WO2012141454A2

WO2012141454A2 - Graft materials derived from mammalian cartilage

Info

Publication number: WO2012141454A2
Application number: PCT/KR2012/002616
Authority: WO
Inventors: Ho Chan Hwang; Ke Won Kang; Jin Young Kim; Jae Hyoung Ahn; Da Mi Choi
Original assignee: Hans Biomed. Cor.
Priority date: 2011-04-12
Filing date: 2012-04-06
Publication date: 2012-10-18
Also published as: WO2012141454A3

Abstract

The present invention relates to a graft material derived from mammalian cartilage. The graft material according to the present invention has significantly lowered risk by eliminating antigenicity, pathogen and cytotoxicity through multi-step processing treatments and inactivating all kinds of pathogens without affecting biomechanics and tissue reconstruction performance. It allows smooth accepting and supplying and low cost manufacturing because of use of mammalian cartilage not human's.

Description

GRAFT MATERIALS DERIVED FROM MAMMALIAN CARTILAGE

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

It has been widely used to treat damaged cartilage or transplant cartilage in the field of plastic surgery. In response to a large demand particularly for nasal augmentation, rhinoplasty has been increased. Various kinds of grafts including autologous cartilage graft have been used to this for several decades. Among them, alloplastic grafts such as gore-tex or silicon have advantages in easiness to handle, saving operation time and eliminating morbidity of donor site. Related prior arts using hetero-materials are KR Publication Nos. 2011-0012807 disclosing artificial cartilage using balloon and shape memory alloy and 2011-0097662 disclosing scaffold for articular cartilage regeneration and process for preparing the same).

However, side effects such as foreign body sensation, infection, pain, and escape and mobility of grafts, etc. have been reported in the long-term follow up. Even though the autograft can provide satisfactory surgery reports with rare side effects due to perfect biocompatibility, it is limited to be used for increased morbidity of donor site, limited choices, delayed operation time, a large amount collection and the like.

As an alternative to overcome the problem associated with autologous cartilage graft, a method for supplying cartilage from allograft or xenograft is provided. In this case, viruses can be transferred with graft and such problems can be solved by processing the selected and non-infected tissues after determining whether a donor has virus infection in the world tissue bank. Since 1972, these kinds of allogeneic cartilage grafts have been used in orthopedic surgery, plastic surgery, ophthalmology, gynecology and urology surgery and do not require additional collection of autologous cartilage for the nasal tip plasty unlike alloplastic graft.

However, since it uses human cartilage, it has some drawbacks such as difficulty to handle a large amount, risk in spread of pathogens all the time due to allograft, and high cost of the final product manufactured for transplantation.

The inventors of the present invention has completed the invention by developing graft materials which not only have significantly low side effects during transplanting but also are easy to prepared in low cost.

An aspect of the present invention is to provide a method for preparing a graft material derived from mammalian.

Another aspect of the present invention is to provide a graft material prepared by the method according to the present invention.

According to an aspect of the present invention, there is provided a method for preparing cartilage graft comprising: preparing mammalian cartilage; treating the prepared cartilage with a sodium chloride hypertonic solution; treating the hypertonic solution-treated cartilage with a virus inactivation solution; treating the virus inactivation solution-treated cartilage with an alkali solution; dewatering treating or freezing treating the alkali solution-treated cartilage; and gamma irradiating the dewatering-treated or freezing-treated cartilage.

According to an embodiment, the mammalian may be at least one selected from the group consisting of pig, human, horse, cow, dog, and mouse.

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

According to an embodiment, the step of preparing mammalian cartilage may further comprise immersing the cartilage in a solution of (acetone or methyl alcohol) : chloroform mixed in a ratio of 1 : 1 (v/v) for 24-72 hours and defatting the result.

According to an embodiment, the mammalian cartilage may be pig cartilage.

According to an embodiment, the pig cartilage may be elastic ear cartilage or rip cartilage.

According to an embodiment, the step of treating with a hypertonic solution may be immersing the cartilage in a 6-12% sodium chloride solution for 4-6 hours while applying ultrasonic wave to the cartilage.

According to an embodiment, the step of treating with a virus inactivation solution may be immersing in a virus inactivation solution comprising 0.5-1.5% of hydrogen peroxide, 0.1-1.0% of peracetic acid, 70-99% of alcohol, and 0.001-3% of sodium hypochlorite(NaOCl) for 1/2 to 1 hour.

According to an embodiment, the step of treating with an alkali solution may be immersing in a 0.25-0.75M sodium hydroxide(NaOH) for 15-45 minutes.

According to an embodiment, the dewatering treatment may be conducted by at least one method selected from the group consisting of immersing sequentially in 50%, 80% and 100% of acetone; immersing in absolute alcohol; immersing sequentially in 50% and 80% of acetone and then absolute alcohol; and immersing first in a solution comprising 0.1-1% of antibiotic and 90-99.9% of glycerol and then in 100% glycerol solution.

According to an embodiment, the freezing treatment may be conducted by freezing the alkali solution-treated cartilage at -70℃ for 1-2 hours and freeze drying for 48-72 hours.

According to another aspect of the present invention, there is provided a cartilage graft prepared by the above-described method.

The graft material according to the present invention can be prepared through multiple steps by eliminating antigenicity, pathogen and cytotoxicity without affecting biomechanics and tissue reconstruction abilities, in which all kinds of pathogens are inactivated. Since it does not use human cartilage but mammalian cartilage, there are advantages in smooth accepting and supplying and low cost of a graft material.

FIG. 1 is photographs of the cut section of fresh pig ear cartilages stained with various stains prior to treatments according to the present invention, observed through a microscope.

(H&E, MT, Safranine O, Alcian blue, VVG)

FIG. 2 is photographs of the cut section of fresh pig rip cartilages stained with various stains prior to treatments according to the present invention, observed through a microscope.

Arrows marks viable chondrocyte.

FIG. 3 is photographs of cut section of final pig ear cartilages stained with various stains after treatments according to the present invention, observed through a microscope.

FIG. 4 is photographs of cut section of final pig rip cartilages stained with various stains after treatments according to the present invention, observed through a microscope.

FIG. 5 is photographs illustrating collagen(A, C), karatan sulfate(B, D) present in fresh(A, B) and final(C, D) pig ear cartilages prior to and after treatments according to the present invention, respectively.

FIG. 6 is photographs illustrating fibronectin(A, C), chondroitin sulfate(B, D) present in fresh(A, B) and final(C, D) pig ear cartilages prior to and after treatments according to the present invention, respectively.

FIG. 7 is SEM of fresh and final pig ear cartilages prior to and after treatments according to the present invention, respectively. (A: fresh cartilage, B: Final product(EEC))

FIG. 8 is a graph of HPLC results illustrating chemical structures of fresh and final pig ear cartilages prior to and after treatments according to the present invention, respectively. (A:before treatment, B: during treatment, C: after treatment)

FIG. 9 illustrates comparison in the thermal property with acetone and glycerol treatment according to the present invention.

-Acetone: Sample dewatered by immersing in sequential acetone treatment(50%>80%>100%) after processing.

-glycerol: sample dewatered by immersing in 100% of glycerol (including an antibiotic) after processing.

FIG. 10 illustrates difference in cytotoxicity according to acetone dewatering compared to glycerol dewatering and difference in cytotoxicity of final cartilage graft.

(OD 570nm, A:DMSO, B:PBS, C:Control, D:Acetone, E:Glycerol)

FIG. 11 illustrates transplanting process through animal experiment in order to evaluate biological stability of the cartilage graft prepared according to the method of the present invention.

FIG. 12 illustrates the result of the tissue obtained after performing transplantation test through an animal experiment in order to determine the biological stability of the cartilage graft prepared by the method of the present invention.

Hereinafter, the present invention is described in more detail.

Here, the mammalian cartilage may be hyaline cartilage, elastic cartilage, or fibrocartilage of mammalian including pig, human, horse, cow, dog, and mouse, preferably pig cartilage. More preferably, it may be elastic ear cartilage or rip cartilage of pig.

Further, the step of preparing mammalian cartilage may further comprise immersing the cartilage in a solution of (acetone or methyl alcohol) : chloroform mixed in a ratio of 1 : 1 (v/v) for 24-72 hours, preferably 48 hours and defatting the result.

When the dewatering step is performed and amount of crude fat is determined by using a chloroform-methanol extraction, the amount of the crude fat of the pig rip cartilage is determined as follows.

Table 1

Category	Analysis Result		Sample weight	Testing method
	Before dewatering	After dewatering
Crude fat	3.4g	0.0g	100g	chloroform-methanol extraction

According to an embodiment, the step of treating with a sodium chloride hypertonic solution may be performed by immersing the cartilage in a 6-12% sodium chloride solution for 4-6 hours while applying ultrasonic wave to the cartilage. When concentration of sodium chloride is less than 6%, the osmotic destruction effect may be week because the osmosis action in the cartilage cells becomes little, while when it is more than 12%, un-dissolved salt may be participated in the cartilage or container. Here, the ultrasonic wave treatment helps the solution penetrating inside the cartilage and enhance osmotic destruction of cells.

According to an embodiment, the step of treating with a virus inactivation solution may be performed by immersing in a virus inactivation solution comprising 0.5-1.5% of hydrogen peroxide, 0.1-1.0% of peracetic acid, 70-99% of alcohol, and 0.001-3% of sodium hypochlorite(NaOCl) for 1/2 to 1 hour. All pathogens of virus particles and prion particles, etc. may be inactivated through the virus inactivation solution treatment. 0.1-0.5% of peracetic acid is preferable and 0.15-0.25% of peracetic acid is the most preferable. When concentration of hydrogen peroxide in the virus inactivation solution is less than 0.5%, inactivation of the pathogens may not be performed sufficiently, while when it is more than 1.5%, it may damage the cartilage. Here, when concentration of alcohol is from more than 95.5% to close to 100%, it may result in undesirable side effects such as degeneration and dehydration of the cartilage.

According to an embodiment, the alkali solution treatment may be performed by immersing in a 0.25-0.75M sodium hydroxide(NaOH) for 15-45 minutes. The viruses that are present in the cartilage or can be added during the process may be inactivated through the alkali solution treatment. It also allows eliminating cartilage cells in the cartilage tissue. When the treatment is performed with less than 0.25M of sodium hydroxide, chemical reactions to inactivate such viruses and eliminate cells may not occur sufficiently, while when it is treated with more than 0.75M of sodium hydroxide, it may damage the cartilage.

According to an embodiment, the dewatering treatment after performing the alkali solution treatment may be conducted by at least one method selected from the group consisting of immersing the cartilage sequentially in 50%, 80% and 100% of acetone; immersing in absolute alcohol; immersing sequentially in 50% and 80% of acetone and then absolute alcohol; and immersing first in a solution comprising 0.1-1% of antibiotic and 90-99.9% of glycerol and then in 100% glycerol solution. When water inside the cells of the cartilage graft is substituted with glycerol, since it improves conservative property, 2 or more of the above methods may be combined.

Also the alkali solution-treated cartilage may be stored, after washing step, at -20℃ or lower, without the dewatering treatment.

According to an embodiment, the freezing treatment is performed by freezing the alkali solution-treated cartilage at -70℃ for 1-2 hours and then freeze drying may be performed for 48-72 hours.

The final cartilage after the treatments may be individually packaged and disinfected with ethylene oxide(EO) gas. Or the final cartilage after the treatments may be gamma irradiated with 10-60kGy, preferably 10-30kGy to sterilize the cartilage graft. Particularly, when the gamma sterilization is performed with 30kGy or higher, the cartilage graft may be prepared by performing only gamma sterilization process by immersing the cartilage graft in a sterilized isotonic solution.

Hereinafter, although more detailed descriptions will be given by examples, those are only for explanation and there is no intention to limit the invention.

Establishment of Optimal Conditions By Process

Example 1. Determination of Optimal Dose of Gamma Rays

Fresh cartilage extracted from a pig ear was immersed in a 9% NaCl solution for 5 hours and irradiated with ultrasonic wave. Cartilage cells were removed through this treatment. Then it was immersed in 1% of hydrogen peroxide as a virus inactivation solution for 45 minutes and stirred. Viruses and other pathogens were inactivated through this treatment. And then, it was immersed in 0.5M sodium hydroxide(NaOH) as an alkali solution for 30 minutes. Before each treatment, the pre-treated solution was completely washed out with a washing solution. Dewatering treatment was performed by immersing sequentially in 50%, 80% and 100% acetone in order to minimize damages of the cartilage tissue, then sealed into a container and gamma-irradiated. The cartilage graft prepared through the above process was gamma-irradiated with a 5kGy unit within a range of 5-30kGy in order to determine optimal dose of gamma rays. Optimal dose of gamma rays is determined by digitizing degree of active cartilage cells depending on the gamma irradiation and amount of other materials forming the cartilage and the result is shown in Table 1.

After the gamma-irradiation, the cartilage cells remained as the activated state were observed and compared through the H&E stain. Since alive cartilage cells may act as an antigen after implantation, it is the most important to inactivate them. Each sample depending on the amount of irradiation was tested to determine the degree of the maintained shape of the cartilage cells. When the shape of the cartilage cells was maintained the most, it was scored as 5 and degree of the maintained shape of the cartilage cells were scored. As a result, the cartilage cells maintaining the original shape were not observed in 25kGy and 30kGy conditions.

Further, according to MT(masson's trichrome) stain and VVG(verhoeff-van gieson) stain, it is noted that a large part of collagen fiber and elastic fiber was maintained. Presence of proteoglycan was determined by the safranine O stain and presence of glycosaminoglycan(GAGs) by the alcian blue stain.

The higher the amount of gamma rays becomes, the more the cartilage cells in the matrix were removed and the more degree of inactivation was increased. Referring to Table 2, when the dose of gamma rays was 25kGy, removal of the cartilage cells was the highest and conservation ratio of collagen, proteoglycan GAGs and elastic fiber was the highest, so that it is noted as an optimal dose. FIG. 5 and FIG. 6 illustrate the result when the amount of gamma rays was 25kGy.

Table 2

Evaluation Category	5kGy	15kGy	25kGy	30kGy
Degree of maintained shape of cartilage cells	5	4	1	1
Collagen fiber	4	4	4	3
Proteoglycan	5	5	4	3
GAGs	5	5	4	4
Elastic fiber	5	5	5	4

Example 2. Comparison of Dewatering

The prepared cartilage was dewatered by using acetone or glycerol treatment. The glycerol treatment was performed by immersing the tissue in glycerol including about 0.11% of antibiotic and then in more than 99.9% of glycerol to substitute water in the tissue with glycerol.

Referring to FIG. 9, after dewatering by using glycerol, thermal properties of the final products were compared. According to FIG. 9 illustrating the thermal property depending on dewatering, the glass transition temperature of acetone was about 99° and that of glycerol was about 96°. Accordingly, it is noted that there was no significant difference and the thermal property of acetone and glycerol did not affect the shape of the samples. According to FIG. 10 illustrating cytotoxicity depending on dewatering, it is also noted that there is no cytotoxicity with the dewatering using acetone or glycerol.

Therefore, the cartilage graft according to the present invention can be dewatered by acetone or glycerol treatment and stored.

Investigation of Selected Process

Example 3. Determination of ECM Factor By Process

As shown in FIG. 5 and FIG. 6, amount of karatan sulfate, collagen II, fibronectin, and chondroitin sulfate from fresh pig ear cartilage and final cartilage graft was determined. Karatan sulfate was observed throughout the matrix, while chondroitin sulfate was observed mainly near the cartilage membrane, and particularly, chondroitin sulfate was concentrated on one side of the cartilage membrane. It is noted that the amount of karatan sulfate, collagen II, fibronectin, and chondroitin sulfate was decreased slightly in the final product but still maintained and maintained.

Example 4. Biopsy of the Final Product-Determination of Inactivation of the Cartilage Cells and Maintenance of Extracellular Matrix(ECM) Factor

The final cartilage graft prepared by performing the above-described treatments was performed for biopsy with the staining agent selective to each factor to determine whether the cartilage cells causing antigenicity to the final cartilage graft existed, and how much other extracellular matrix were maintained.

Referring to FIG. 3 and FIG. 4, it is noted that any alive cartilage cell was not observed in the final cartilage graft and all cartilage cells in the matrix were inactivated when it was determined using a hematoxylin & eosin(H&E) stain. This was distinct from the result shown in FIG. 1 and FIG. 2 illustrating observation of the live cartilage cells. According to the result of MT(Masson's Trichrome) and VVG(Verhoeff-Van Gieson) stain in FIG. 3 and FIG. 4, it is noted that a large part of collagen fiber and Elastic fiber were maintained. The presence of proteoglycan was determined through safranine O stain and that of glycosaminoglycans(GAGs) through alcian blue stain.

Example 7. Determination of 3-D Porous Structure

3-D structure of the final product was observed with a scanning electron microscope. As shown in FIG. 8, the porous structure and 3-D structure of original cartilage tissue was well maintained.

Example 8. Determination of Chemical Characteristics

Chemical characteristics of the cartilage prior to the processing (red line), the cartilage during the processing (blue line) and the final product (purple line) were determined. As shown in FIG. 9, it is noted that chemical characteristics were not changed by the processing and sterilization since each sample showed identical peaks.

Example 9. Determination of Biological Stability

7-Week-old sprague dawley rat (SD-rat, male) were received and fed for 1 week prior to testing to observe general health conditions and acclimatization. After this, test was performed by selecting objects having an average weight. Anesthesia was induced by intramuscular injection of zoletile (20mg/kg) and xylazine (5mg/kg) and then, it was cut in 2cm in left/right of the spine horizontally. A pocket was made by separating fascia and dermis in order to implant a testing material and the cartilage graft pre-prepared in a 0.5cm size was implanted into the muscle. After implantation of the graft, the defect area was stitched by a simple interrupted suture using 4-0 nylon. After 3 months and 4 months of the implantation test, the implanted area including normal tissues was obtained by sacrificing the object and tested.

According to biopsy at 3 months, very weak inflammation, which is commonly observed for implantation, was observed around the graft, but it is noted that the initial implant was existed as it was without inflammatory reaction or foreign body reaction since nucleus of inflammatory cells were not active. The biopsy result at 4 months showing very weak inflammatory reaction around the cartilage graft was similar to that at 3 months. This is very common in implantation of graft and specific pathologic phenomena were not observed. It is noted that all inactivated cartilage cells were existed without the strain inside the implanted graft and other extracellular matrixes were also existed without the strain. Accordingly, the stability of the cartilage graft of the present invention was observed through the animal test.

The cartilage graft of the present invention was prepared by eliminating antigenicity, pathogen and cytotoxicity through multi-step processing treatments and inactivating all kinds of pathogens without affecting biomechanics and tissue reconstruction performance. It allows smooth accepting and supplying and low manufacturing cost because of use of mammalian cartilage not human’s.

While the present invention has been described with reference to particular embodiments, it is to be appreciated that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, as defined by the appended claims and their equivalents.

Claims

A method for preparing cartilage graft comprising:

preparing mammalian cartilage;

treating the prepared cartilage with a sodium chloride hypertonic solution;

treating the hypertonic solution-treated cartilage with a virus inactivation solution;

treating the virus inactivation solution-treated cartilage with an alkali solution;

dewatering treating or freezing treating the alkali solution-treated cartilage; and

gamma irradiating the dewatering-treated or freezing-treated cartilage.
The method for preparing cartilage graft of claim 1, wherein the mammalian is at least one selected from the group consisting of pig, human, horse, cow, dog, and mouse.
The method for preparing cartilage graft of claim 1, wherein the cartilage is at least one selected from the group consisting of hyaline cartilage, elastic cartilage and fibrocartilage.
The method for preparing cartilage graft of claim 1, wherein the step of preparing mammalian cartilage further comprises immersing the cartilage in a solution of (acetone or methyl alcohol) : chloroform mixed in a ratio of 1 : 1 (v/v) for 24-72 hours and defatting the result.
The method for preparing cartilage graft of claim 1, wherein the mammalian cartilage is pig cartilage.
The method for preparing cartilage graft of claim 4, wherein the pig cartilage is elastic ear cartilage or rip cartilage.
The method for preparing cartilage graft of claim 1, wherein the step of treating with a hypertonic solution is immersing the cartilage in a 6-12% sodium chloride solution for 4-6 hours while applying ultrasonic wave to the cartilage.
The method for preparing cartilage graft of claim 1, wherein the step of treating with a virus inactivation solution is immersing in a virus inactivation solution comprising 0.5-1.5% of hydrogen peroxide, 0.1-1.0% of peracetic acid, 70-99% of alcohol, and 0.001-3% of sodium hypochlorite(NaOCl) for 1/2 to 1 hour.
The method for preparing cartilage graft of claim 1, wherein the step of treating with an alkali solution is immersing in a 0.25-0.75M sodium hydroxide(NaOH) for 15-45 minutes.
The method for preparing cartilage graft of claim 1, wherein the dewatering treatment is conducted by at least one method selected from the group consisting of immersing sequentially in 50%, 80% and 100% of acetone; immersing in absolute alcohol; immersing sequentially in 50% and 80% of acetone and then absolute alcohol; and immersing first in a solution comprising 0.1-1% of antibiotic and 90-99.9% of glycerol and then in 100% glycerol solution.
The method for preparing cartilage graft of claim 1, wherein the freezing treatment is conducted by freezing the alkali solution-treated cartilage at -70℃ for 1-2 hours and freeze drying for 48-72 hours.
A cartilage graft prepared according to any one of claim 1 to claim 11.