generally observed in the transplantation around the implant. It was confirmed that the initial transplantation state as it exists. At 4 months, the biopsy was similar to that of 3 months, and a slight inflammatory response was observed around the cartilage transplant. These are natural phenomena due to implantation and no specific histopathological phenomena other than a weak inflammatory response have been observed. In addition, it was confirmed that inactivated chondrocytes remained intact and all other extracellular matrix remained intact. As a result, the safety of heterologous cartilage transplants to be developed in the present invention was observed through animal experiments.

finally. The biograft according to the present invention is prepared with a multi-step treatment process to remove antigenicity, pathogens and cytotoxicity, and to inactivate all kinds of pathogenicity without affecting biomechanics and tissue reconstitution ability. . Using cartilage tissue of non-human animals (mammals) has the advantage that the acquisition and supply is smooth and the cost is low when applied as a living transplant material.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (12)

Preparing cartilage tissue of mammals other than humans;
Treating the prepared cartilage tissue with sodium chloride hypertonic solution;
Treating cartilage tissue treated with the hypertonic solution with a virus inactivation solution;
Treating cartilage tissue treated with the virus inactivation solution with an alkaline solution;
Dewatering or freezing the cartilage tissue treated with the alkaline solution; And
Method of producing a cartilage transplant comprising the step of irradiating the dehydrated or frozen cartilage tissue with gamma rays.
The method of claim 1, wherein the mammals other than humans are one or more selected from the group consisting of pigs, horses, cows, dogs, and mice.
The method of claim 1, wherein the cartilage tissue is at least one selected from the group consisting of free cartilage, elastic cartilage and fibrocartilage.
The method of claim 1, wherein preparing cartilage tissues of mammals other than humans is performed in a solution in which the collected cartilage tissues are mixed in a ratio of [acetone or methyl alcohol]: chloroform = 1: 1 (v / v). Method for producing cartilage graft material further comprising the step of defatting by soaking for 72 hours.
The method of claim 1, wherein the cartilage tissue of mammals other than humans is pig cartilage tissue.
The method of claim 5, wherein the cartilage tissue of the pig is an ear cartilage (Elastic ear cartilage) or rib cartilage (rip cartilage).
The method of claim 1, wherein the treating the hypertonic solution is immersed in 6-12% sodium chloride solution for 4-6 hours while applying ultrasonic waves to the cartilage tissue.
The method of claim 1, wherein the treating of the virus inactivation solution comprises 0.5-1.5% hydrogen peroxide, 0.1-1.0% peracetic acid, 70-99% alcohol, and 0.001-3% sodium hypochlorite. (NaOCl) A method for producing cartilage graft material, characterized in that immersed in virus inactivation solution containing for 30 minutes to 1 hour.
The method of claim 1, wherein the treatment of the alkaline solution is a method for producing cartilage graft, characterized in that immersed in 0.25 ~ 0.75M sodium hydroxide (NaOH) for 15 to 45 minutes.
The method of claim 1, wherein the dehydration treatment is sequentially immersed in acetone at 50%, 80% and 100% concentration, immersed in anhydrous alcohol (absolute alcohol), or sequentially immersed in acetone at 50%, 80% concentration Then immersed in anhydrous alcohol or firstly immersed cartilage in a solution containing 0.1-1% antibiotic and 90-99.9% glycerol, and then transferred to 100% glycerol solution for the second immersion. Method for producing cartilage transplant, characterized in that one or more.
The method of claim 1, wherein the freezing treatment freezes the cartilage tissue treated in the alkaline solution at −70 ° C. for 1 to 2 hours, and lyophilizes for 48 to 72 hours.
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