A method for preparing acellular cartilage matrix material derived from pig rib cartilage

Abstract

The present invention relates to the field of biomedical materials, and in particular to a method for preparing a decellularized cartilage matrix material derived from pig rib cartilage, comprising the following steps: (1) freezing and thawing the pig rib cartilage for 5 times; (2) soaking the pig rib cartilage frozen and thawed in step (1) in a sodium borohydride solution; (3) soaking the pig rib cartilage treated with sodium borohydride in step (2) in a sodium dodecyl sulfate solution; and (4) enzymolyzing the pig rib cartilage obtained in step (3) with a mixed solution of DNase and RNase. The present invention is suitable for decellularization of cartilage tissue with a thickness of 300 microns, and utilizes the spontaneous generation of hydrogen by the sodium borohydride solution to effectively expand the cartilage tissue, and then the surfactant SDS fully destroys the cell membrane, effectively removes the genetic material, and retains the effective components or extracellular matrix of the natural cartilage to the greatest extent.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a method for preparing a decellularized cartilage matrix material derived from pig rib cartilage. Background Art

[0002] Decellularized matrix is ​​a commonly used tissue repair material. Common sources of xenogeneic cartilage tissue include pig ear cartilage and articular cartilage. Natural xenogeneic or allogeneic cartilage tissue is decellularized to remove the main genetic material and reduce its immunogenicity, retaining the main extracellular matrix of natural cartilage, including collagen, glycosaminoglycans and some bioactive factors. Decellularized cartilage matrix can be used for a variety of organ repairs, such as ear reconstruction and articular cartilage repair. Currently, commonly used tissue decellularization methods include physical methods (freeze-thaw and ultrasonic treatment, etc.), chemical methods (acids, alkalis and ionic surfactants, etc.) and biological enzymes (collagenase, pancreatic enzymes and nucleases, etc.).

[0003] Although there are many decellularization methods, given that cartilage tissue is relatively dense, a single decellularization method often cannot achieve the goal of decellularization, and most decellularization schemes use a combination of physical and chemical methods. In addition, in order to facilitate full contact of the solvent during the decellularization process, the cartilage tissue often needs to be crushed or cut into slices of about 50 microns or less in advance before decellularization. However, such cartilage debris and cartilage slices less than 50 microns will lose a large amount of extracellular matrix, such as collagen and glycosaminoglycans, during the decellularization process due to their small size. Therefore, how to efficiently prepare cartilage decellularized matrices of larger volume or thickness is a difficult problem in the field of tissue engineering. Summary of the invention

[0004] The invention provides a method for decellularization of pig rib cartilage, which can effectively remove genetic material-nucleic acid in the pig rib cartilage while retaining extracellular matrix.

[0005] In order to solve the above technical problems, the present invention provides a method for decellularization of pig rib cartilage, comprising the following steps: (1) freezing and thawing the pig rib cartilage 5 times; (2) soaking the pig rib cartilage frozen and thawed in step (1) in a sodium borohydride solution; (3) soaking the pig rib cartilage treated with sodium borohydride in step (2) in a sodium dodecyl sulfate solution; (4) enzymatically hydrolyzing the pig rib cartilage obtained in step (3) with a mixed solution of DNase and RNase;

[0006] The mass concentration of the sodium borohydride solution is 0.5-2%, and the immersion time of the sodium borohydride solution is 12-36 hours;

[0007] The mass concentration of the sodium dodecyl sulfate solution is 1-3%, and the immersion time of the sodium dodecyl sulfate (SDS) solution is 12-36 hours;

[0008] The concentration of the DNase is 50-200 U / ml, the concentration of the RNase is 0.5-2 mg / ml, and the enzymolysis time is 24 h.

[0009] Preferably, the mass concentration of the sodium borohydride solution is 1%, and the immersion time of the sodium borohydride solution is 12 hours as one cycle, and the immersion period is 1-3 cycles, and fresh sodium borohydride solution is replaced in each immersion cycle.

[0010] Preferably, the sodium borohydride solution is immersed for 2 cycles, with a cumulative immersion time of 24 hours.

[0011] Preferably, the mass concentration of the sodium dodecyl sulfate solution is 2%, and the immersion time in the SDS solution is 24 hours.

[0012] Preferably, the volume of the sodium borohydride solution is 50-100 times the volume of the pig rib cartilage slices.

[0013] Preferably, the volume of the SDS solution is 20-50 times that of the pig rib cartilage slices.

[0014] Preferably, the freeze-thaw process is to immerse the pig rib cartilage in water, freeze it in liquid nitrogen for more than 5-10 minutes until the liquid is completely frozen, and then thaw it in a 37° C. water bath for more than 10-20 minutes until the ice is completely melted.

[0015] Preferably, the concentration of DNase is 100 U / ml, and the concentration of RNase is 1 mg / ml.

[0016] Preferably, the SDS solution immersion and enzymatic hydrolysis process is carried out on a 37° C. constant temperature shaker with a rotation speed of 100-120 rpm.

[0017] Preferably, the pork rib cartilage comes from pigs weighing 150 kg and under 4 months of age.

[0018] Preferably, the thickness of the pig rib cartilage is 300 microns.

[0019] Preferably, the method further comprises a washing step: before freezing and thawing, washing the cartilage slices with sterile pure water until no obvious foam is generated.

[0020] Preferably, the specific step of washing is to use 20-50 times the volume of pure water to wash the pig rib cartilage, place it in a constant temperature shaker at 37°C, rotate at 100-120 rpm, change it every 3 hours, and wash for 2-3 days until no obvious foam is generated in the washing water.

[0021] Preferably, before soaking in the SDS solution, the pig rib cartilage is ultrasonically cleaned with pure water for 3 times, then freeze-dried for 24 hours, and then sterilized with an ultraviolet lamp for 2 hours.

[0022] The present invention is aimed at the fact that the existing cartilage decellularization scheme is not suitable for cartilage tissue with a thickness of more than 100 microns. The cartilage decellularization matrix often needs to cut the cartilage tissue into thin slices of about 50 microns or grind it into particles before decellularization. The biggest disadvantage of this type of thin layer and granular decellularized cartilage is that it lacks the large volume of natural cartilage; the present invention is suitable for the decellularization of cartilage tissue with a thickness of 300 microns. It utilizes the spontaneous generation of hydrogen by sodium borohydride solution to effectively expand the cartilage tissue. After that, the surfactant SDS fully destroys the cell membrane, effectively removes the genetic material, and retains the effective components or extracellular matrix of natural cartilage to the greatest extent.

[0023] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of HE staining and aniline blue staining of fresh pig rib cartilage tissue.

[0025] Figure 2 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0026] Figure 3 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0027] Figure 4 This is the histological staining result of the decellularized matrix of the costal cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 36 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0028] Figure 5 The figure is a bar graph showing the quantitative analysis of DNA content in decellularized costal cartilage.

[0029] Figure 6This is the histological staining result of the decellularized matrix of the costal cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0030] Figure 7 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0031] Figure 8 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 36 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0032] Fig. 9 This is the histological staining result of the decellularized matrix of the costal cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 6 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0033] Fig.10 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0034] Fig.11 This is the histological staining result of the decellularized matrix of the rib cartilage; the specific decellularization operation includes 5 freeze-thaw cycles, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours. DETAILED DESCRIPTION

[0035] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0036] Example 1 Preparation and Histological Staining of Fresh Pork Rib Cartilage Slices

[0037] The preparation steps of fresh pork rib cartilage slices are as follows:

[0038] (1) Remove the attached muscles, soft tissues and perichondrium from fresh pig rib cartilage and wash it three times with phosphate buffer;

[0039] (2) Use a hard tissue slicer to cut fresh pig rib cartilage into slices with a thickness of 300 μm and wash them three times with PBS;

[0040] (3) Fresh cartilage slices were fixed with 4% paraformaldehyde for 24 hours, then dehydrated with gradient alcohol, transparentized with xylene, and embedded in paraffin. The paraffin tissue was cut into 5-μm-thick tissue slices for histological staining.

[0041] (4) HE staining steps: conventional HE staining steps are used, see Table 1 below;

[0042] Table 1 HE staining

[0043] Serial number step time 1 60℃ baking sheet 30min 2 Dewaxed xylene① 20min 3 Dewaxing xylene② 20min 4 100% ethanol① 5min 5 100% ethanol② 5min 6 95% ethanol 5min 7 90% ethanol 5min 8 80% ethanol 5min 9 70% ethanol 5min 10 Hematoxylin staining 10min 11 Tap water rinse 10min 12 0.5% hydrochloric acid alcohol differentiation 5s 13 Washing with tap water turns blue 10min 14 70% ethanol 5min 15 80% ethanol 5min 16 0.5% eosin (prepared with 90% ethanol) 15s 17 90% ethanol 3min 18 95% ethanol① 3min 19 95% ethanol② 5min 20 100% ethanol① 5min 21 100% ethanol② 5min 22 Transparent xylene① 7min 23 Transparent xylene② 7min 24 Neutral resin sealant /

[0044] (5) The steps of aniline blue staining are shown in Table 2 below;

[0045] Table 2 Aniline blue staining steps

[0046] Serial number step time 1 60℃ baking sheet 30min 2 Dewaxed xylene① 20min 3 Dewaxing xylene② 20min 4 100% ethanol① 5min 5 100% ethanol② 5min 6 95% ethanol 5min 7 90% ethanol 5min 8 80% ethanol 5min 9 70% ethanol 5min 10 Pure water 5min 11 Aniline blue 5min 12 90% ethanol 5min 13 95% ethanol① 5min 14 95% ethanol② 5min 15 100% ethanol① 5min 16 100% ethanol② 5min 17 Transparent xylene① 7min 18 Transparent xylene② 7min 19 Neutral resin sealant /

[0047] (6) After sealing with neutral resin, take photos. The images will be used for histological observation and analysis.

[0048] The results of HE staining and aniline blue staining of fresh pig rib cartilage are shown in the figure Figure 1 As shown, HE staining showed that a large number of cell nuclei existed in the cartilage lacunae, and aniline blue staining showed that fresh pig rib cartilage contained rich collagen matrix.

[0049] Example 2 Preparation and Histological Staining of Costal Cartilage Acellular Matrix

[0050] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100 U / ml) and RNase (1 mg / ml) for 24 hours.

[0051] The steps for preparing the costal cartilage acellular matrix are as follows:

[0052] (1) Prepare cartilage slices in the same manner as (1) and (2) in Example 1;

[0053] (2) Place the cartilage slices in a centrifuge tube, add pure water to cover the sample, and place the centrifuge tube in liquid nitrogen for more than 5-10 minutes until the liquid is completely frozen; then, place the centrifuge tube in a 37°C water bath and thaw for more than 10-20 minutes until the ice is completely melted. Repeat the freeze-thaw cycle 5 times;

[0054] (3) Place the frozen and thawed cartilage slices in a 1% (mass fraction) sodium borohydride solution, the volume of the solution being 50-100 times the volume of the cartilage slices. Immerse the cartilage slices in the sodium borohydride solution for 12 hours, then replace with a fresh sodium borohydride solution and continue immersing for 12 hours. The cartilage slices are immersed in the 1% sodium borohydride solution for a total of 24 hours. Afterwards, discard the sodium borohydride solution, use pure water ultrasonic cleaning for 3 times, freeze-dry the cartilage slices for 24 hours, and sterilize them with ultraviolet light for 2 hours.

[0055] (4) After freeze-drying, the cartilage slices were placed in a 2% mass fraction sterile SDS solution for 12 hours. The volume of the SDS solution was 20-50 times that of the cartilage slices. The solution was continuously shaken on a constant temperature shaker at 37°C for 24 hours at a speed of 100-120 rpm.

[0056] (5) Afterwards, wash the cartilage slices with sterile pure water until no obvious foam is generated. Use 20-50 times the volume of pure water for washing, place in a 37°C constant temperature shaker, rotate at 100-120 rpm, change every 3 hours, and wash for 2-3 days until no obvious foam is generated in the washing water;

[0057] (6) After freeze-drying the cartilage slices for 24 hours, immerse them in a mixed solution of DNase (100 U / ml) and RNase (1 mg / ml) for 24 hours. The solution needs to completely submerge the cartilage slices. The solution is continuously shaken on a constant temperature shaker at 37°C for 24 hours at a speed of 100-120 rpm, and washed with sterile pure water three times.

[0058] (7) The decellularized cartilage slices were subjected to tissue dehydration, paraffin embedding, sectioning, HE staining, and aniline blue staining according to (3) to (5) in Example 1.

[0059] (8) Safranin staining: Paraffin sections were dewaxed and hydrated according to Table 2. Afterwards, the sections were stained with bone tissue safranin stain for 2-5 minutes, and then rapidly dehydrated in four cylinders of anhydrous ethanol for 3-5 seconds each time. The sections were transparentized in two cylinders of xylene for 7 minutes each time, and then sealed with neutral gum.

[0060] (9) Alcian blue staining: Paraffin sections were dewaxed and hydrated according to Table 2. Afterwards, the sections were immersed in Alcian blue acidification solution for 3 min, then immersed in Alcian blue staining solution (pH 1.0) for 30 min, rinsed with running water for 5 min, dehydrated with gradient ethanol, transparentized with xylene, and sealed with neutral resin.

[0061] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 2 As shown, HE staining images show that there are almost no cell nuclei in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining images show that there are no obvious cell nuclei in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained; safranin staining images show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining images show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0062] Example 3 Preparation and Histological Staining of Costal Cartilage Acellular Matrix

[0063] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100 U / ml) and RNase (1 mg / ml) for 24 hours.

[0064] The steps for preparing the costal cartilage acellular matrix are as follows:

[0065] The difference from Example 2 is that in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 24 hours.

[0066] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 3 As shown, HE staining images show that there are almost no cell nuclei in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining images show that there are no obvious cell nuclei in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained; safranin staining images show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining images show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0067] Example 4 Preparation and Histological Staining of Costal Cartilage Acellular Matrix

[0068] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 24 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 36 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0069] The steps for preparing the costal cartilage acellular matrix are as follows:

[0070] The difference from Example 2 is that in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 36 hours.

[0071] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 4As shown, HE staining images show that there are almost no cell nuclei in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining images show that there are no obvious cell nuclei in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained; safranin staining images show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining images show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0072] Determination of residual DNA content

[0073] Take 20 mg of tissue from each of the decellularized cartilage group (Example 2, Example 3 and Example 4) and the fresh rib cartilage tissue group (Example 1), cut into pieces, add 200 ul of buffer GA, shake and mix, add 20 ul of ProteinaseK solution, place in a 56°C oven to dissolve for 2 hours, and then briefly centrifuge. Add 200 ul of buffer GB, mix, place at 70°C for 10 minutes, and briefly centrifuge. Add 200 ul of ethanol, mix and shake for 15 seconds. Add the resulting solution to the adsorption column CB3, place the adsorption column in a collection tube, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid, and return the adsorption column CB3 to the collection tube. Add 500 ul of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid, and return the adsorption column CB3 to the collection tube. Add 600 ul of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and repeat once. Place at room temperature for 4 minutes, transfer the adsorption column to a new centrifuge tube, add 200ul elution buffer TE, place at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, repeat once to improve the DNA recovery rate. Use a spectrophotometer to measure the DNA content.

[0074] The quantitative analysis of DNA content in the tissue of decellularized rib cartilage of Example 1 (fresh pig rib cartilage), Example 2, Example 3 and Example 4 (n=3 or 4) is shown in the bar graph. Figure 5 As shown, compared with Example 1, the DNA content in the tissues of Example 2, Example 3 and Example 4 was significantly reduced after decellularization, reaching the application standard of decellularized biomaterials (<50 ng / mg).

[0075] Comparative Example 1

[0076] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100 U / ml) and RNase (1 mg / ml) for 24 hours.

[0077] The steps for preparing the costal cartilage acellular matrix are as follows:

[0078] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 12 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 12 hours.

[0079] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 6 As shown, HE staining pictures show that some cell nuclei remain in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining pictures show that a small number of cell nuclei are found in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained in the extracellular matrix; safranin staining pictures show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining pictures show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0080] Comparative Example 2

[0081] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0082] The steps for preparing the costal cartilage acellular matrix are as follows:

[0083] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 12 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 24 hours.

[0084] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 7 As shown, HE staining pictures show that some cell nuclei remain in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining pictures show that a small number of cell nuclei are found in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained in the extracellular matrix; safranin staining pictures show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining pictures show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0085] Comparative Example 3

[0086] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 12 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 36 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0087] The steps for preparing the costal cartilage acellular matrix are as follows:

[0088] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 12 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 36 hours.

[0089] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Figure 8 As shown, HE staining pictures show that some cell nuclei remain in the cartilage lacunas and the extracellular matrix is ​​rich; aniline blue staining pictures show that a small number of cell nuclei are found in the decellularized cartilage matrix and a large amount of collagen matrix is ​​retained in the extracellular matrix; safranin staining pictures show that the decellularized cartilage matrix contains proteoglycans; Alcian blue staining pictures show that the decellularized cartilage matrix contains acidic mucopolysaccharides.

[0090] Comparative Example 4

[0091] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 6 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0092] The steps for preparing the costal cartilage acellular matrix are as follows:

[0093] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 36 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 6 hours.

[0094] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Fig. 9 As shown, the HE staining pictures show that some cell nuclei remain in the cartilage lacunas, the extracellular matrix is ​​rich, and the thickness of the cartilage sheet is thinner than that of Example 2, Example 3, and Example 4; the aniline blue staining pictures show that the collagen matrix retained in the extracellular matrix is ​​reduced compared with that of Example 2, Example 3, and Example 4; the safranin staining pictures show that the proteoglycans retained in the extracellular matrix are reduced compared with that of Example 2, Example 3, and Example 4; the Alcian blue staining pictures show that the acidic mucopolysaccharides retained in the extracellular matrix are reduced compared with that of Example 2, Example 3, and Example 4.

[0095] Comparative Example 5

[0096] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 12 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0097] The steps for preparing the costal cartilage acellular matrix are as follows:

[0098] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 36 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 12 hours.

[0099] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Fig.10 As shown, the HE staining pictures show that some cell nuclei remain in the cartilage lacunas, the extracellular matrix is ​​rich, and the thickness of the cartilage sheet is thinner than that of Example 2, Example 3, and Example 4; the aniline blue staining pictures show that the collagen matrix retained in the extracellular matrix is ​​reduced compared with that of Example 2, Example 3, and Example 4; the safranin staining pictures show that the proteoglycans retained in the extracellular matrix are reduced compared with that of Example 2, Example 3, and Example 4; the Alcian blue staining pictures show that the acidic mucopolysaccharides retained in the extracellular matrix are reduced compared with that of Example 2, Example 3, and Example 4.

[0100] Comparative Example 6

[0101] Decellularization protocol: freeze-thaw cycles 5 times, immersion in 1% sodium borohydride for 36 hours, immersion in 2% sodium dodecyl sulfate (SDS) for 24 hours, and immersion in a mixed solution of DNase (100U / ml) and RNase (1mg / ml) for 24 hours.

[0102] The steps for preparing the costal cartilage acellular matrix are as follows:

[0103] The difference from Example 2 is that in step (3), the cartilage slices are immersed in the sodium borohydride solution for 36 hours, and in step (4), the freeze-dried cartilage slices are placed in a 2% by mass sterile SDS solution for 24 hours.

[0104] The histological staining results of the decellularized matrix of the rib cartilage are shown in the figure Fig.11 As shown, the HE staining pictures show that some cell nuclei remain in the cartilage lacunas and the extracellular matrix is ​​rich. Compared with Example 2, Example 3, and Example 4, the thickness of the cartilage sheet is significantly thinner and smaller; the aniline blue staining pictures show that compared with Example 2, Example 3, and Example 4, the collagen matrix retained in the extracellular matrix is ​​significantly reduced; the safranin staining pictures show that compared with Example 2, Example 3, and Example 4, the proteoglycans retained in the extracellular matrix are significantly reduced; the Alcian blue staining pictures show that compared with Example 2, Example 3, and Example 4, the acidic mucopolysaccharides retained in the extracellular matrix are significantly reduced.

Claims

1. A method for decellularization of pig rib cartilage, comprising the following steps: (1) Freeze and thaw the pig rib cartilage 5 times; (2) soaking the frozen-thawed pig rib cartilage in step (1) in a sodium borohydride solution; (3) soaking the pig rib cartilage treated with sodium borohydride in step (2) in an SDS solution; (4) enzymatically hydrolyzing the pig rib cartilage obtained in step (3) using a mixed solution of DNase and RNase; The mass concentration of the SDS solution is 1-3%, and the soaking time of the SDS solution is 12-36 hours; The concentration of the DNase is 100 U / ml, the concentration of the RNase is 1 mg / ml, and the enzymolysis time is 24 h; The mass concentration of the sodium borohydride solution is 1%, and the immersion time of the sodium borohydride solution is 12 hours as one cycle, and the immersion is 2 cycles, and fresh sodium borohydride solution is replaced in each immersion cycle, and the cumulative immersion time is 24 hours; The thickness of the pig rib cartilage is 300 microns.

2. The method for decellularization of pig rib cartilage according to claim 1, characterized in that: The mass concentration of the SDS solution is 2%, and the immersion time of the SDS solution is 24 hours.

3. The method for decellularization of pig rib cartilage according to claim 1, characterized in that: The volume of the sodium borohydride solution is 50-100 times the volume of the pig rib cartilage slices.

4. The method for decellularization of pig rib cartilage according to claim 1, characterized in that: The volume of the SDS solution is 20-50 times that of the pig rib cartilage slices.

5. The method for decellularization of pig rib cartilage according to claim 1, characterized in that: The freeze-thaw process is to immerse the pig rib cartilage in water, freeze it in liquid nitrogen for more than 5-10 minutes until the liquid is completely frozen, and then thaw it in a 37° C. water bath for more than 10-20 minutes until the ice is completely melted.

Images