A method for quantitatively detecting the amount of residual DNA and the amount of glycosaminoglycan remaining in a cartilage tissue engineering sample

Abstract

This invention relates to a method for quantitatively detecting residual DNA and glycosaminoglycan (GAG) levels in cartilage tissue-engineered samples. The method involves enzymatically digesting decellularized cartilage matrix samples using a combination enzyme, wherein the combination enzyme consists of papain and bromelain, with a mass ratio of papain to bromelain of 1-3:1. The digested sample is then centrifuged and ultrafiltered to obtain a test liquid containing DNA and GAG. DNA and GAG detection are then performed on the test liquid containing DNA and GAG, respectively. This invention, through the selection of enzyme types, allows the digested sample to be used for both DNA and GAG detection, improving detection efficiency and reducing the amount of tissue-engineered sample required, thus lowering sample usage costs.

Description

Technical Field

[0001] This invention relates to a method for quantitatively detecting the amount of residual DNA and glycosaminoglycan (GAG) in cartilage tissue engineering samples. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the field of cartilage tissue engineering, the extracellular matrix obtained after decellularizing cartilage has been incorporated into research on cartilage injury repair. DNA residue is a key indicator of the completeness of cartilage decellularization and whether the product quality meets requirements. Cartilage tissue is relatively dense, and maximizing the retention of effective components such as GAGs while achieving complete decellularization often requires extensive experimentation with different decellularization methods. Rapid and simple quantitative detection of DNA residue and GAG retention is crucial for optimizing processes and improving product quality.

[0004] Currently, there are two main methods for detecting residual DNA in the industry: First, DNA is extracted and purified from the sample using a specialized extraction kit, followed by primer-mediated amplification for detection. Second, DNA is extracted using an extraction kit, and then the specific binding of fluorescent dyes to the DNA is used for quantitative fluorescence detection. However, both methods have significant technical limitations: they rely on specialized extraction kits, increasing testing costs and requiring cumbersome manual procedures. These methods demand high levels of expertise and adherence to standardized operating procedures, and even minor deviations can affect the accuracy of results. For personnel without relevant experience, errors are easily made during DNA extraction, leading to insufficient DNA recovery rates and impacting the reliability of results. This also hinders the rapid popularization and widespread application of these methods, making them unsuitable for efficient and convenient testing, especially for rapid on-site testing in grassroots laboratories. Meanwhile, the most widely used existing method for GAG detection is to first enzymatically digest the sample to fully release the GAG, then specifically react the digested sample with 1,9-dimethylmethylene blue (DMMB), and finally detect the absorbance of the reaction product using a spectrophotometer to achieve quantitative analysis of GAG.

[0005] Furthermore, the synergistic defects of the two detection methods are more pronounced in the context of cartilage tissue engineering sample testing: the enzymes in the DNA kits used for DNA testing have low enzymatic digestion efficiency in cartilage tissue engineering samples, resulting in incomplete release of GAG components and making accurate detection of GAG in the sample difficult; while the enzymes used for GAG detection have high enzymatic digestion efficiency in cartilage tissue engineering samples, the detection process does not involve DNA purification, thus hindering accurate detection of DNA in the sample. More importantly, the two detection methods cannot simultaneously detect DNA and GAG. If two detection methods are used to detect DNA and GAG in the sample separately, it will lead to an increase in operational steps, making the detection process cumbersome and significantly reducing the detection efficiency of the two components in the sample, failing to meet the actual needs of efficient detection in the field of cartilage tissue engineering. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for quantitatively detecting residual DNA and glycosaminoglycan (GAG) content in cartilage tissue engineering samples. By selecting appropriate enzymes, the present invention enables rapid enzymatic digestion of proteins in cartilage tissue engineering samples, making the digested samples suitable for both DNA and GAG detection. This not only improves detection efficiency but also reduces the amount of tissue engineering samples used, thereby lowering sample usage costs.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, an enzymatic digestion method for decellularized cartilage matrix samples includes the following steps: A combination enzyme was used to enzymatically digest decellularized cartilage matrix samples; wherein the combination enzyme consisted of papain and bromelain, and the mass ratio of papain to bromelain was 1~3:1.

[0008] This invention, through experiments using various enzymes to enzymatically digest decellularized cartilage matrix samples, found that papain and bromelain have higher enzymatic digestion efficiency compared to existing DNA extraction kits; however, the digestion still requires 1 hour, resulting in relatively low detection efficiency. Further experiments unexpectedly revealed that when papain and bromelain are combined in a mass ratio of 1-3:1 to form a combined enzyme, the digestion efficiency is significantly improved, and the digestion time can be reduced to as low as half an hour.

[0009] Secondly, a method for quantitatively detecting residual DNA and glycosaminoglycans in engineered cartilage tissue samples includes the following steps: The enzymatically hydrolyzed sample was obtained using the enzymatic hydrolysis method described in the first aspect of the present invention; The enzymatically digested sample was centrifuged and ultrafiltered to obtain the test liquid containing DNA and glycosaminoglycans; DNA and glycosaminoglycans were detected separately in the test liquid containing DNA and glycosaminoglycans, respectively.

[0010] After enzymatic digestion, large protein molecules are converted into small peptides, releasing residual DNA and a large amount of GAG from the tissue-engineered cartilage. Under the action of centrifugation and ultrafiltration, DNA can be retained by the ultrafiltration membrane, while GAG, small peptides and protease are centrifuged to the lower collection tube. The DNA retained by the ultrafiltration membrane can be further washed with TE buffer solution to obtain the test liquid containing DNA, thereby achieving the purpose of simultaneously detecting DNA and GAG in one enzymatic digestion of the sample.

[0011] Thirdly, a kit for quantitatively detecting residual DNA and glycosaminoglycans in engineered cartilage tissue samples includes: The combined enzyme, composed of papain and bromelain in a mass ratio of 1 to 3:1, is used for enzymatic hydrolysis of decellularized cartilage matrix samples. Combination enzyme solvent, used to dissolve combination enzyme solutions; Ultrafiltration centrifuge tubes are used to centrifuge and ultrafilter enzymatically digested samples. TE buffer is used to rinse the ultrafiltration membrane after centrifugation and ultrafiltration to obtain the test liquid containing DNA. DNA detection dyes are used to stain and detect DNA-containing test liquids after centrifugation and ultrafiltration. Glycosaminoglycan detection dyes are used to stain and detect test liquids containing glycosaminoglycans after centrifugation and ultrafiltration.

[0012] The beneficial effects of this invention are as follows: (1) The present invention can significantly improve detection efficiency and versatility. Compared with the enzymatic digestion method of DNA extraction kits, the present invention uses a combined enzymatic digestion system composed of papain and bromelain, which can greatly shorten the digestion time and achieve efficient and rapid digestion of decellularized cartilage matrix.

[0013] (2) This invention greatly simplifies the extraction process and reduces the technical threshold and operational errors. This invention uses a combination enzyme formed by papain and bromelain for rapid enzymatic digestion, combined with centrifugation and ultrafiltration to replace the complex purification steps of traditional DNA extraction kits. This not only enables rapid separation of GAG and DNA from the sample, but also yields DNA with high purity and recovery rate, significantly reducing operational steps and simplifying the process. Moreover, it requires less technical background from the operators, and even those without a molecular biology experimental background can easily complete the process. This effectively avoids the problem of unstable DNA recovery rate caused by complex operations, and improves the reliability and repeatability of the detection.

[0014] (3) The present invention can significantly improve the versatility of the test samples. The present invention uses a combination enzyme formed by papain and bromelain for rapid enzymatic digestion. The same sample after enzymatic digestion can be used simultaneously for the detection of two key indicators: DNA residue and GAG retention. This completely changes the situation in the traditional method where the two tests need to be performed independently and the samples cannot be used interchangeably. This greatly improves the detection efficiency, saves the amount of raw materials used for cartilage tissue engineering samples, and increases the utilization rate of the test samples. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The following is a diagram of the state of the sample after 0h of enzymatic hydrolysis in Example 1 of the present invention: (1) enzymatic hydrolysis was performed according to step (1); (2) enzymatic hydrolysis was performed according to step (2); (3) enzymatic hydrolysis was performed according to step (3); (4) enzymatic hydrolysis was performed according to step (4); (5) enzymatic hydrolysis was performed according to step (5); and (6) enzymatic hydrolysis was performed according to step (6). Figure 2 The following is a diagram of the state of the sample after 1 hour of enzymatic hydrolysis in Example 1 of the present invention: (1) enzymatic hydrolysis was performed according to step 2 (1); (2) enzymatic hydrolysis was performed according to step 2 (2); (3) enzymatic hydrolysis was performed according to step 2 (3); (4) enzymatic hydrolysis was performed according to step 2 (4); (5) enzymatic hydrolysis was performed according to step 2 (5); and (6) enzymatic hydrolysis was performed according to step 2 (6). Figure 3 The following is a diagram of the state of the sample after 2 hours of enzymatic hydrolysis in Example 1 of the present invention: (1) enzymatic hydrolysis was performed according to step 2 (1); (2) enzymatic hydrolysis was performed according to step 2 (2); (3) enzymatic hydrolysis was performed according to step 2 (3); (4) enzymatic hydrolysis was performed according to step 2 (4); (5) enzymatic hydrolysis was performed according to step 2 (5); and (6) enzymatic hydrolysis was performed according to step 2 (6). Figure 4 The following is a sample state diagram after 0.5h of enzymatic hydrolysis in Example 2 of the present invention: (1) shows enzymatic hydrolysis performed according to step (1) of step 2; (2) shows enzymatic hydrolysis performed according to step (2) of step 2. Figure 5 The DNA standard curve constructed in Example 3 of this invention; Figure 6 The GAG ​​standard curve constructed for Embodiment 3 of the present invention; Figure 7 These are agarose gel electrophoresis images of DNA before and after purification in Example 7 of this invention. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] Given that existing technologies require separate detection of residual DNA and GAG retention in decellularized cartilage matrix, resulting in low extraction efficiency, complex procedures, high costs, and low sample utilization, this invention proposes a method for quantitatively detecting residual DNA and GAG retention in cartilage tissue engineering samples to address these technical problems.

[0020] A typical embodiment of the present invention provides an enzymatic digestion method for decellularized cartilage matrix samples, comprising the following steps: A combination enzyme was used to enzymatically digest decellularized cartilage matrix samples; wherein the combination enzyme consisted of papain and bromelain, and the mass ratio of papain to bromelain was 1~3:1.

[0021] In some embodiments, the decellularized cartilage matrix sample is in powder form. Processing the powdered decellularized cartilage matrix sample improves the contact efficiency between the sample and the combined enzyme, thereby further enhancing its enzymatic hydrolysis efficiency. Specifically, the particle size of the decellularized cartilage matrix sample is no higher than 30 nm.

[0022] Specifically, the cartilage particles were decellularized, washed, and freeze-dried. The freeze-dried cartilage particles were then pulverized to obtain a powdered decellularized cartilage matrix sample. More specifically, the cartilage particles were decellularized using Triton X-100 solution. The mass concentration of the Triton X-100 solution was 2.5–3.5%.

[0023] In some embodiments, the mass ratio of the combined enzyme to the decellularized cartilage matrix sample is 2 to 5:5, preferably 2:5.

[0024] In some embodiments, the combined enzyme is prepared into a combined enzyme solution and added to a decellularized cartilage matrix sample for enzymatic digestion. Specifically, the solvent for preparing the combined enzyme solution is a cysteine-containing PBS solution (phosphate buffered solution). More specifically, the pH of the solvent for preparing the combined enzyme solution is 6.5–7.5. Specifically, the total mass concentration of the enzyme in the combined enzyme solution is 1–3 mg / mL.

[0025] In some embodiments, the temperature during enzymatic hydrolysis is 60-65°C, and the hydrolysis time is 25-35 min.

[0026] Another embodiment of the present invention provides a method for quantitatively detecting residual DNA and glycosaminoglycan retention in cartilage tissue engineering samples, comprising the following steps: The enzymatically hydrolyzed sample was obtained using the above enzymatic hydrolysis method; The enzymatically digested sample was centrifuged and ultrafiltered to obtain the test liquid containing DNA and glycosaminoglycans; DNA and glycosaminoglycans were detected separately in the test liquid containing DNA and glycosaminoglycans, respectively.

[0027] In some embodiments, during the centrifugal ultrafiltration process, the rotation speed is 5000~8000 rpm and the time is 5~10 min.

[0028] In some embodiments, during the centrifugal ultrafiltration process, the collected lower ultrafiltrate is used as the test liquid containing glycosaminoglycans; the ultrafiltration membrane after centrifugation and ultrafiltration is rinsed at least twice with TE buffer, and the lower washing liquid is combined as the test liquid containing DNA.

[0029] In some embodiments, when detecting DNA, a DNA dye is added to the test liquid, followed by optical detection. Specifically, the DNA dye is Goldview type II nucleic acid dye or Hoechst 33258 fluorescent dye. Goldview type II nucleic acid dye has good stability, low cost, and relatively flexible storage time and conditions; storage at 4°C is sufficient. Currently, DNA dyes are generally fluorescent dyes; therefore, the optical detection used for DNA detection is fluorescence detection.

[0030] In some embodiments, when detecting glycosaminoglycans, a glycosaminoglycan dye is added to the test liquid, followed by optical detection. Specifically, the glycosaminoglycan dye is DMMB. When using DMMB as the dye to detect glycosaminoglycans, a spectrophotometer is used for detection.

[0031] In some embodiments, the step of establishing a DNA standard curve is also included: DNA standard solutions of different concentrations were prepared, DNA dye was added to the DNA standard solutions of different concentrations, optical detection was performed, and a DNA standard curve was established based on the optical intensity.

[0032] Specifically, DNA standard solutions were prepared using TE buffer. Specifically, the DNA dye used in establishing the DNA standard curve was the same as the DNA dye used to treat the test solution.

[0033] In some embodiments, the step of establishing a glycosaminoglycan standard curve is also included: Chondroitin sulfate standard solutions of different concentrations were prepared, and glycosaminoglycan dyes were added to the chondroitin sulfate standard solutions of different concentrations. Optical detection was performed, and a glycosaminoglycan standard curve was established based on the optical intensity.

[0034] Specifically, a chondroitin sulfate standard solution was prepared using a combination enzyme solution. Specifically, the glycosaminoglycan dye used in establishing the glycosaminoglycan standard curve was the same as the glycosaminoglycan dye used to treat the test liquid.

[0035] The preferred steps of the method are as follows: (1) Sample enzymatic digestion The cartilage tissue sample was digested using a combination of papain and bromelain (in a mass ratio of 3 to 1:1, with a final enzyme concentration of 1 to 3 mg / mL, dissolved in PBS solution containing cysteine ​​at pH 6.5 to 7.5) to ensure complete digestion. (2) Sample purification After enzymatic digestion, the sample is transferred to an ultrafiltration tube (30kD~50kD) for centrifugation purification (centrifugation conditions: 5000~8000rpm; 5min~10min). After enzymatic digestion, large protein molecules are converted into small peptide molecules, releasing residual DNA and a large amount of GAG from the tissue-engineered cartilage. Under the action of the ultrafiltration tube, DNA can be retained by the ultrafiltration membrane, while GAG, small peptide molecules and protease are centrifuged to the lower collection tube to obtain the test liquid containing GAG. Replace the lower collection tube and rinse the ultrafiltration membrane three times with slightly warmed TE buffer (quantitative rinsing). Collect and mix the lower rinsing solution to obtain the DNA-containing test liquid for subsequent quantitative analysis.

[0036] (3) Establishment of standard curve Establishment of DNA Standard Curve Using standard DNA as a known concentration of standard control, a standard curve was established after dilution with TE buffer. For example, the concentrations were diluted to 0 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL. 100 μL of the sample at each of these concentrations was added to 100 μL of 1x Goldview II nucleic acid dye, incubated in the dark for 5 min, and the fluorescence value was detected on a fluorometer. A standard curve was constructed with concentration on the x-axis and fluorescence value on the y-axis. The curve R... 2 It should be greater than 0.99; Establishment of GAG Standard Curve A series of chondroitin 6-sulfate solutions of 0 µg / mL, 3.125 µg / mL, 6.25 µg / mL, 12.5 µg / mL, 25 µg / mL, 50 µg / mL, and 100 µg / mL were prepared using the extraction solution (a combination enzyme solution of papain and bromelain). 0.1 mL of the standard was added to 1 mL of DMMB solution, mixed well, and measured using the 530 nm cuvette method. (4) Sample testing DNA testing Take the purified sample and add 1x Goldview Type II nucleic acid dye (diluted 10,000 times) at a volume ratio of 1:1. After incubation in the dark, detect the fluorescence value on a fluorometer or ELISA reader. Ensure that the fluorescence value falls within the standard curve range. Use the purified group without sample as a blank sample and subtract the fluorescence background value. GAG testing Add 0.1 mL of the purified sample to 2 mL of DMMB solution, mix well, and measure using the 530 nm cuvette method.

[0037] A third embodiment of the present invention provides a kit for quantitatively detecting residual DNA and glycosaminoglycan retention in engineered cartilage tissue samples, comprising: The combined enzyme, composed of papain and bromelain in a mass ratio of 1 to 3:1, is used for enzymatic hydrolysis of decellularized cartilage matrix samples. Combination enzyme solvent, used to dissolve combination enzyme solutions; Ultrafiltration centrifuge tubes are used to centrifuge and ultrafilter enzymatically digested samples. TE buffer is used to rinse the ultrafiltration membrane after centrifugation and ultrafiltration to obtain the test liquid containing DNA. DNA detection dyes are used to stain and detect DNA-containing test liquids after centrifugation and ultrafiltration. Glycosaminoglycan detection dyes are used to stain and detect test liquids containing glycosaminoglycans after centrifugation and ultrafiltration.

[0038] In some embodiments, DNA standards are also included for use with DNA detection dyes and TE buffer to construct a DNA standard curve.

[0039] In some embodiments, the system further includes glycosaminoglycan standards for use in conjunction with glycosaminoglycan detection dyes and combination enzyme solutions to construct a glycosaminoglycan standard curve. Specifically, the glycosaminoglycan standards may be chondroitin sulfate standards, such as chondroitin 6-sulfate standards.

[0040] In some embodiments, the enzyme solvent is a PBS solution containing cysteine. Specifically, the pH of the enzyme solvent is 6.5–7.5.

[0041] In some embodiments, the DNA detection dye is Goldview type II nucleic acid dye or Hoechst 33258 fluorescent dye, preferably Goldview type II nucleic acid dye.

[0042] In some embodiments, the glycosaminoglycan detection dye is DMMB.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0044] Example 1 Enzyme Selection Step 1: Preparation of cartilage tissue engineering samples Cartilage particles were decellularized with 3% Triton X-100. After washing the decellularized sample, it was freeze-dried. The freeze-dried cartilage particles were then pulverized to a particle size of less than 30 nm to obtain cartilage matrix powder material.

[0045] Step 2: Sample enzymatic digestion: (1) 2 mg / mL papain (solvent is PBS solution containing cysteine ​​at pH 7.4) was enzymatically hydrolyzed at 60 °C.

[0046] (2) 2 mg / mL bromelain (solvent is PBS solution containing cysteine ​​at pH 7.4), enzymatically hydrolyzed at 60°C.

[0047] (3) 2 mg / mL type II collagenase (solvent is PBS solution with pH 7.4), enzymatic hydrolysis was performed at 37°C.

[0048] (4) The enzymatic digestion solution provided with the DNA extraction kit is used for enzymatic digestion at 65°C.

[0049] (5) 2 mg / mL trypsin (solvent is PBS solution with pH 7.4), enzymatic hydrolysis was carried out at 37°C.

[0050] (6) 2 mg / mL pepsin (solvent is hydrochloric acid aqueous solution with pH 2), enzymatic hydrolysis at 37°C.

[0051] Accurately weigh 5.0-5.1 mg of cartilage matrix powder and place it in 1 mL of the above 6 enzyme solutions. Observe the enzymatic hydrolysis status of the sample every 0.5 h.

[0052] The results are as follows Figures 1-3 As shown, both papain and bromelain exhibited faster enzymatic hydrolysis efficiency than the DNA extraction kit, with both becoming clear and transparent after approximately 1 hour of enzymatic hydrolysis. In contrast, the DNA extraction kit still showed suspended matter in the solution after 1 hour of enzymatic hydrolysis, and the solution only became clear and transparent after 2 hours of enzymatic hydrolysis.

[0053] Example 2 Combined enzymatic hydrolysis of papain and bromelain Step 1: Preparation of cartilage tissue engineering samples Cartilage particles were decellularized with 3% Triton X-100. After washing the decellularized sample, it was freeze-dried. The freeze-dried cartilage particles were then pulverized to a particle size of less than 30 nm to obtain cartilage matrix powder material.

[0054] Step 2: Sample enzymatic digestion: (1) 2 mg / mL of combined enzyme (papain and bromelain in a mass ratio of 3:1, in PBS solution containing cysteine ​​at pH 7.4) was used for enzymatic hydrolysis at 60°C.

[0055] (2) 2 mg / mL of combined enzyme (papain and bromelain in a mass ratio of 1:1, in PBS solution containing cysteine ​​at pH 7.4) was used for enzymatic hydrolysis at 60°C.

[0056] Accurately weigh 5.0-5.1 mg of cartilage matrix powder and place it in 1 mL of the above two enzyme solutions. Observe the enzymatic hydrolysis status of the sample after 0.5 h.

[0057] The results are as follows Figure 4 As shown, the combined enzyme can achieve complete enzymatic hydrolysis after 0.5 hours, and the solution is clear and transparent. It can be seen that the combined enzyme can effectively improve the enzymatic hydrolysis efficiency compared with the single enzyme.

[0058] Example 3: Establishment of the Standard Curve I. Establishment of DNA Standard Curve A standard curve was established using λDNA as a known concentration standard. Dilutes were made to 0 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL. 100 μL of each sample at these concentrations was added to 100 μL of 1x Goldview II nucleic acid dye (diluted 10000 times), and incubated in the dark for 5 min. Fluorescence values ​​were then measured using a fluorometer. A standard curve was constructed with concentration on the x-axis and fluorescence value on the y-axis. The curve R0 was calculated. 2 It should be greater than 0.99. The established DNA standard curve is as follows: Figure 5 As shown.

[0059] II. Establishment of the GAG ​​Standard Curve A series of chondroitin 6-sulfate solutions were prepared using a papain and bromelain combinatorial enzyme extract (mass ratio 1:1) at concentrations of 0 µg / mL, 3.125 µg / mL, 6.25 µg / mL, 12.5 µg / mL, 25 µg / mL, 50 µg / mL, and 100 µg / mL. 0.1 mL of the extracted sample or standard was added to 1 mL of DMMB solution, mixed thoroughly, and the absorbance at 530 nm (525 nm–535 nm) was measured immediately (within 3 minutes of mixing) using a spectrophotometer via the cuvette method. The established GAG standard curve is shown below. Figure 6 As shown.

[0060] Example 4 A method for quantitatively detecting residual DNA and glycosaminoglycan retention in engineered cartilage tissue samples, comprising the following steps: S1. Decellularize cartilage particles with 3% Triton X-100. After washing the decellularized sample, freeze-dry it. Then, pulverize the freeze-dried cartilage particles to a particle size of less than 30 nm to obtain cartilage matrix powder material.

[0061] S2. Papain and bromelain were combined in a mass ratio of 1:1 and added to a cysteine-containing PBS solution at pH 7.4 to prepare an enzyme solution with a total enzyme concentration of 2 mg / mL. 5.0-5.1 mg of cartilage matrix powder was placed in the enzyme solution and enzymatically digested at 60°C for 0.5 h until the sample was completely digested.

[0062] S3. Add the enzymatically digested sample obtained in step S2 to a micro ultrafiltration centrifuge tube with a molecular weight cutoff of 30kD. Centrifuge at 5000rpm for 5min and take out the lower layer liquid as the GAG ​​test sample. Replace with a new clean lower layer collection tube, rinse the ultrafiltration membrane with 0.5mL TE buffer (preheated at 56℃), let it stand naturally, and then take out the liquid in the ultrafiltration membrane. Repeat 3 times, combine the liquids, and use it as the DNA test sample.

[0063] S4. Add 1x Goldview Type II nucleic acid dye (diluted 10,000 times) to the DNA sample to be tested at a volume ratio of 1:1. After incubation in the dark, detect the fluorescence value on a fluorescence microplate reader. Use the DNA standard curve established in Example 3 to obtain the concentration of the DNA sample to be tested at this fluorescence value.

[0064] S5. Add 0.1 mL of the GAG ​​test sample to 2 mL of DMMB solution, mix well, and measure using the 530 nm cuvette method. Obtain the concentration of the GAG ​​test sample at this fluorescence value using the GAG ​​standard curve established in Example 3.

[0065] The test results are shown in Table 1.

[0066] Table 1. Detection results of this embodiment

[0067] Example 5 This embodiment is the same as Embodiment 4, except that in step S3, the centrifugation speed is 5000 rpm. The test results are shown in Table 2.

[0068] Table 2. Detection results of this embodiment

[0069] Analysis of the results in Table 1 of Example 4 and Table 2 of this example shows that there is basically no statistical difference in the detection results at centrifugation speeds of 5000 rpm and 8000 rpm.

[0070] Example 6 This embodiment is the same as Embodiment 4, except that in step S3, the centrifugation time is 10 minutes. The test results are shown in Table 3.

[0071] Table 3. Detection results of this embodiment

[0072] As can be seen from the results in Table 1 of Example 4 and Table 3 of this Example, there is basically no statistical difference in the detection results for centrifugation times of 5 min and 10 min.

[0073] Example 7 Recovery rate verification Steps S1-S3 were repeated according to Example 4. Because the molecular weight of the standard λDNA was too large, agarose gel electrophoresis was used to verify the amount of DNA before and after sample purification.

[0074] The results are as follows Figure 7 As shown in the electrophoresis diagram, the brightness of the sample before and after purification is basically the same, which proves that the DNA recovery rate is high. Combined with the grayscale analysis of ImageJ software, as shown in Table 4, the DNA recovery rate reached 95.86%, which meets the relevant standard requirements, indicating that the ultrafiltration centrifugation purification step does not affect the DNA content in the sample.

[0075] Table 4 Gray-scale analysis results

[0076] Example 8 This embodiment is the same as Embodiment 4, except that the 1x Goldview type II nucleic acid dye in step S4 is replaced with Hoechst 33258 fluorescent dye.

[0077] The test results are shown in Table 5.

[0078] Table 5 Comparison of detection results for different DNA dyes

[0079] Note: The data for Example 4 were obtained by repeating Example 4 three times, and the data for Example 8 were obtained by repeating Example 8 three times.

[0080] Table 5 shows that there is no difference in fluorescence values ​​between the two, but compared with Hoechst 33258 fluorescent dye, Goldview type II nucleic acid dye has better stability and more lenient storage time and conditions.

[0081] Comparative Example 1: DNA kit for sample extraction and purification Follow the instructions of the corresponding kit as follows: Take 5.0-5.1 mg of cartilage tissue sample, add the enzyme digestion solution in the kit, and digest at 65°C until no visible particles are visible. Add lysis buffer and incubate at 75°C for 15 min; add isopropanol and vortex to mix, then add an appropriate amount of magnetic beads and vortex to mix; allow the magnetic beads to adhere to the magnetic rack, discard the liquid, add buffer to wash the magnetic beads, and then elute the DNA (eluting it from the magnetic beads) with the elution buffer in the kit, 50 µL each time, repeating the elution 3 times.

[0082] The test results are shown in Table 6.

[0083] Comparative Example 2 This comparative example is the same as Example 4, except that step S3 is omitted. Instead, 1x Goldview Type II nucleic acid dye is added directly to the enzymatically digested sample at a volume ratio of 1:1. After incubation in the dark, the fluorescence value is detected on a fluorescence microplate reader.

[0084] The test results are shown in Table 6.

[0085] Table 6 Comparison of DNA content detection values ​​using different methods

[0086] Note: The data of this invention group were obtained by repeating Example 4 three times, the data of Comparative Example 1 group were obtained by repeating Comparative Example 1 three times, and the data of Comparative Example 2 group were obtained by repeating Comparative Example 2 three times.

[0087] Table 6 shows that the fluorescence values ​​of samples purified using micro-ultrafiltration centrifuge tubes were slightly higher than those measured after extraction and purification using DNA kits, but there was no statistically significant difference between the two. Samples that did not undergo purification had high fluorescence values ​​and poor parallelism due to the interference of a large number of enzyme components.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enzymatic digestion method for decellularized cartilage matrix samples, characterized in that, Includes the following steps: A combination enzyme was used to enzymatically digest decellularized cartilage matrix samples; wherein the combination enzyme consisted of papain and bromelain, and the mass ratio of papain to bromelain was 1~3:

1.

2. The enzymatic hydrolysis method as described in claim 1, characterized in that, The mass ratio of the combined enzyme to the decellularized cartilage matrix sample was 2-5:

5.

3. The enzymatic hydrolysis method as described in claim 1, characterized in that, During enzymatic hydrolysis, the temperature is 55~65℃.

4. A method for quantitatively detecting residual DNA and glycosaminoglycan retention in engineered cartilage tissue samples, characterized in that, Includes the following steps: The enzymatically hydrolyzed sample was obtained using the enzymatic hydrolysis method described in claims 1-3; The enzymatically digested sample was centrifuged and ultrafiltered to obtain the test liquid containing DNA and glycosaminoglycans; DNA and glycosaminoglycans were detected separately in the test liquid containing DNA and glycosaminoglycans, respectively.

5. The method as described in claim 4, characterized in that, During the centrifugal ultrafiltration process, the rotation speed is 5000~8000 rpm and the time is 5~10 min; Alternatively, during the centrifugation and ultrafiltration process, the collected lower ultrafiltrate is used as the test liquid containing glycosaminoglycans; the ultrafiltration membrane after centrifugation and ultrafiltration is rinsed at least twice with TE buffer, and the lower washing liquid is combined as the test liquid containing DNA.

6. The method as described in claim 4, characterized in that, When detecting DNA, a DNA dye is added to the test liquid, followed by optical detection; or, the DNA dye is Goldview type II nucleic acid dye or Hoechst 33258 fluorescent dye.

7. The method as described in claim 4, characterized in that, When detecting glycosaminoglycans, a glycosaminoglycan dye is added to the liquid to be tested, and then optical detection is performed; or, the glycosaminoglycan dye is DMMB.

8. The method as described in claim 4, characterized in that, It also includes the step of establishing a DNA standard curve: Prepare DNA standard solutions of different concentrations, add DNA dye to the DNA standard solutions of different concentrations, perform optical detection, and establish a DNA standard curve based on the optical intensity. Alternatively, it may also include the step of establishing a glycosaminoglycan standard curve: Chondroitin sulfate standard solutions of different concentrations were prepared, and glycosaminoglycan dyes were added to the chondroitin sulfate standard solutions of different concentrations. Optical detection was performed, and a glycosaminoglycan standard curve was established based on the optical intensity.

9. A kit for quantitatively detecting residual DNA and glycosaminoglycan retention in engineered cartilage tissue samples, characterized in that, include: The combined enzyme, composed of papain and bromelain in a mass ratio of 1 to 3:1, is used for enzymatic hydrolysis of decellularized cartilage matrix samples. Combination enzyme solvent, used to dissolve combination enzyme solutions; Ultrafiltration centrifuge tubes are used to centrifuge and ultrafilter enzymatically digested samples. TE buffer is used to rinse the ultrafiltration membrane after centrifugation and ultrafiltration to obtain the test liquid containing DNA. DNA detection dyes are used to stain and detect DNA-containing test liquids after centrifugation and ultrafiltration. Glycosaminoglycan detection dyes are used to stain and detect test liquids containing glycosaminoglycans after centrifugation and ultrafiltration.

10. The kit according to claim 9, characterized in that, It also includes DNA standards, which are used in conjunction with DNA detection dyes and TE buffer to construct DNA standard curves; Alternatively, it may also include glycosaminoglycan standards, which are used in conjunction with glycosaminoglycan detection dyes and combination enzyme solutions to construct glycosaminoglycan standard curves; Alternatively, the enzyme solvent may be a PBS solution containing cysteine; Alternatively, the pH of the combined enzyme solvent is 6.5–7.5; Alternatively, the DNA detection dye may be Goldview type II nucleic acid dye or Hoechst 33258 fluorescent dye; Alternatively, the glycosaminoglycan detection dye may be DMMB.

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