CN113599577A - A kind of acellular cartilage material from porcine costal cartilage and preparation method and application - Google Patents
A kind of acellular cartilage material from porcine costal cartilage and preparation method and application Download PDFInfo
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- CN113599577A CN113599577A CN202110901794.9A CN202110901794A CN113599577A CN 113599577 A CN113599577 A CN 113599577A CN 202110901794 A CN202110901794 A CN 202110901794A CN 113599577 A CN113599577 A CN 113599577A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The invention relates to a acellular cartilage material from pig costal cartilage and a preparation method and application thereof, wherein the preparation method comprises the step of acellular, phosphate buffer solution containing trypsin and ethylene diamine tetraacetic acid is adopted as acellular solution, the costal cartilage material is added into the acellular solution and is placed into a shaking table, the volume ratio of the acellular solution to the costal cartilage material is 20:1, the shaking table is set to rotate at 150r/min for 72 hours, the temperature is 37 ℃, and the acellular solution is replaced every 24 hours. The acellular cartilage material prepared by the invention basically reserves the structure and function of an extracellular matrix, provides a stable environment for cell proliferation and tissue regeneration, reserves most of collagen fiber components in the extracellular matrix, can induce cell migration and differentiation and tissue regeneration in the defect repair process of costal cartilage, provides mechanical support, and realizes the effect of improving local thoracic cavity depression deformity.
Description
Technical Field
The invention relates to a decellularized cartilage material from pig costal cartilage, a preparation method and application thereof, belonging to the technical field of biological materials.
Background
By virtue of the advantages of no immunological rejection, sufficient quantity, good plasticity, good histocompatibility, stable form and the like, the autologous costal cartilage is widely applied to the field of plastic surgery, such as auricle reconstruction operation and costal cartilage augmentation nasal operation. In the reconstruction operation of auricle reconstruction, 2-3 autologous costal cartilages are required to be carved and spliced into a support in an auricle form, cartilage loss is left after the autologous costal cartilages are adopted, and more obvious abnormal thoracic form may be caused by the loss of a plurality of costal cartilages, such as local chest wall depression, asymmetric bilateral thoracic development and the like, so that the attractiveness is influenced. In order to reduce the local concave deformation of the thorax, the remaining costal cartilage fragments are usually carved and transplanted back to the cartilage collecting area in the operation, and the cartilage membrane is reserved. At present, the operation design scheme is optimized according to the size of an auricle in the operation, so that costal cartilage as little as possible is adopted, the quantity of residual materials in the operation is small, and the residual materials are mostly fragmented cartilage and cannot be completely filled in the costal cartilage defect part. Therefore, the difficulty in repairing the defect by using the residual autologous costal cartilage material in the operation is high, the search for a proper material which can replace the costal cartilage and has sufficient quantity is an important solution, the search for the proper material to be filled in the costal cartilage deletion part and the reconstruction of the local chest wall shape is an important aspect for reducing the occurrence of the thoracic deformity.
At present, the acellular material is an ideal biological material, cell components are removed from the material, extracellular matrix structures, signal molecules and functional proteins are reserved, and the extracellular matrix structures are highly conserved in different species, so that the extracellular matrix from heterogeneous sources can be transplanted and applied, immune rejection is small, and cell adhesion and tissue regeneration induction can be promoted. The acellular material is widely applied to clinical operations, such as joint and bronchial operations, acellular dermal matrix materials from human sources, acellular heart valves from pig sources and the like, and related researches on the application of the acellular cartilage material filling to costal cartilage defect areas do not exist.
The pig-derived biomaterial is one of the best choices for human tissue and organ repair, has sufficient costal cartilage materials and easily obtained materials, and is convenient for subsequent industrial production.
In the preparation process of the acellular cartilage material, the damage effect of different acellular methods on extracellular matrix is fully considered, and the search for a more mild and effective acellular technology is an important aspect for preparing the acellular cartilage material.
Disclosure of Invention
The invention aims to solve the technical problem of providing a decellularized cartilage material from pig costal cartilage and a preparation method and application thereof.
The technical scheme for solving the technical problems is as follows: a method for preparing acellular cartilage material from costal cartilage of pig comprises the following steps: (1) extracting costal cartilage material from costal cartilage of pig; (2) primarily treating costal cartilage materials; (3) inactivating viruses; (4) cleaning for the first time; (5) removing cells; (6) secondary cleaning; (7) cleaning with injection water; (8) sterilizing;
wherein the step (5) is specifically as follows: adopting phosphate buffer solution containing trypsin and disodium ethylene diamine tetraacetate as decellularization solution, adding costal cartilage material into the decellularization solution, and placing into a shaking table, wherein the volume ratio of the decellularization solution to the costal cartilage material is 20:1, the shaking table is set to rotate at a speed of 150r/min, the action is continuously carried out for 72 hours, the temperature is 37 ℃, and the decellularization solution is replaced every 24 hours.
The invention has the beneficial effects that: the invention utilizes trypsin and disodium ethylene diamine tetraacetate to carry out acellular treatment, is a milder and effective acellular method, the obtained acellular cartilage material has small residual DNA content, meets the basic requirement of acellular, can also effectively remove alpha-Gal antigen, obviously reduces the immune rejection risk of later implantation in vivo, and in addition, cell components causing inflammatory reaction in the acellular cartilage matrix are effectively removed, and can inhibit the polarization reaction of macrophages.
Based on the special decellularization step, the decellularized cartilage material prepared by the invention basically reserves the structure and function of an extracellular matrix, provides a stable environment for cell proliferation and tissue regeneration, most of collagen fiber components in the extracellular matrix are reserved, and in the costal cartilage defect repair process, the acellular cartilage material can induce cell migration and differentiation and tissue regeneration, provides mechanical support and realizes the effect of improving local thoracic cavity deformity.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the step (1) is specifically as follows: removing hair of operation area after pig general anesthesia, sterilizing with iodine tincture, removing iodine with alcohol, and spreading sterile towel; after local infiltration anesthesia, a 10cm incision is made on the right side of the lower end of the sternum by using a circular knife, the tangent line is parallel to the costal arch, the skin, the subcutaneous layer and the muscle layer are cut in layers, the 6 th, 7 th and 8 th costal cartilages are exposed, the 3 costal cartilages are completely cut off and placed in normal saline for later use; after sufficient hemostasis, the incision is sutured layer by layer, the incision is disinfected again, the antibiotic ointment is externally applied locally, and the intramuscular injection of penicillin is given for three consecutive days after the operation to prevent infection.
Further, the step (2) is specifically as follows: removing perichondrium and connective tissue from the costal cartilage material obtained in the step (1), cutting into a proper size, and washing with purified water until the surface is free from stains. Wherein suitable size means a diameter of less than 2cm and a thickness of no more than 3 mm.
Further, the step (3) is specifically as follows: treating with a mixed solution of peroxyacetic acid and ethanol, placing costal cartilage material in the mixed solution, wherein the volume ratio of the costal cartilage material to the mixed solution is 1:20, the inactivation time is 4 hours, and the inactivation temperature is 20 ℃.
Furthermore, in the mixed solution of the peroxyacetic acid and the ethanol, the volume ratio of the peroxyacetic acid to the ethanol is 1: 24.
Further, the step (4) and the step (6) have the same operation, specifically: cleaning with phosphate buffer solution with pH of 6-8 at 10-30 deg.C for 15 min at least 3 times until the pH of the cleaned flushing liquid is 6-8 (generally, pH is in accordance with the requirement after 3 times of cleaning, and if pH is not in accordance with the requirement, the flushing is continued), cleaning with purified water at 10-30 deg.C for 15 min at 30:1 for 1 time; the step (7) is specifically as follows: washing with injection water for 4 times, each time for 15 minutes, wherein the volume ratio of the injection water to the costal cartilage material is 30:1, the temperature range is 10-30 ℃; the step (8) is specifically as follows: the solution was sterilized by cobalt 60 irradiation and then sealed in a sterile vial.
Furthermore, each cleaning step is performed by using an ultrasonic cleaning machine with power of more than 3000W and frequency of 40 kHz.
The further scheme has the beneficial effects that based on the special cell removing step, ultrasonic cleaning and oscillating equipment can be used in a matched manner to repeatedly wash materials, so that the cytotoxicity of the residual reagent is reduced, and the effect is better.
Further, the cell removing solution is phosphate buffer solution containing 2.5g/L of trypsin and 0.5mM of disodium ethylene diamine tetraacetate, and the pH value is 6-8.
The beneficial effect of adopting the further scheme is that through various experimental comparison and accumulated experiences, the invention obtains that the cell removing mode and the used reagent have the best effect.
The invention also relates to the decellularized cartilage material from the costal cartilage of the pig prepared by the preparation method.
The invention also relates to application of the acellular cartilage material from the pig costal cartilage, which is used for repairing costal cartilage defects.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1: preparation of acellular cartilage material from pig costal cartilage
6 Bama mini pigs, 5 months old, male, 12-13kg body weight were selected. All animals were provided by the animal center of the plastic surgery hospital, were renowned and loved in this experiment, and were approved by the animal ethics committee of the hospital.
The acellular cartilage material from the costal cartilage of the pig is prepared by the following preparation method:
(1) removing hair of operation area after pig general anesthesia, sterilizing with iodine tincture, removing iodine with alcohol, and spreading sterile towel;
after local infiltration anesthesia, a 10cm incision is made on the right side of the lower end of the sternum by using a circular knife, the tangent line is parallel to the costal arch, the skin, the subcutaneous layer and the muscle layer are cut in layers, the 6 th, 7 th and 8 th costal cartilages are exposed, the 3 costal cartilages are completely cut off and placed in normal saline for later use; after sufficient hemostasis, the incision is sutured layer by layer, the incision is disinfected again, the antibiotic ointment is externally applied locally, and the intramuscular injection of penicillin is given for three consecutive days after the operation to prevent infection.
(2) Removing perichondrium and connective tissue from the costal cartilage material obtained in the step (1), cutting into a proper size (the diameter is less than 2cm, and the thickness is not more than 3mm), and washing with purified water until the surface is free of stains.
(3) Treating with a mixed solution of peroxyacetic acid and ethanol, placing costal cartilage material in the mixed solution, wherein the volume ratio of the costal cartilage material to the mixed solution is 1:20, the inactivation time is 4 hours, and the inactivation temperature is 20 ℃. Wherein, in the mixed solution of the peroxyacetic acid and the ethanol, the volume ratio of the peroxyacetic acid to the ethanol is 1: 24.
(4) Cleaning with phosphate buffer solution with pH of 6-8 at 10-30 deg.C for 15 min at least 3 times until the pH of the cleaned flushing liquid is 6-8, cleaning with purified water at 10-30 deg.C for 15 min for 1 time.
(5) Adopting phosphate buffer solution containing trypsin and disodium ethylene diamine tetraacetate as decellularization solution, adding costal cartilage material into the decellularization solution, and placing into a shaking table, wherein the volume ratio of the decellularization solution to the costal cartilage material is 20:1, the shaking table is set to rotate at a speed of 150r/min, the action is continuously carried out for 72 hours, the temperature is 37 ℃, and the decellularization solution is replaced every 24 hours. Wherein the cell removing solution is phosphate buffer solution containing 2.5g/L of trypsin and 0.5mM of disodium ethylene diamine tetraacetate, and the pH value is between 6 and 8.
(6) Cleaning with phosphate buffer solution with pH of 6-8 at 10-30 deg.C for 15 min at least 3 times until the pH of the cleaned flushing liquid is 6-8, cleaning with purified water at 10-30 deg.C for 15 min for 1 time.
(7) Washing with injection water for 4 times, each time for 15 minutes, wherein the volume ratio of the injection water to the costal cartilage material is 30:1, the temperature range is 10-30 ℃.
(8) The solution was sterilized by cobalt 60 irradiation and then sealed in a sterile vial. The obtained acellular cartilage material from the costal cartilage of the pig.
The cleaning steps are recommended to be carried out by using an ultrasonic cleaning machine, wherein the power is more than 3000W, and the frequency is 40 kHz. The washing process can also be carried out in a shaker.
Example 2: acellular cartilage material from pig costal cartilage implanted into costal cartilage defect area
Selecting 18 healthy New Zealand white rabbits, male, 3 months old, and 2kg body weight. All animals were provided by the animal center of the plastic surgery hospital, were renowned and loved in this experiment, and were approved by the animal ethics committee of the hospital.
(1) Grouping
In order to compare the difference between the decellularized cartilage material prepared by the present invention and the prior art, two experimental examples were set, experimental example 1 being the decellularized cartilage material prepared according to the preparation method of example 1, and experimental example 2 being the decellularized cartilage material prepared according to the conventional method in the prior art, wherein the decellularization step uses an alkali solution as a decellularization solution, such as a sodium hydroxide solution.
18 rabbits were divided into 3 groups, blank control, filled according to the invention and filled according to the prior art.
(2) Surgical operation
Exposing the operated area after the whole rabbit is anesthetized, and disinfecting with iodine tincture and alcohol. After local infiltration anesthesia, a 10cm incision is made on the right side of the lower end of the sternum by a circular knife, and the tangent is parallel to the costal arch. The skin, subcutaneous and muscle layers were dissected in layers to expose the 5 th, 6 th and 7 th costal cartilages, the perichondrium was dissected in the middle of the cartilage, the perichondrium was slowly pushed away from the middle to both sides, leaving the perichondrium intact as much as possible, and the 3 costal cartilages were excised intact. In the placebo group, the perichondrium was directly sutured again. In both filling groups, the decellularized cartilage material of experimental example 1 and the decellularized cartilage material of experimental example 2 were filled in the cartilage defect region, respectively, and the perichondrium was sutured again to wrap the decellularized cartilage. After sufficient hemostasis, the incision is sutured layer by layer, and the antibiotic ointment is applied topically. Intramuscular injection of penicillin three consecutive days post-surgery was used to prevent infection. After 4 months, the experimental animals were general anesthetized and subjected to thoracic CT scanning. Then stripping and taking out the new tissues in the costal cartilage defect area according to the original operation and the assistance of CT reconstructed image, and respectively storing the new tissues in paraformaldehyde and liquid nitrogen for subsequent research.
Example 3
The following properties were measured for the blank control group and the two experimental example-filled groups, respectively: CT reconstruction of costal cartilage, DNA content determination, total collagen content determination, glycosaminoglycan (GAG) content determination, alpha-Gal content determination, degradation rate and porosity determination, histological examination (including hematoxylin eosin HE staining, Alisin Blue staining, Alizarin Red Alizarin Red staining, Masson staining, Safranin green fixation Safranin O and fast green staining), scanning electron microscopy measurement of the pore size of acellular cartilage material, culture and passage of rabbit costal cartilage cells, toxicity detection of acellular cartilage material, Live/Dead cell activity detection, macrophage polarization reaction (including preparation of reagents, PMA induction of THP-1 cells to M0, THP-1 induction of polarization to M1/M2, ELISA detection of related proteins in cell supernatant, flow cytometry detection of cell surface markers), PCR detection (including total RNA, total DNA, and recombinant DNA), PCR detection, and PCR, Reverse transcription, quantitative PCR, PCR result processing).
The detection method adopts the common technology in the field, can be the following specific method, and can also be in a similar mode, and the experimental result can be verified.
1. Costal cartilage CT reconstruction
The X-ray CT examination of the thoracic cage after the operation is carried out by using 64 rows of CT in the radiology department of the plastic surgery hospital, and the specific parameters are as follows: brilliance CT 64 line, tube voltage 120kV, tube current 220mA, layer thickness 1mm, rotation time 0.75s, pixel matrix 512X 512. Data from Extended BrillianceTMAnd processing by the workstation to obtain a reconstructed three-dimensional image.
2. Determination of DNA content
(1) Placing the acellular cartilage material in liquid nitrogen, grinding into powder, adding 200ul buffer GA and 20mg of powder tissue into a 2ml centrifuge tube, and shaking and uniformly mixing.
(2) Adding 20ul proteinase K (10mg/ml), fully shaking and mixing uniformly, then bathing in water at 56 ℃ for 60 minutes, shaking for 2-3 times to promote complete digestion and lysis. Adding 200ul of buffer solution GB into the fully lysed sample, uniformly mixing the fully lysed sample by oscillation, carrying out water bath at 70 ℃ for 10 minutes, and fully and uniformly mixing the fully lysed sample.
(3) After the centrifugal tube is cooled, 200ul of absolute ethyl alcohol is added, the mixture is fully and uniformly mixed for 15 seconds, and the content is added into an adsorption column CB 3. The tube was centrifuged at 12000rpm for 30 seconds to remove waste from the tube.
(4) 500ul of buffer GD was added to adsorption column CB3, and centrifuged at 12000rpm for 30 seconds to discard the waste liquid from the collection tube.
(5) 600ul of the rinsing solution PW was added, and the tube was centrifuged at 12000rpm for 30 seconds to remove the waste from the tube.
(6) After repeating the previous step again, the adsorption column CB3 was returned to the collection tube, centrifuged at 12000rpm for 2 minutes, and then air-dried at room temperature for several minutes.
(7) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu l of elution buffer TE into the middle part of the adsorption film, standing for 4 minutes at room temperature, centrifuging at 12000rpm for 2 minutes, and collecting the solution into the centrifuge tube. Storing at 4 deg.C, and detecting DNA content with micro-spectrophotometer.
3. Total collagen content determination
(1) Preparing standard solution with the concentration of 5 mug/ml hydroxyproline for the reagent I, the reagent II, the reagent III and the kit according to the kit specification.
(2) Firstly, sample treatment is carried out: grinding the decellularized cartilage material into powder, weighing 80mg, adding 1ml of 6mol/L hydrochloric acid, and standing and hydrolyzing for 5 hours in an oven at 100 ℃.
(3) Mu.l of indicator, 1.5ml of PH solution A and 0.2ml of PH solution B are added into each tube in a unified manner. Continuously dropwise adding the solution B into each tube, shaking uniformly, and adjusting the pH value to 6.0-6.8.
(4) 10ml of double distilled water was added to each tube for dilution. Taking 3ml of the diluted sample, adding 20mg of activated carbon, mixing uniformly, centrifuging at 3500rpm for 10 minutes, taking 1ml of the supernatant as a solution to be detected, and transferring the solution to a measuring tube.
(5) Additionally, a blank tube and a standard tube were set, and 1ml of double distilled water and 1ml of hydroxyproline standard solution were added, respectively.
(6) The detection is carried out according to the following operation flow:
(7)
4. glycosaminoglycan (GAG) content determination
(1) Weighing 0.2g of acellular cartilage material, grinding the acellular cartilage material into powder tissue, putting the powder tissue into a 1.5ml centrifuge tube, adding 500ul of extract, shaking for 1 minute, respectively placing the powder tissue in a 56 ℃ constant temperature water bath for 16 hours and a 90 ℃ constant temperature water bath for 10 minutes, then centrifuging the powder tissue at the speed of 2000rpm for 10 minutes, and taking supernatant out of the centrifuge tube of 1.5 ml.
(2) And (3) preparing No. 1-5 tubes of the standard sample in sequence according to a method indicated by the instruction, and constructing a standard curve after dyeing treatment.
(3) 50ul of sample to be tested is put into a 1.5ml centrifuge tube, 1ml of staining solution is added, and after shaking and incubation treatment, the sample is centrifuged at 12000rpm for 10 minutes. And (4) sucking the supernatant, adding 1ml of dissociation solution, and carrying out shaking and incubation treatment. And (3) detecting by using a spectrophotometer under the parameter of 656nm wavelength to obtain the light absorption value of the sample to be detected, and obtaining the concentration of the corresponding sample to be detected according to the standard curve.
5. Determination of alpha-Gal content
(1) Weighing 30mg of acellular cartilage material, grinding the acellular cartilage material into powder tissue, adding PBS according to the proportion of 1:9, fully homogenizing and cracking on ice, finally centrifuging the homogenate for 10 minutes at the speed of 5000g, and taking supernatant for detection.
(2) Standard holes, blank holes and sample holes are arranged, and standard substances with different concentrations are added into the standard substance holes for 50 ul. 50ul of sample to be detected is added into the sample hole, and 50ul of sample diluent is added into the blank hole. 100ul of detection antibody labeled with horseradish peroxidase was added to each well, the reaction wells were sealed with a sealing plate, and incubated at 37 ℃ for 60 minutes. After discarding the liquid and washing the plate 5 times, 50ul of each of the reaction substrates A, B was added to each well and incubated for 15 minutes at 37 ℃ in the absence of light.
(3) Adding 50ul of stop solution into each hole, detecting the OD value of each hole at the wavelength of 450nm, and obtaining the concentration of the corresponding sample to be detected according to the standard curve.
6. Determination of degradation and porosity
(1) The mass of each low-temperature dried acellular cartilage material is measured in advance to be W0。
(2) The decellularized cartilage material was placed in a centrifuge tube containing 10ml of PBS, and subjected to shaking for 4, 8, 12, and 16 weeks with the parameters set at 120rpm and 37 ℃.
(3) Taking out a sample, washing with deionized water, drying at low temperature, and measuring the mass Wm. Determining the residue according to the formulam/W0×100％。
(4) Measuring porosity of the decellularized cartilage material by a drainage method, placing the material into a centrifuge tube containing 3ml of absolute ethyl alcohol, standing for 24 hours, and measuring the total volume to be V1。
(5) After removal of the material, the volume of the remaining solution was determined to be V2Porosity ═ 3-V2)/(V1–V2) X 100%. Each of the above tests was performed 5 times per sample.
7. Histological examination
Dehydrating the sample tissue to be detected, embedding paraffin, and preparing a 5 mu m paraffin section in the pathology department of the plastic surgery hospital.
7.1 Hematoxylin Eosin (HE) staining
(1) Putting the slices into an oven at 70 ℃ for baking for 1-2 hours;
(2) putting the slices into a dimethylbenzene solution in sequence, soaking for 15 minutes for dewaxing, and then soaking for 5 minutes in alcohol with different concentrations from high concentration to low concentration in a gradient manner;
(3) washing the slices with PBS for 2 minutes, staining with hematoxylin solution for 10 minutes, and washing with tap water for 1 minute;
(4) rapidly differentiating with 0.5% hydrochloric acid alcohol (1:1, mixing hydrochloric acid with mass fraction of 0.5% and ethanol with mass fraction of 0.5%, the same below), and turning blue for 1 min;
(5) eosin staining for 1.5 min;
(6) 70 percent (volume fraction, the same below), 85 percent, 95 percent and 100 percent gradient alcohol are quickly placed in sequence;
(7) and (5) treating the mixture by using dimethylbenzene for 5 minutes, and sealing the mixture by using neutral resin.
7.2 staining with Alisin Blue (Alcian Blue)
(1) Baking slices, dewaxing and hydrating, and dyeing with hematoxylin and eosin;
(2) putting the mixture into an alisin blue dye solution for treatment for 15 minutes, and washing the mixture for 1 minute by tap water;
(3) treating in dimethylbenzene for 5 minutes, and sealing the piece by neutral resin;
(4) under microscope observation, chondrocytes and acidic mucus substance are blue.
7.3 Alizarin Red (Alizarin Red) staining
(1) Baking slices, dewaxing and hydrating, and dyeing with hematoxylin and eosin;
(2) placing the slices into alizarin red dye solution for treatment for 5 minutes, and washing with tap water for 1 minute;
(3) after the slices are placed in an oven to be dried, the slices are treated for 5 minutes in dimethylbenzene, and the neutral resin is sealed;
(4) under microscope observation, the calcium salt deposition part is dark red, and the background is light red or nearly colorless.
7.4 Masson staining
(1) Baking slices, dewaxing and hydrating, and dyeing with hematoxylin and eosin;
(2) soaking the slices in Masson A solution overnight, and soaking the slices in Masson A solution in an oven at 37 ℃ for 30 minutes;
(3) washing with tap water for 30 seconds;
(4) mixing Masson B solution and Masson C solution in equal volume before use, placing the slices into the mixed solution for treatment for 1 minute, and flushing with running water;
(5) 1% hydrochloric acid alcohol is differentiated for 1 minute, the nucleus is gray black, and the background is almost colorless or light gray;
(6) slightly washing with tap water, draining, treating with Masson D solution for 6 min to obtain bright red tissue;
(7) draining the water as much as possible, and treating the mixture by Masson E liquid for about 1 minute;
(8) slightly draining the Masson E liquid, and directly putting the Masson F liquid for dyeing for 10 seconds;
(9) rinsing and differentiating in 1% glacial acetic acid, and treating for three times, wherein each time lasts for about 5 seconds;
(10) after dehydration treatment with absolute ethyl alcohol, treatment with xylene for 5 minutes, and sealing with neutral resin;
(11) under microscope observation, the blue part is collagen fiber, and the muscle fiber is red.
7.5 Safranin O and fast green staining
(1) Baking slices, dewaxing and hydrating, and dyeing with hematoxylin and eosin;
(2) dip-dyeing with a solid green dyeing solution for 5 minutes, and washing with tap water for 1 minute;
(3) dip dyeing with safranin dyeing liquid for 1 minute, washing with tap water for 1 minute;
(4) 0.5% hydrochloric acid alcohol is differentiated for several seconds, and washed with tap water for 10 minutes;
(5) treating the slices with acetic acid solution for 1.5 minutes, removing redundant solid green dye solution, and washing with tap water for 1 minute;
(6) after the gradient ethanol solution is dehydrated, xylene is treated for 5 minutes, and a neutral resin is sealed;
(7) under microscope observation, cartilage is red or orange-red, and background is green.
8. Scanning electron microscope for measuring the aperture of acellular cartilage material
(1) Placing the acellular cartilage material into a solution containing 2% glutaraldehyde, and standing and fixing for 24 hours;
(2) fixed with 1% osmic acid solution for 0.5 h;
(3) gradient dehydration with 85%, 95% and 100% alcohol;
(4) replacing the mixture with isoamyl acetate solution for 4 hours;
(5) drying the sample by using a carbon dioxide critical dryer;
(6) sputtering platinum ions for 60 seconds in vacuum;
(7) scanning electron microscope pictures were taken, 10 samples were measured for each decellularized cartilage, and 50 pore sizes were randomly measured for each sample.
9. Culture and passage of rabbit costal chondrocytes
(1) Obtaining rabbit-derived costal cartilage tissue by adopting steps according to the costal cartilage;
(2) spreading sterile towel on super clean bench, and cutting cartilage to 1mm with circular knife3Size;
(3) repeatedly washing a large amount of PBS, centrifuging at 1200rpm for 5 minutes, removing supernatant, and adding 0.25% trypsin with 5 times volume;
(4) treating in a constant temperature shaking table at 37 deg.C for 30 min, centrifuging at 1200rpm for 5 min, and removing supernatant;
(5) adding 5 times of collagen II enzyme with 0.25% volume to digest cartilage tissue, and shaking at 37 deg.C for 8-12 hr;
(6) standing, filtering with a 70 μm filter screen, and collecting cell suspension;
(7) after washing the resuspended cell pellet with PBS solution, 2ml of complete medium was added to resuspend the cell pellet;
(8) cell viability was observed under microscope, counted, in 10cm dishes at 5X104Seeding chondrocytes at a density of/ml;
(9) culturing in an incubator for 48-72 hours, and replacing the complete culture medium when 80% of chondrocytes adhere to the wall, and replacing the culture medium every 2-3 days;
(10) after the cell growth reaches 90%, passage is carried out, the original culture medium is sucked out, and the cell is washed for 2 times by using 3ml of PBS;
(11) adding 2ml pancreatin, digesting the cells at 37 ℃ for 15 seconds, observing cell rounding and separating, adding 2ml complete culture medium to stop digestion, and gently blowing and beating;
(12) centrifuging at 1200rpm for 5 min, and removing supernatant;
(13) after washing the resuspended cell pellet with PBS solution, 2ml of complete medium was added to the cell pellet to prepare a cell suspension, and the cells were cultured according to the above procedure.
10. Toxicity testing of decellularized cartilage material
(1) Taking 10mmx10mm acellular cartilage material, putting the acellular cartilage material into a complete culture medium for 12 hours in advance, taking the acellular cartilage material out when the acellular cartilage material is used, cleaning the culture medium on the surface of the material by using a negative pressure suction tube, and then putting the acellular cartilage material into a 6-hole plate;
(2) cell density of 4.5x107Per ml of P2 generation costal chondrocyte suspension 80ul, and the cell suspension was dropped on the surface of the decellularized cartilage material. The procedure was carried out in 2-3 times to avoid the cell suspension flowing down the surface of the cartilage pieces, and then 1ml of complete medium was added around the material without exceeding the surface of the material.
(3) Placing the culture plate in an incubator for 2 hours for standing, adding 1ml of complete culture medium into each hole, and periodically replacing the culture solution;
(4) respectively arranging a blank group, the acellular cartilage material group and the acellular cartilage material group in the prior art, wherein the blank group does not contain materials according to a conventional cartilage cell culture method;
(5) at 1, 3, 7, 9, 14d, after aspirating the old medium, 1mL of complete medium containing CCK-8 (volume ratio 1: 9) was added to each well;
(6) the plates were placed in an incubator for 2 hours, 100ul of liquid was taken per well into a 96-well plate, and the OD at 450nm was measured. Three groups of samples were run in triplicate 3 times, 5 wells per fluid.
11. Live/Dead cell activity assay
(1) The process of cocultivation of chondrocytes with decellularized cartilage material is the same as in the previous section 9 (1-4);
(2) taking out the Live/Dead cell activity kit, adding 20 mu l of the component B into 10ml of PBS for uniformly mixing, then adding 5 mu l of the component A for uniformly mixing, and preparing to obtain a working solution;
(3) the experimental group is characterized in that the bracket is soaked in the working solution, the working solution is directly added into the blank group culture plate, and the blank group culture plate is respectively incubated in the incubator for 30 minutes;
(4) the distribution and activity of chondrocytes were observed using a fluorescence confocal microscope and photographed.
12. Macrophage polarization reaction
12.1 preparation of reagents
(1)0.5mg/ml PMA solution: to 5mg PMA was added 10mL DMSO solution and mixed well.
(2) THP-1 cell complete medium: 1640 basic medium + 10% FBS +0.05mM beta-mercaptoethanol + 1% double antibody.
(3) PMA inducing solution: THP-1 cell complete medium 500ml + PMA (0.5mg/ml)30 ul.
(4) M1 polarization-inducing solution: THP-1 cell complete medium 50ml + LPS (5ug/ml)100ul + IFN-. gamma. (10ug/ml)100 ul.
(5) M2 polarization-inducing solution: THP-1 cell complete medium 50ml + IL-4(10ug/ml)100ul + IL-13(10ug/ml)100 ul.
12.2PMA induces THP-1 cells as M0
(1) Culturing THP-1 cells in 6-well plate until the cell amount reaches 5 × 105At the time of ml, centrifuging for 5 minutes at the speed of 1000rpm, and discarding the supernatant;
(2) the cells are resuspended by PMA inducing solution, evenly mixed and evenly spread in a 6-well plate, each well is 2mL, and the mixture is placed in an incubator at 37 ℃ and 5% CO2 for static culture for 72 h.
12.3THP-1 induces polarization to M1, M2
(1) Collecting cell suspension, centrifuging at 1000rpm for 5 min, discarding supernatant, resuspending cells according to experimental groups by using corresponding induction culture medium, uniformly spreading the cells in a 6-well plate with 2mL per well, and standing and culturing in a 5% CO2 incubator at 37 ℃.
(2) The experimental groups were as follows: group M0: m0+ THP-1 cell complete medium; group M1: m0+ M1 inducer; group M2: m0+ M2 inducer; m1+ group of decellularized cartilage material of the invention: m0+ M1 inducing solution + the set of acellular cartilage materials of the present invention; m1+ group of prior art decellularized cartilage material: m0+ M1 inducing solution + prior art acellular cartilage material group; m2+ group of decellularized cartilage material of the invention: m0+ M2 inducing solution + the set of acellular cartilage materials of the present invention; m2+ group of prior art decellularized cartilage material: m0+ M2 inducing solution + prior art acellular cartilage material group;
(3) after 72h of culture, cell supernatants were collected and stored at-80 ℃ for ELISA detection. Sterile PBS buffer was added to the cell pellet for flow assay.
12.4 detection of proteins associated with cell supernatants by ELISA
(1) Centrifuging the cell supernatant for 10 minutes at the speed of 3000rpm, and sucking the supernatant into a centrifuge tube for later use;
(2) setting a blank hole, a standard hole and a sample hole to be detected, respectively adding 100ul of corresponding standard solution or sample to be detected, and incubating for 120 minutes at 37 ℃;
(3) adding biotinylated antibody working solution, enzyme conjugate working solution, chromogenic substrate solution and termination solution in several times according to the detection instruction, washing plate, incubating and other treatment;
(4) immediately, the OD value of each hole is measured sequentially by a microplate reader at 450nm, and the concentration of the sample to be measured is calculated according to a standard curve.
12.5 flow cytometry detection of cell surface markers
(1) Resuspending the cell pellet with PBS and adjusting the cell density to 5X106Taking 5 mul of cell suspension in an EP tube;
(2) adding 100 μ l of direct standard antibody diluent (antibody and PBS mixed at a ratio of 1: 10), mixing with cells, incubating at 4 deg.C for 40 min in dark;
(3) adding 1ml of flow analysis solution into each tube, centrifuging at 1500rpm for 5 minutes, and removing supernatant;
(4) resuspending the cells with 500. mu.l of pre-cooled flow assay, and mixing by aspiration;
(5) immediately, the cells were tested on the machine and analyzed for positive rate by FlowJo.
13. PCR detection
13.1 Total RNA extraction
(1) Taking a homogenate tube, adding 1ml of Trizol, and placing on ice for precooling;
(2) taking 100mg of tissues, and adding the tissues into a homogenizing tube;
(3) fully grinding by a homogenizer until no tissue block is visible;
(4) centrifuging at 12000rpm for 10 min to obtain supernatant;
(5) adding 250 mul of trichloromethane, uniformly mixing for 20s, and standing for 4 minutes;
(6) centrifuging at 12000rpm for 10 min at 4 ℃;
(7) taking 400 mu l of supernatant into a centrifuge tube, adding isopropanol, wherein the volume ratio of the supernatant to the isopropanol is 1: 0.8. Fully and uniformly mixing, and standing for 15 minutes at the temperature of minus 20 ℃;
(8) centrifuging at 12000rpm for 10 min to obtain white precipitate as RNA;
(9) carefully remove the liquid by suction, add 1.5ml of 75% ethanol to wash the precipitate;
(10) centrifuging at 12000rpm for 5 min at 4 ℃;
(11) carefully sucking and discarding the supernatant, and placing the supernatant on an ultra-clean bench for drying at room temperature for 5 minutes;
(12) adding 30 μ l of RNAse-free DEPC water to dissolve RNA, and incubating for 5 minutes at 55 ℃;
(13) detecting light absorption value OD of RNA sample to be detected260/280The purity is higher at 1.8-2.0;
(14) the extent of RNA degradation and the presence of contamination were analyzed using agarose gel electrophoresis.
13.2 reverse transcription
(1) Architecture configuration
(2) Water bath at 37 ℃ for 30 minutes;
(3) inactivating DNase I in water bath at 65 ℃ for 10 minutes;
(4) architecture configuration
(5) Mixing, and centrifuging;
(6) water bath at 42 ℃ for 60 minutes;
(7) water bath at 85 deg.c for 10 min to deactivate reverse transcriptase.
13.3 quantitative PCR
(1) Primer information
(2) PCR System configuration
(3) The above reaction system was added to a 0.2ml EP tube and mixed well and centrifuged briefly.
(4) The above solution was added to 8-bank tubes and amplification was performed according to the following reaction procedure.
PCR reaction Programming
13.4PCR results processing
Normalizing with GAPDH as reference gene, and using 2-△△Ct method calculates data.
Data analysis statistics were performed using software SPSS 25.0(SPSS, USA), quantitative indices are expressed as mean ± standard deviation, comparisons between two experimental example cohorts were analyzed using independent sample T-test, comparisons over two experimental example cohorts were analyzed using one-way anova, P <0.05 indicates significant differences, and GraphPad Prism 8 was used to plot the statistics.
And (4) analyzing results:
at present, the transplantation of xenogenic or xenogenic materials is primarily aimed at solving the immunological rejection of the recipient, which is also the basic requirement for the application of materials of biological origin. Cellular components and α -Gal antigens within the decellularized cartilage material may cause hyperacute immune rejection and chronic immune toxicity.
According to the detection result, after the acellular treatment, the residual DNA content of the acellular cartilage material is less than 40ng/mg, the basic requirement of acellular is met, 80% of alpha-Gal antigen is effectively removed, and the immune rejection risk of later-stage implantation in vivo is obviously reduced. After the acellular treatment of the prior conventional technology, the residual DNA content of the acellular cartilage material is more than 40ng/mg, the basic requirement of the acellular cartilage material is not met, and other matched steps are required.
The decellularization process also destroys extracellular matrix components while removing cellular DNA components, and inevitably removes part of the matrix components. After decellularization, Elisa and masson staining analysis showed that the collagen fiber component of the extracellular matrix was largely retained. Collagen fibers are important structural components in cartilage extracellular matrix, can induce cell migration and differentiation and tissue regeneration during the repair process of costal cartilage defects, and provide mechanical support. It has been shown that the elution of glycosaminoglycans (GAG) is evident during the decellularization process, and after the relatively mild decellularization treatment of the invention, Elisa results show that about 50% of GAG is retained by the decellularized cartilage material, and the staining of Alisin blue is pale blue. GAGs in the chondrocyte extracellular matrix have the effect of promoting cartilage differentiation and endochondral ossification. Therefore, in the preparation process of the decellularized cartilage, the damage effect of different decellularization methods on extracellular matrix is fully considered, and the search for a more mild and effective decellularization technology is an important aspect of the preparation of decellularized materials.
The complete structure and special internal microenvironment of the extracellular matrix provide a site for cell migration and tissue remodeling, and the degradation rate of the extracellular matrix also significantly affects the progress of tissue remodeling. The relatively slow degradation rate allows the decellularized cartilage material to provide a durable mechanical support at the defect to reduce local deformation and provide a long-term stable environment for accommodating cell migration and tissue replacement remodeling. Due to the influence of various factors such as local immune phagocytosis reaction, inflammatory factors and the like, the decomposition speed of the acellular cartilage material in vivo is obviously accelerated, and the acellular cartilage material is gradually replaced by the surrounding new tissues. After 4 months of in vivo animal experiments, only a small amount of residual acellular cartilage material was found in a few specimens and surrounding tissues.
In the conventional preparation process of the acellular material, a physical method, a chemical method and an enzymatic method are involved. Where residual chemicals and enzymes are cytotoxic, affecting the clinical use of the material. According to the invention, a milder cell removal treatment method is applied, pancreatin containing EDTA is used for continuous treatment for 72 hours, ultrasonic cleaning and oscillation equipment is used in a matched manner, and materials are repeatedly washed, so that the cytotoxicity of the remaining reagent is reduced. Fluorescence staining of live and dead cells showed that no significant dead cells were seen in both decellularized cartilage groups and the blank group. Further CCK-8 results indicate that the decellularized cartilage material using the decellularization method of the invention does not inhibit cell proliferation, but rather cell proliferation exceeds that of the blank control group at some point in time, which may be due to the effect of promoting cartilage cell proliferation of cytokines slowly released from the extracellular matrix, such as TGF-beta and IGF-1.
The local immune response can be caused by surgical trauma and filling of the xenograft, the implanted material attracts local macrophages and neutrophils to aggregate at the early stage of the inflammatory response, and then secretes a plurality of chemotactic factors such as IL-8 and the like, so that more inflammatory cells (dendritic cells and lymphocytes) are chemotactic and activated to aggregate, the local response mainly takes the macrophages as the main part along with the fading of inflammation and the starting of tissue repair and reconstruction, apoptotic cells and tissue fragments are phagocytosed and treated gradually, and therefore, the macrophages have important roles in regulating the local immune response and repairing and reconstructing foreign body materials. Macrophages can be recruited to sites of inflammation, and under the influence of various cytokines, there is a polarization from M0 type macrophages to M1 type macrophages that promote inflammatory responses or M2 type macrophages that inhibit inflammation. The M1 type macrophage can continuously cause local inflammatory reaction, and release various inflammatory factors to regulate cell proliferation, differentiation and apoptosis. The M1 type macrophage may inhibit tissue repair, causing rejection of foreign materials. The M2 type macrophage has the function of inhibiting inflammatory reaction, can phagocytize local necrotic tissue fragments, release inhibitory inflammatory factors and promote local wound healing. The research shows that for degradable biological materials, M1 type macrophages promote the material degradation in the early stage, and M2 type macrophages promote the material to fuse and regenerate with tissues in the later stage, but the process is influenced by different materials and can generate obvious opposite effects. Therefore, the polarization reaction of the acellular cartilage material on macrophages is evaluated, and the filling material with light local inflammatory reaction is selected, so that the method has important significance on tissue repair and regeneration.
Macrophages are formed by the stimulation and differentiation of monocytes through various cytokines, and currently, phorbol ester (PMA) is commonly used for stimulating the directional differentiation of a human monocyte line THP-1 to obtain an in vitro macrophage model. The invention uses the mode of co-culture of macrophages and decellularized cartilage to evaluate the polarization reaction in an in vitro environment. After induction, THP-1 cell is differentiated to M0 type macrophage, and then M0 type macrophage is differentiated to M1 type macrophage or M2 type macrophage through LPS + IFN-gamma or IL-4+ IL-13 drug treatment. In the acellular cartilage co-culture group, the acellular cartilage material obviously inhibits the differentiation from M0 type to M1 type macrophage, the expression of cell surface markers CD80 and CD86 is obviously reduced, and the secretion of inflammatory factors TNF-alpha and IL-1 beta is obviously reduced. In addition, the decellularized cartilage material of the present invention also exhibits an effect of inhibiting differentiation from M0 type to M2 type macrophages. And the possibility of the decellularized cartilage material inhibiting the proliferation of macrophages is eliminated by the CCK-8 experiment. Therefore, the acellular cartilage material can inhibit the polarization reaction of macrophages, particularly the activation of M1 type macrophages, wherein the physiological mechanism is not clear, further deep analysis is needed, and the inhibition of a JNK pathway and the activation of a STAT 6/PPAR-gamma pathway in immune response are probably involved, so that further molecular mechanism research is awaited. The active molecules released by the graft material and the signal molecules existing at the graft site can regulate macrophage polarization from various aspects such as cell recruitment and differentiation, and research indicates that type II collagen in extracellular matrix has the function of regulating M1 type macrophages so as to inhibit inflammatory response. Micro-RNAs for regulating and controlling inflammatory reaction by loading materials can inhibit macrophage activation and promote local tissue regeneration and repair. In addition, the regulation of macrophage polarization response also indicates that the cellular components of the acellular cartilage matrix that cause the inflammatory response are effectively removed. Research indicates that the physicochemical properties of the material can regulate the polarization of macrophages, meanwhile, the macrophages can also react to the mechanical performance of the implant, small-pore and soft materials can promote the differentiation of the macrophages to proinflammatory types, and large-pore, hard and thick-fiber-diameter materials can promote the differentiation of the macrophages to anti-inflammatory types.
After the costal cartilage is defected, a local cavity structure is formed and is filled with tissues such as intercostal muscles, blood clots, perichondrium and the like after operation. Moreover, the site is continuously affected by muscle and bone traction during regenerative repair, further increasing the difficulty and uncertainty of tissue repair. Therefore, it is desirable to implant decellularized cartilage material back into the cartilage defect site to assess its effect on local thoracic morphology and tissue repair regeneration. The costal cartilage CT reconstruction can evaluate the local thoracic morphology and the tissue regeneration condition before an operation, provide visual image recognition and guide the follow-up operation to explore and draw materials. In the invention, the anatomical exploration result is consistent with the CT reconstruction result, a large amount of scattered new tissues can be seen at the material replanting position and are distributed along the original costal cartilage region, and the sunken situation of the defect position at the affected side is obviously reduced, which shows that the acellular cartilage material filling group can promote tissue regeneration and improve local thoracic cavity sunken deformity.
After filling the cartilage defect site, the decellularized cartilage material is fused with the surrounding tissues, and the composition of the new tissues is affected by various factors, such as the autologous components of the decellularized cartilage matrix, the cytokines of the surrounding environment, the muscle and the cartilage membrane, and the like. Histological staining and PCR results indicated that the regeneration tissue of the blank consisted primarily of fibrous tissue, with no obvious chondrocytes and cartilage-specific matrix. In the decellularized cartilage material-filled group, the morphology of cartilage components and high expression of cartilage-related genes were observed, and at the same time, a large amount of non-cartilage components such as fibrous tissues and osteogenic tissues were also found. Among them, chondrocytes may be derived from peripheral mesenchymal stem cells or cartilage precursor cells within the proliferation layer of perichondrium. On the aspect of bone formation, calcified nodules and bone trabecular structures can be seen in the acellular cartilage material filling group, and the relatively high expression of the osteogenic gene Runx2 is accompanied, especially in the acellular costal cartilage group of the invention, probably due to the material property of costal cartilage which is easy to calcify and the microenvironment of the costal cartilage defect area. Therefore, under the influence of various inflammatory factors and various signal molecules, the acellular cartilage material provided by the invention is used as a scaffold structure to attract various cells and peripheral tissues for fusion and reconstruction, and the regenerated tissue structure is complex and is composed of various components such as cartilage, osteogenesis, fibrous connective tissue and the like.
At present, most cartilage regeneration tissue engineering experiments involve the planting of chondrocytes on the surface of a material, and transplanting the chondrocytes into the subcutaneous tissues of animals after in vitro culture for 1-2 months. Mature and stable cartilage structures can be obtained by this method, but there are some limitations, such as limited source of chondrocytes, fibrosis of chondrocytes, long-term and demanding experimental procedures. In the invention, the acellular cartilage material without any cell load can be immediately filled in the defect area when the costal cartilage is taken out, so that the preparation time is greatly shortened, and the additional operation is also reduced.
Therefore, the acellular cartilage material basically keeps the structure and the function of an extracellular matrix and provides a stable environment for cell proliferation and tissue regeneration, wherein the acellular cartilage material prepared by adopting the special acellular method limited by the invention has better applicability in various aspects, is expected to be deeply researched, and is further applied to clinical research of cartilage repair and tissue regeneration.
While embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and not to be construed as limiting the present invention, which can be made by one of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A method for preparing acellular cartilage material from costal cartilage of a pig, which is characterized by comprising the following steps: (1) extracting costal cartilage material from costal cartilage of pig; (2) primarily treating costal cartilage materials; (3) inactivating viruses; (4) cleaning for the first time; (5) removing cells; (6) secondary cleaning; (7) cleaning with injection water; (8) sterilizing;
wherein the step (5) is specifically as follows: adopting phosphate buffer solution containing trypsin and disodium ethylene diamine tetraacetate as decellularization solution, adding costal cartilage material into the decellularization solution, and placing into a shaking table, wherein the volume ratio of the decellularization solution to the costal cartilage material is 20:1, the shaking table is set to rotate at a speed of 150r/min, the action is continuously carried out for 72 hours, the temperature is 37 ℃, and the decellularization solution is replaced every 24 hours.
2. The method for preparing the acellular cartilage material from the costal cartilage of the pig according to claim 1, wherein the step (1) is specifically as follows: removing hair of operation area after pig general anesthesia, sterilizing with iodine tincture, removing iodine with alcohol, and spreading sterile towel; after local infiltration anesthesia, a 10cm incision is made on the right side of the lower end of the sternum by using a circular knife, the tangent line is parallel to the costal arch, the skin, the subcutaneous layer and the muscle layer are cut in layers, the 6 th, 7 th and 8 th costal cartilages are exposed, the 3 costal cartilages are completely cut off and placed in normal saline for later use; after sufficient hemostasis, the incision is sutured layer by layer, the incision is disinfected again, the antibiotic ointment is externally applied locally, and the intramuscular injection of penicillin is given for three consecutive days after the operation to prevent infection.
3. The method for preparing the acellular cartilage material from the costal cartilage of the pig according to claim 1, wherein the step (2) is specifically as follows: removing perichondrium and connective tissue from the costal cartilage material obtained in the step (1), cutting into a proper size, and washing with purified water until the surface is free from stains.
4. The method for preparing the acellular cartilage material from the costal cartilage of the pig according to claim 1, wherein the step (3) is specifically as follows: treating with a mixed solution of peroxyacetic acid and ethanol, placing costal cartilage material in the mixed solution, wherein the volume ratio of the costal cartilage material to the mixed solution is 1:20, the inactivation time is 4 hours, and the inactivation temperature is 20 ℃.
5. The method for preparing acellular cartilage material from costal cartilage of pig according to claim 4, wherein the volume ratio of the peroxyacetic acid to the ethanol in the mixed solution of the peroxyacetic acid and the ethanol is 1: 24.
6. The method for preparing the acellular cartilage material from the costal cartilage of the pig according to claim 1, wherein the step (4) and the step (6) are operated in the same way, and specifically comprise the following steps: cleaning with phosphate buffer solution with pH of 6-8 at 10-30 deg.C for 15 min at least 3 times until the pH of the cleaned flushing liquid is 6-8, cleaning with purified water at 10-30 deg.C for 15 min for 1 time; the step (7) is specifically as follows: washing with injection water for 4 times, each time for 15 minutes, wherein the volume ratio of the injection water to the costal cartilage material is 30:1, the temperature range is 10-30 ℃; the step (8) is specifically as follows: the solution was sterilized by cobalt 60 irradiation and then sealed in a sterile vial.
7. The method according to claim 6, wherein the washing step is carried out using an ultrasonic washer at a power of 3000W or more and a frequency of 40 kHz.
8. The method for preparing a decellularized cartilage material derived from costal cartilage of pig according to any one of claims 1 to 7, wherein the decellularized solution is phosphate buffered solution containing 2.5g/L trypsin and 0.5mM disodium EDTA, and the pH is 6-8.
9. A decellularized cartilage material derived from costal cartilage of swine prepared by the preparation method of any one of claims 1 to 8.
10. Use of the decellularized cartilage material derived from pig costal cartilage according to claim 9 for the repair of costal cartilage defects.
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