CN109646719A - The articular cartilage remediation composition of the matrix particles containing regenerating tissues - Google Patents

The articular cartilage remediation composition of the matrix particles containing regenerating tissues Download PDF

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CN109646719A
CN109646719A CN201910084415.4A CN201910084415A CN109646719A CN 109646719 A CN109646719 A CN 109646719A CN 201910084415 A CN201910084415 A CN 201910084415A CN 109646719 A CN109646719 A CN 109646719A
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semi
prp
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articular cartilage
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CN109646719B (en
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莫丽影
孙善凤
石卫华
王姣
王正元
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Beijing Haomei Cell Gene Biotechnology Co Ltd
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Beijing Haomei Cell Gene Biotechnology Co Ltd
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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
    • A61L27/3604—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 human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3616—Blood, e.g. platelet-rich plasma
    • 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
    • A61L27/3604—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 human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • 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
    • 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
    • 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 present invention proposes a kind of articular cartilage remediation composition of matrix particles containing regenerating tissues.The articular cartilage remediation composition, includes regenerating tissues matrix particles and PRP, and the PRP is to contain platelet concentration (1200~1600) × 109The platelet rich plasma of/L.The present invention also proposes the preparation method and application of the articular cartilage remediation composition.Composition proposed by the present invention, based on regenerating tissues matrix particles particle size it is uniform, loss late is lower, DNA remains the characteristic of structure that is low and maintaining regenerating tissues matrix, to maintain curative effect time is long and safety, plasticity are good composition, as joint repair agent, test proves that this composition treats gonitis and has better long-term efficacy.

Description

The articular cartilage remediation composition of the matrix particles containing regenerating tissues
Technical field
The invention belongs to regenerative medicine fields, and in particular to a kind of regenerating tissues matrix particles implant and its preparation side Method and application.
Background technique
Cartilage defect is clinically common disease, and pathogenesis includes cartilage damage caused by various wounds and with bone Property arthritis be representative cartilage degradation etc..The power of regeneration of cartilage is weak, and self-regeneration is relatively difficult after damage, therefore its Treatment is the research topic that presently relevant subject compares concern, and research fundamental has seed cell, timbering material and training Support system three parts.And in timbering material, syringeability timbering material gradually shows it because its wound is small, plasticity is strong Unique superiority.
The extracellular matrix of cartilage is mainly made of collagen and proteoglycans (PG), and effect is to protect cartilage cell from each Kind damage.Articular cartilage collagen has 5 kinds, wherein II, IV, VI Collagen Type VI is that articular cartilage is distinctive.II Collagen Type VI composition is extracellular The basic framework of matrix, the 90%~95% of the total collagen content of Zhan, 20~300 μm of diameter.II Collagen Type VI network guarantees that joint is soft The good ductility of bone resists shearing force.Type Ⅳ collagen accounts for the 1%~2% of cartilage collagen total amount, it adjust II Collagen Type VI and It plays an important role in terms of proteoglycans function.VI Collagen Type VI accounts for about the 2%~3% of mature articular cartilage collagen content, it can It can be related with the diameter of II type collagen fiber of control or growth.Proteoglycans clogs the reticular structure constituted in II Collagen Type VI In, monomer is made of a core protein and one to up to a hundred aminoglycan chains.Proteoglycans acidic-group containing there are many, can inhale A large amount of moisture content is received, to guarantee the good viscoplasticity of articular cartilage, renitency.
Summary of the invention
The present invention is in order to solve the above technical problems, provide a kind of matrix containing regenerating tissues that can be used for intraarticular injection The articular cartilage remediation composition of particle.
The present invention also proposes the preparation method of the articular cartilage remediation composition of the matrix particles containing regenerating tissues.
Present invention further propose that the application of the articular cartilage remediation composition of the matrix particles containing regenerating tissues.
Realize the technical solution of above-mentioned purpose of the present invention are as follows:
A kind of articular cartilage remediation composition of the matrix particles containing regenerating tissues, include regenerating tissues matrix particles and PRP, The PRP is to contain platelet concentration (1200~1600) × 109The platelet rich plasma of/L.
In the composition of regenerating tissues matrix particles and PRP, if PRP dosage is too big, injection site is easy thrombus, dosage The effect for accelerating joint repair, therefore, the mass body of the present invention preferably described regenerating tissues matrix particles and PRP are not had at least Product is than being 1g:4~6mL.
It is highly preferred that DNA residual quantity is in 2.0ng/mg hereinafter, the regenerating tissues base in the regenerating tissues matrix particles The partial size of matter particle is in 1.2mm hereinafter, D50 is between 0.018~1.0mm.
The present invention also proposes the preparation method of the joint repair composition of the matrix particles containing regenerating tissues.The pass Save the preparation method of repair of cartilage composition, comprising the following steps:
1) by the glycerite of allograft skin raw material investment alkali, oscillation treatment is impregnated, semi-finished product A is obtained；
2) it after semi-finished product A being removed epidermis, puts into surfactant solution, ultrasonic immersion treatment obtains semi-finished product B；
3) semi-finished product B is cut into small skin graft, 3~10min in liquid nitrogen is immersed in a manner of immersing, taken out as semi-finished product C；
4) under the action of high speed rotation centrifugal force, semi-finished product C is cut with rotor, obtains semi-finished product D；
5) semi-finished product D is placed in D- amino acid solution and is impregnated, separation of solid and liquid obtains semi-finished product E；The D- amino acid Solution is selected from one or more of aspartic acid, alanine, glutamic acid, serine；
6) semi-finished product E is mixed with PRP to get implant.
Wherein, alkali described in step 1) is sodium hydroxide and/or potassium hydroxide, and the concentration of the alkali is 0.5~5%；Into The concentration of the preferred lye of one step is 1~3%；
And/or in the glycerite of the allograft skin raw material investment alkali, 1~10h is impregnated simultaneously preferably at 25~35 DEG C Oscillation treatment.
Wherein, surfactant described in step 2) is SDS Triton X-100；The surfactant solution Concentration is 0.1~0.3g/L；
Ultrasonic soaking conditions are as follows: 20~45KHz, temperature are 1~20 DEG C, 1~5min of ultrasound, impregnate 1~4h, preferably repeat Ultrasound is with immersion treatment 2~6 times, the surfactant solution renewed before repeating.
Further, semi-finished product B is crosslinked with crosslinking agent, is then cut out；
The crosslinking agent is the genipin solution of mass concentration 0.1~0.6%, time of crosslinking is 2~for 24 hours.
Wherein, semi-finished product D described in step 4) are 50~100g/min by feed inlet admission velocity, it is preferable that 50~ 80g/min；
Preferably, step 5) is respectively 0.8~1.4 using the concentration of aspartic acid, alanine, glutamic acid, serine, The amino acid solution of 1.2~1.8,0.4~0.6,4~10.8 μm of ol/L, example is 1:(0.8~1.2 by volume): (0.8~ 1.2): (0.8~1.2) is prepared.
Wherein, it is described PRP's the preparation method comprises the following steps: acquisition autologous vein blood in anticoagulant tube, first time relative low speeds centrifugation, Blood plasma and blood platelet is set to be divided into three layers with red blood cell and leucocyte；Middle layer is taken to carry out second of relatively high speed centrifugation, further PRP and separation thrombocyte plasma component is concentrated, obtains the PRP of middle layer higher concentration；It is activated with calcium chloride.
Wherein, the parameter being centrifuged for the first time are as follows: 120~150 × g is centrifuged 5~15min；The parameter of second of centrifugation are as follows: 400~600 × g is centrifuged 10~25min；Preferably；10% calcium chloride solution that 1/10 volume is added is activated.
Present invention further propose that repair materials of the joint repair composition in preparation treatment articular cartilage damage In application
The beneficial effects of the present invention are:
Composition proposed by the present invention, the uniform and loss late based on regenerating tissues matrix particles particle size are lower. DNA remains the characteristic of structure that is low and maintaining regenerating tissues matrix, and to maintain, curative effect time is long and safety, plasticity are good Composition as joint repair agent.Regenerating tissues matrix particles implant can substantially reduced gonalgia, compared to injection PRP group treatment gonitis has better long-term efficacy.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Operation involved in embodiment It unless otherwise specified, is this field customary technical operation.
Embodiment 1
1) allograft skin raw material investment alkali glycerite in, in 1-10 hours oscillation treatments of 25~35 DEG C of immersion, obtain partly at Product A；
2) after semi-finished product A being removed epidermis, 0.1~5.0min is rinsed with sterile pure water, is put into surfactant solution, In supersonic frequency 20-45KHz, ultrasonic temperature is 1-20 DEG C, 1-5 minutes min of ultrasonic time, impregnates 1~4h, the surface more renewed Activator solution is impregnated and is repeated 0~4 time, and obtaining semi-finished product B, (specifically, embodiment 1 is 2.5h tetra- times.Embodiment 2 is 2h tetra- times. Embodiment 3 is 2.5h bis- times.Embodiment 4 is 2h primary.Embodiment 5 is 1h primary).
3) semi-finished product B sterile pure water is rinsed into 0.1~5.0min, put into the aqueous solution of 0~0.6% Geniposide Crosslinking (pH value of solution is 5~7), rinses 1~5h with sterile pure water.
It 4) is (0.3~1) × (0.3~1) cm by the size of the small skin graft being cut into of the semi-finished product B Jing Guo step 3)2, with Immersion mode is immersed in 5min in liquid nitrogen, obtains semi-finished product C.
It 5) is 50-80g/min by feed inlet admission velocity by semi-finished product C, in rotor with the tune of 8000-15000 turns/min Under the action of frequency speed high speed rotation centrifugal force, rotor cuts semi-finished product C, obtains partial size less than ring grizzly aperture 2mm's Particle is collected in collector by ring grizzly and obtains semi-finished product D.
6) semi-finished product D sterile pure water is rinsed into 5.0min, is placed in D- amino acid solution and impregnates 3-5min, was centrifuged Filter obtains semi-finished product E.
7) preparation process of PRP: venous puncture Venous Blood rear merging high speed in the centrifuge tube containing sodium citrate Centrifuge will extract visible blood after blood is centrifuged for the first time and be divided into supernatant plasma layer, boundary leucocyte platelet layer and lower part Red blood cell layer.Syringe needle extracts a little red blood cell under supernatant, interlayer, interlayer, moves into sterile centrifugation tube, for the second time Platelet rich plasma is obtained after removing upper layer platelet poor plasma with syringe needle after centrifugation.By 0.2ml platelet rich plasma Platelet concentration in inspection examination platelet rich plasma after being diluted to 2mL with 0.9% sodium chloride injection, will according to each group concentration Platelet rich plasma obtained is diluted to after target zone with 0.9% sodium chloride injection and leaves and takes 2mL, and 10% calcium chloride is added 0.2ml activates the blood platelet in platelet rich plasma.
Preparing according to 1 gram of semi-finished product E and being added to the platelet concentration of 5mL is (1900~2500) × 109In the PRP of/L.
Embodiment 2-5
The step of preparation, is shown in Table 1 with embodiment 1, each step design parameter.
Each parameter in 1 embodiment 1-5 test procedure of table
DNA residue detection
Respectively implemented according to the standard detection of 3.6 clauses in " decellularized vascular matrix matrix " medical device product technical requirements DNA residual quantity in the semi-finished product E (regenerating tissues matrix particles) of example, the results are shown in Table 2, DNA residual quantity 0.3~0.7ng/mg it Between.
The DNA retention analysis of 2 embodiment 1-5 semi-finished product E of table
Embodiment 1 Embodiment 2 Embodiment 3 Embodiment 4 Embodiment 5
DNA residual quantity (ng/mg) 0.3 0.4 0.5 0.6 0.7
Malvern Particle Size Analyzer measures partial size
When being measured using Malvern Particle Size Analyzer, D50 indicates that the cumulative particle sizes percentile of a sample reaches Corresponding partial size when 50%.D50 is commonly used to indicate the average particle size of powder.The cumulative particle sizes of mono- sample of D95 are distributed percentage Number reaches partial size corresponding when 95%.Its physical significance is that partial size is less than its particle and accounts for 97%.Mono- sample of D10 tires out Meter size distribution percentage reaches partial size corresponding when 10%.D10 and D95 is commonly used to indicate that the granularity at powder thickness both ends refers to Mark.
Other embodiments product is equally detected, the diameter of particle of embodiment 1-5 semi-finished product E is in 1.2mm hereinafter, its D50 Between 0.018~1.0mm, D95 is between 0.025~1.2mm, and D10 is at 0.45~20 μm.
Application Example
Injecting method
Before injection, patient's horizontal position is suffered from kneepad pillow and is slightly bent, loosens knee joint, sufficient neutral position.Partly sterilised chooses kneecap Lateral border and upper limb interface point are entry point puncture, have been entered when having to break through sense or can extract hydrops articuli prompt syringe needle out Suprapatellar bursa articular cavity injects 1~2ml of implant of embodiment 1.Passive flexion and extension active patient knee joint 3~5 times, wear after injection Thorn point aseptic dressing covering wrapping.And weekly knee joint injection treatment, continue six weeks.
Effective percentage evaluation
Medical Patients with Knee Osteoarthritis 132 is taken, 1 group of PRP group and test are randomly divided into, 2 groups of test is (of the invention Regenerating tissues matrix implant group), wherein 44 people of PRP group patient, the intracavitary injection PRP 2ml of patient articular；Test 1 group of patient 44 People, the intracavitary 1 implant 1ml of the injection embodiment of the present invention of patient articular；2 groups of 44 people of patient are tested, the intracavitary injection of patient articular is originally 2 implant 1ml of inventive embodiments.Three groups are injected 1 time, the course for the treatment of 6 weeks weekly totally.Before treatment and 3,6,9 months after the course for the treatment of, Patient symptom is assessed using Western and McMaster University Osteoarthritis Index (WOMAC), WOMAC scoring is higher to be shown Symptom is more serious.
It is handled with SPSS17.00 statistics software, is scored, examined using t, as a result such as using WOMAC Osteoarthritis Index Table 3.
3 two groups of patient's different time follow-up record WOMAC scorings of table are compared
Group Before treatment 3 months 6 months 9 months
PRP group 40.36±11.28 20.15±2.32 25.66±6.32 30.87±7.22
Test 1 group 40.57±10.83 10.34±2.19 11.86±4.57 15.69±6.14
Test 2 groups 40.53±10.23 10.44±2.09 11.96±4.37 15.89±6.04
t 0.021 1.563 1.842 4.657
P 0.883 0.124 0.101 0
Conclusion: 1 group of PRP group and test before treating test 2 groups of WOMAC scorings without significant difference, 1 group, examination are tested after treatment Test 2 groups than PRP group WOMAC score it is low, therapeutic effect is significant；6th month after treatment, the 9th month follow-up, compared with PRP group WOMAC scoring is significantly better than that PRP group；Test 1 group, 2 groups of patients of test follow-up WOMAC scoring in the 3rd, 6,9 month after the treatment Compare, scoring is improved over time, still remains notable difference with preceding WOMAC scoring is treated.PRP group and test 1 Group, 2 groups of test have certain clinical efficacy for improvements of gonitis patient symptom, test group treats knee pass compared with PRP group Section inflammation has better long-term efficacy.
Above embodiment is only that preferred embodiments of the present invention will be described, is not carried out to the scope of the present invention It limits, without departing from the spirit of the design of the present invention, this field ordinary engineering and technical personnel is to technical solution of the present invention The all variations and modifications made, should fall within the scope of protection determined by the claims of the present invention.

Claims (11)

1. a kind of articular cartilage remediation composition of matrix particles containing regenerating tissues, which is characterized in that include regenerating tissues matrix Particle and PRP, the PRP are to contain platelet concentration (1200~1600) × 109The platelet rich plasma of/L.
2. articular cartilage remediation composition according to claim 1, which is characterized in that the regenerating tissues matrix particles and The mass volume ratio of PRP is 1g:4~6mL.
3. articular cartilage remediation composition according to claim 1, which is characterized in that in the regenerating tissues matrix particles DNA residual quantity in 2.0ng/mg hereinafter, the partial size of the regenerating tissues matrix particles in 1.2mm hereinafter, D50 0.018~ Between 1.0mm.
4. the preparation method of the described in any item articular cartilage remediation compositions of claims 1 to 3, which is characterized in that including with Lower step:
1) by the glycerite of allograft skin raw material investment alkali, oscillation treatment is impregnated, semi-finished product A is obtained；
2) it after semi-finished product A being removed epidermis, puts into surfactant solution, ultrasonic immersion treatment obtains semi-finished product B；
3) semi-finished product B is cut into small skin graft, 3~10min in liquid nitrogen is immersed in a manner of immersing, taken out as semi-finished product C；
4) under the action of high speed rotation centrifugal force, semi-finished product C is cut with rotor, obtains semi-finished product D；
5) semi-finished product D is placed in D- amino acid solution and is impregnated, separation of solid and liquid obtains semi-finished product E；The D- amino acid solution Selected from one or more of aspartic acid, alanine, glutamic acid, serine；
6) semi-finished product E is mixed with PRP to get implant.
5. the preparation method according to claim 4, which is characterized in that alkali described in step 1) is sodium hydroxide and/or hydrogen Potassium oxide, the concentration of the alkali are 0.5~5%；The concentration of further preferred alkali is 1~3%；
And/or in the glycerite of the allograft skin raw material investment alkali, 1~10h is impregnated preferably at 25~35 DEG C and is vibrated Processing.
6. the preparation method according to claim 4, which is characterized in that surfactant described in step 2) be SDS or Triton X-100；The concentration of the surfactant solution is 0.1~0.3g/L；
Ultrasonic soaking conditions are as follows: 20~45KHz, temperature are 1~20 DEG C, 1~5min of ultrasound, impregnate 1~4h, preferably repeat ultrasound With immersion treatment 2~6 times, the surfactant solution renewed before repeating.
7. the preparation method according to claim 4, which is characterized in that semi-finished product B is crosslinked with crosslinking agent, then into Row is cut out；
The crosslinking agent is the genipin solution of mass concentration 0.1~0.6%, time of crosslinking is 2~for 24 hours.
8. the preparation method according to claim 4, which is characterized in that semi-finished product D described in step 4) by feed inlet into Entering speed is 50~100g/min, it is preferable that 50~80g/min；
Preferably, step 5) using the concentration of aspartic acid, alanine, glutamic acid, serine be respectively 0.8~1.4,1.2~ The amino acid solution of 1.8,0.4~0.6,4~10.8 μm of ol/L, example is 1:(0.8~1.2 by volume): (0.8~1.2): (0.8~1.2) it prepares.
9. according to the described in any item preparation methods of claim 4~8, which is characterized in that
It is described PRP's the preparation method comprises the following steps: acquisition autologous vein blood in anticoagulant tube, first time relative low speeds centrifugation, make blood plasma and Blood platelet is divided into three layers with red blood cell and leucocyte；Take middle layer to carry out second relatively high speed centrifugation, be further concentrated PRP and Thrombocyte plasma component is separated, the PRP of middle layer higher concentration is obtained；It is activated with calcium chloride.
10. preparation method according to claim 8, which is characterized in that the parameter of centrifugation for the first time are as follows: 120~150 × g, It is centrifuged 5~15min；The parameter of second of centrifugation are as follows: 400~600 × g is centrifuged 10~25min；Preferably；1/10 volume is added 10% calcium chloride solution activated.
11. the described in any item joint repair compositions of claims 1 to 3 are in the repair materials of preparation treatment articular cartilage damage In application.
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