CN103272258A - Novel freeze-drying platelet-rich fibrin, as well as preparation and application thereof - Google Patents
Novel freeze-drying platelet-rich fibrin, as well as preparation and application thereof Download PDFInfo
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- CN103272258A CN103272258A CN2013101802842A CN201310180284A CN103272258A CN 103272258 A CN103272258 A CN 103272258A CN 2013101802842 A CN2013101802842 A CN 2013101802842A CN 201310180284 A CN201310180284 A CN 201310180284A CN 103272258 A CN103272258 A CN 103272258A
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Abstract
The invention discloses a preparation method of a novel freeze-drying platelet-rich fibrin. The method comprises steps of: centrifuging the fresh blood to obtain the gel-like platelet-rich fibrin (PRF), freeze drying the gel-like platelet-rich fibrin for 24 hours in the vacuum low-temperature environment of 400 Pa and minus 45-minus 4 DEG C so as to obtain the freeze-drying platelet-rich fibrin (PRF) with bioactivity, and preparing the sheet, granular or powdery freeze-drying platelet-rich fibrin (PRF) according requirements so as to be used for preparing trauma auxiliary materials, tissue engineering scaffolds or other biological materials for recombination use. The method can maintain the activity of the fresh PRF, so that the PRF can be easily and refrigerated for a long time, is convenient and flexible to use, can be used as the trauma auxiliary material or the scaffold material to be compounded with other tissue engineering materials and can be applied to war injury repair and tissue defect repair.
Description
Technical field
The invention belongs to biomaterial and field of tissue engineering technology, relate to the technology of preparing of biomaterial, particularly a kind of new lyophilizing rich platelet fibrin and preparation and application.
Background technology
French scientist Choukroun proposed a kind of a new generation in 2000 and is rich in platelet concentrate, be rich platelet fibrin (platelet-rich fibrin, PRF), to have manufacture method simple, with low cost because of it, be rich in somatomedin, long action time, certain tare effect, and plurality of advantages such as certain infection and immunologic function, in the wound of various soft tissues and sclerous tissues and defect repair research, paid close attention to widely, and obtained gratifying effect.But, be subjected to the limitation of PRF preparation method at present, use the existing usefulness that only limits to individuation at it and now get, namely gather fresh blood after, must be centrifugal rapidly in a few minutes, then, isolate gelatinous PRF.This mode, use clinically is very inconvenient, need prepare centrifuge at operative site, also brings very big inconvenience to operation; The popularization of PRF and the marketization, commercialization are brought great inconvenience.If purifying laboratory or purifying factory, adopt freeze-drying to make biologically active, dry, loose porous membrane-like, microgranular or pulverous PRF fresh PRF, be suitable for the cryopreserved biomaterial of long term seal.Like this, will greatly provide clinical ease of use, enrich the purposes of PRF, promote large-scale production, promote lyophilizing PRF to enter clinical practice from laboratory.At present, by consulting domestic and foreign literature, do not find the research report of this respect as yet.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, the object of the present invention is to provide a kind of new lyophilizing rich platelet fibrin and preparation and application, the activity that can keep fresh PRF, be easier to long-term cold preservation, easy to use and flexible, can be compound as wound adjuvant or timbering material and other tissue engineering materials, repair in trauma and tissue defect reparation are applied to fight.
To achieve these goals, the technical solution used in the present invention is:
A kind of new fibrinous preparation method of lyophilizing rich platelet, from the centrifugal acquisition gel of fresh blood rich platelet fibrin (PRF), at 400Pa, lyophilization is 24 hours in-45 to-4 ℃ the vacuum low-temperature environment, obtains the lyophilizing rich platelet fibrin (PRF) of biologically active.
Described centrifugation step comprises:
Extract blood with asepsis injector from human or animal's vein, add the sterilization centrifuge tube rapidly, 3000r/min is centrifugal 10 minutes immediately, left standstill 30 minutes at 4 ℃ of refrigerators, after treating blood coagulation, the blood coagulation thing is taken out, with the blood clot excision of bottom, keep light yellow gel shape material, i.e. rich platelet fibrin (PRF).
Described gel rich platelet fibrin (PRF) is directly made loose porous lyophilizing rich platelet fibrin (PRF) through the lyophilization processing.
Described gel rich platelet fibrin (PRF) with the back extruding of sterile gauze parcel, is extruded liquid wherein, formed a membrane-like object, handle through lyophilization afterwards and make chaffy lyophilizing rich platelet fibrin (PRF)
Described gel rich platelet fibrin (PRF) is cut into microgranule, handle through lyophilization afterwards and make particulate lyophilizing rich platelet fibrin (PRF).
Described particulate lyophilizing rich platelet fibrin (PRF) is made pulverous lyophilizing rich platelet fibrin (PRF) through grinding.
Can in described lyophilizing rich platelet fibrin (PRF), sneak into from body ADSCs, for example in every 500mg lyophilizing rich platelet fibrin (PRF), sneak into 1.0x10
7From body ADSCs.
Described new lyophilizing rich platelet fibrin can be used for preparing wound adjuvant or tissue engineering bracket material or with the compound use of other biomaterial.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the preparation raw material sources of lyophilizing PRF have the advantage wide, that cost is low (with respect to the biotic factor preparation) of originating in human or animal's blood;
(2) lyophilizing PRF has the biological activity of fresh gel PRF, have fibrin scaffold which effect and the effect of slow release biotic factor, contain multiple biotic factor, for example, platelet-derived growth factor (PDGFs), vascular endothelial cell growth factor (VEGF), transforming growth factor-1(TGF β-1), fibroblast growth factor (FGF), insulin like growth factor (IGFs) etc., and have the ability of good promotion soft or hard tissue regeneration.The tissue repair effect of PRF mainly realizes by two aspects, i.e. the regulating action of cytokine and fibrinous cytoskeleton effect;
(3) lyophilizing PRF has good safety.Freeze-drying is the conventional method of preserving biological product, and simultaneously, freeze-drying can also reduce the immunogenicity of biomaterial.Lyophilizing PRF implants and can not cause immunological rejection;
(4) lyophilizing PRF preparation method is easy, can large-scale production, be easy to apply;
(5) lyophilizing PRF is easy to use, and existing usefulness is now done on the bed side again.Also can be prepared into the lyophilizing PRF of membrane-like, graininess or powdered according to application demand, be easy to storage and transportation.
(6) lyophilizing PRF can with other wounds and tissue defect repair materials Application of composite.
In a word, the lyophilizing PRF of the inventive method preparation have the source enrich, obtain conveniently, cheap, preparation is simple, safe and effective, do not have immunologic rejection, can with characteristics such as other wounds and tissue defect repair materials Application of composite.Preparation method and the using method of lyophilizing PRF are of universal significance, and be easy to utilize.
Description of drawings
Fig. 1 is the fresh PRF outline drawing of the present invention.
Fig. 2 is that the present invention press dry back PRF outline drawing.
Fig. 3 is PRF outline drawing after the lyophilization of the present invention.
Fig. 4 is lyophilizing PRF scanning electron microscope aspect graph of the present invention.
Fig. 5 is that the present invention tests two IL-2 measurement result canonical plottings.
Fig. 6 is that the present invention tests two IL-4 measurement result canonical plottings.
Fig. 7 is that the present invention tests two from body lyophilizing PRF and allosome lyophilizing PRF transplanting back rabbit skin and subcutaneous tissue HE colored graph.
Fig. 8 is that the present invention tests the fresh PRF of two xenogenesis and xenogenesis lyophilizing PRF transplants back rabbit skin and subcutaneous tissue HE colored graph.
Fig. 9 is that the present invention tests three third generation ADSCs HE colored graphs.
Figure 10 is that the present invention tests three third generation ASCs and becomes fat to induce 3 all oil red O stain figure.
Figure 11 is that the present invention tests three third generation ASCs osteogenic inductions, 3 all alizarin red colored graphs.
Figure 12 is that the present invention tests three and respectively organizes rabbit ear cartilage defect repair tissue A Erxinlan dyeing sketch map.
Figure 13 is that the present invention tests three ADSCs+PRF group cartilage defect in March repair tissue A Erxinlan dyeing sketch map, and arrow is depicted as the newborn cartilaginous tissue of the same area.
The specific embodiment
Describe embodiments of the present invention in detail below in conjunction with drawings and Examples.
The preparation method of rich platelet fibrin (PRF) biomaterial of freeze-drying of the present invention may further comprise the steps:
The first step extracts fresh blood (reaching the non-anticoagulation that individual state extracts certain volume as required) with asepsis injector from the human or animal, adds the sterilization centrifuge tube rapidly, and 3000r/min is centrifugal 10 minutes immediately, places 4 ℃ of refrigerators to leave standstill 30 minutes.
Second step, after treating blood coagulation, the blood coagulation thing is taken out, and the blood clot excision with the bottom keeps light yellow gel shape material, it is the rich platelet fibrin, with the back extruding of sterile gauze parcel, liquid is wherein extruded, form a membrane-like object, put into aseptic plate, place-80 ℃ of refrigerators standby.
The 3rd step, from cryogenic refrigerator, take out freezing PRF, be placed on then in the freezer dryer, open plate, temperature sensor is contacted freezing PRF, close hatch door, (400Pa under the vacuum and low temperature condition,-45 to-4 ℃) lyophilization 24 hours, be the PRF of lyophilization shape, M weighs
4-M
0, then, the sealing stored refrigerated.
The detailed process of vacuum lyophilization can be set as follows:
Freezing dry process: 24h
Freezing :-40 ℃, 120min (R) at the uniform velocity ,-40 ℃, 30min (H) fast cooling
Condense :-45 ℃, need extra 90min, guarantee that all samples temperature is identical.
Vacuum: 400pa
Dry :-40 ℃, 120min, H
-35℃，120min,R
-30℃，120min,H
-25℃，120min,R
-20℃，120min,H
-15℃，120min,R
-10℃，120min
-4℃，120min
4℃，120min
Redrying: 4 ℃, 180min
Can be prepared into various ways as required, from the gelatinous PRF of the centrifugal acquisition of fresh blood, liquid is wherein extruded in the back extruding of sterile gauze parcel, forms a membrane-like object, through handling through freeze-drying, makes chaffy PRF again; Also can discharge moisture without extruding from the gelatinous PRF of the centrifugal acquisition of fresh blood, directly carry out lyophilization, make loose PRF; As required, fresh gelatinous PRF can be cut into fine particles, carry out lyophilization, make particulate PRF; As required, with cryodesiccated PRF microgranule, through suitably grinding, make pulverous PRF.
For verifying safety and the effectiveness of lyophilization PRF biomaterial of the present invention, carry out following test:
Experiment one, the preparation of lyophilizing PRF and the experimentation of characteristic thereof
The experimentation of experiment two, lyophilizing PRF animal subcutaneous transplantation
Experiment three, lyophilizing PRF repair the experimentation of rabbit ear cartilage defect
Experiment one, the preparation of lyophilizing PRF and the experimentation of characteristic thereof
This experiment purpose is intended to prepare lyophilization PRF and the characteristic of its ultrastructure and release somatomedin is inquired into.
1 materials and methods
1.1 laboratory animal
10 6 monthly age healthy rabbits, body weight is 2～3kg, purchases the animal center in The Fourth Military Medical University, Stomatological Hospital, Military Surgeon Univ. No. 4's Experimental Animal Center cleaning level barrier environment is raised and experiment.
1.2 main experiment reagent, equipment and equipment
Rabbit VEGF ELISA test kit (west, Shanghai Tang bio tech ltd, Chinese Shanghai).
Glass centrifuge tube, syringe, sterile gauze, culture dish, aseptic tweezer, sterile scissors.
Centrifuge (Hunan, Hunan instrument, TDZ5-WS), general refrigerator (company of Haier), cryogenic refrigerator (U.S. power ﹠ light company), freezer dryer (ADPXL3, the U.S.), scanning electron microscope (S-4800 HIT), microscope (German Leica company), enzyme-linked immunosorbent assay instrument (TECAN GENIOS company, Switzerland).
1.3 the preparation method of lyophilizing PRF
(1) get rabbit handstand position and fix, do not give anaesthetic, from ear medium-sized artery blood drawing 10-20ml, put into the glass centrifuge tube immediately, 3000r/min, 10min is centrifugal, places 4 ℃ of refrigerators to leave standstill 30 minutes.
(2) treat blood coagulation after, the blood coagulation thing is taken out, blood clot excision with the bottom, keep light yellow gel shape material, namely the rich platelet fibrin pushes with sterile gauze parcel back, liquid is wherein extruded, form a membrane-like object, put into aseptic plate, place-80 ℃ of refrigerators standby.
(3) from cryogenic refrigerator, take out freezing PRF, be placed in the freezer dryer then, open plate, temperature sensor is contacted freezing PRF, close hatch door, (400Pa under the vacuum and low temperature condition,-45 to-4 ℃) lyophilization 24 hours, be the PRF of lyophilization shape, M4-M0 weighs, then, the sealing stored refrigerated is standby.
1.4 the scanning electron microscopic observation of lyophilizing PRF
Lyophilizing PRF is sheared suitably size, carry out metal spraying routinely, with the ultrastructure of scanning electron microscopic observation lyophilizing PRF.
1.5PRF discharge the VEGF sample detection
With fresh PRF diaphragm and the lyophilizing PRF diaphragm for preparing, and have in the aseptic centrifuge tube of the aseptic DMEM culture fluid of 4ml in a PRF diaphragm is positioned over respectively, collect sample respectively by seven different time points then, i.e. 20min, 4h, 24h, 72h, 120h, and168h.After different time points finishes, the PRF diaphragm is transferred in another new centrifuge tube that 4ml PBS is housed, and places-70 ℃ of preservations to be detected 4mlPBS collection liquid before, and the like.Experiment repeats 3 times.
Elisa (ELISA) detects step:
Application of sample: every hole adds standard substance and PRF discharges sample 100ul, with the rearmounted 37 ℃ of 2h of the abundant mixing of Sptting plate.
Wash plate: with cleaning mixture Sptting plate is fully washed 4-6 time, seal is done on the filter paper.
Add first antibody working solution 100ul in every hole.With the abundant mixing of Sptting plate rearmounted 37 ℃ 60 minutes.
Wash plate: the same.
Every hole adds enzyme labelled antibody working solution 100ul.With Sptting plate put 37 ℃ 30 minutes.
Wash plate: the same.
Every hole adds substrate working solution 100ul, puts 37 ℃ of dark place reactions 15 minutes.
Every hole adds 100ul stop buffer mixing.
Survey light absorption value at the 450nm place with microplate reader in 30 minutes.
A collected data Curve Expert1.4 computed in software time point VEGF content.
2 results and analysis
Visible three layerings in the centrifuge tube of centrifugal back: basecoat is the erythrocyte layer, middle one deck is milky shape PRF gel, last layer is limpid supernatant, then with aseptic nipper with the PRF gel from managing interior taking-up, its shape is as shown in Figure 1, unnecessary liquid is blotted in the extruding of double-layer sterile gauze, the PRF diaphragm can prepare, and its shape is made dry PRF diaphragm as shown in Figure 2 behind lyophilization 24h, its shape as shown in Figure 3, the sealing cold preservation standby.
Scanning electron microscopic observation lyophilizing PRF is loose porous shape structure, and as shown in Figure 4, this structure is conducive to adhering to of cell and grows.
The fresh PRF of table 1. and lyophilizing PRF discharge the characteristic (pg/ml) of VEGF
Lyophilizing PRF and fresh PRF compare, P〉0.05
The ELISA testing result shows, no matter is fresh PRF diaphragm, or lyophilizing PRF diaphragm, and it discharges VEGF and presents certain regularity.PRF continue to discharge VEGF in a week, the speed that 4h discharged before its characteristics presented is very fast, 1-3d rate of release and then slow down, and 3～7d still continues release VEGF, but speed obviously slows down.Different time points PRF accumulative total discharges the VEGF amount to be increased gradually, and maximum can reach the 1700pg/ml level, and is as shown in table 1.Lyophilizing PRF diaphragm discharges the characteristic of VEGF and the fresh PRF of contrast compares, difference does not have significant statistical significance (P〉0.05), show that lyophilizing PRF still keeps discharging the ability of somatomedin, this specific character is for promoting wound healing and damaged injury repairing that positive meaning is arranged.
The experimentation of experiment two, lyophilization PRF animal subcutaneous transplantation
This experiment purpose is intended to verify whether lyophilization PRF can cause immunological rejection at allogeneic or heterogenous animal subcutaneous transplantation.
1 materials and methods
1.1 laboratory animal
10 6 monthly age healthy rabbits, body weight is 2～3kg; 5 10 age in week healthy BALB/c mouse, body weight 20～28g purchases the animal center in The Fourth Military Medical University, Stomatological Hospital, Military Surgeon Univ. No. 4's Experimental Animal Center cleaning level barrier environment is raised and experiment.
1.2 main agents and instrument
Pentobarbital sodium (section's sky is biological)
Freezer dryer (ADPXL3, the U.S.), optical microscope (Olympus, Japan), flow cytometer (BD FACSAria, the U.S.).
1.3 the preparation of lyophilizing PRF
With experiment one, the concrete form of lyophilizing PRF does not influence this result of experiment.
1.4 the rabbit subcutaneous transplantation of lyophilizing PRF experiment
Get six of the healthy rabbits of 6 months sizes, male and female are not limit, weigh, ear's depilation, the sterilization auricular vein, 3% pentobarbital sodium is in 1ml/kg ratio auricular vein injecting anesthetic rabbit, after waiting to anaesthetize produce effects, omoplate district, back preserved skin, iodophor disinfection, the shop aseptic towel is single, and knife blade cuts the skin holostrome, and passivity is separated makes the skin below form the PRF that a little gap holds lyophilizing altogether, implant the lyophilizing PRF500mg for preparing subcutaneous, tight sew up wound, and carry out labelling on the surface, implantation region, experiment grouping and consumption are as shown in table 2.
Table 2 experiment grouping and implant consumption
1.5 rabbit experiment observation index
1.5.1 gross examination of skeletal muscle
Implant the back at lyophilizing PRF and observe the skin implantation region every day, with skin color, have or not redness, fester, it is observation item that the part has or not downright bad, and record.
1.5.2 the rabbit lymphocyte subgroup is measured
Get respectively transplant before, transplant back 1w, 2w, 3w gathers laboratory animal rabbit vein blood 2ml, anticoagulant; Adopt the fluidic cell detection method to measure lymphocyte subpopulation CD4 and CD8, calculate CD4/CD8 ratio;
Flow cytometry lymphocyte method:
1. get respectively transplant before, transplant back 1w, 2w, 4w laboratory animal rabbit vein blood 2ml, anticoagulant;
2. get wherein two parts of 100ul whole bloods, add CD4, CD8 antibody 10ul respectively, room temperature lucifuge reaction 30 minutes;
3. add erythrocyte cracked liquid 2ml, room temperature lucifuge reflection 10 minutes;
4. centrifuge 1000rpmin, 5min;
5. PBS washed twice, each 2ml, the centrifugal 1000rpmin of centrifuge, 5min;
6. add the 500ul1% paraformaldehyde and fix, or 500ulPBS, detect with flow cytometer in 2 hours.
1.5.3 the rabbit humoral immunization detects: IL-2, IL-4 detect
Respectively at postoperative 1w, 2w, 3w, auricular vein is gathered experimental rabbit venous blood 2ml, 1000r/min, and 5min, centrifugal, collect blood plasma ,-20 ℃ of refrigerators are preserved, row IL-2, IL-4, ELISA detects.
Concrete detection method:
1. the plasma sample of collecting is at room temperature thawed, to be measured;
2. application of sample: every hole adds standard substance or testing sample (sampling blood plasma) 100ul respectively, and low speed shaking table 2min makes the abundant mixing of sample, and 37 ℃, 40 minutes;
3. wash plate: every hole is filled it up with cleaning mixture Sptting plate is fully washed 5 times at least, is inverted on the filter paper and blots;
4. every hole adding primary antibodie working solution and each 50ul(blank well of distilled water do not add).Low speed shaking table 2min makes the rearmounted 37 ℃ of reactions of the abundant mixing of sample 20 minutes;
5. wash plate: method is the same;
6. get enzyme labelled antibody working solution 100ul and add in the hand-hole, 37 ℃ of reaction 10min;
7. wash plate: method is the same;
8. get substrate working solution 100ul and add each hole, 37 ℃ of lucifuges were reacted 15 minutes;
9. the 100ul stop buffer adds each hole mixing cessation reaction;
10. microplate reader 450nm place measures light absorption value (in 30 minutes).
1.5.4 rabbit PRF graft area tectology is observed
Inject 3% pentobarbital sodium 1ml/kg through the rabbit auricular vein, the anesthesia rabbit is excised the skin holostrome with scalpel in tame rabbit back PRF implantation region, tight sew up wound, partial smearing erythromycin eye ointment prevention infection.Subsequently specimen is placed fixedly 24h of 4% formalin, dehydration, transparent, paraffin embedding, the section of making 5 μ m bed thickness.With slice row HE dyeing, observe lyophilizing PRF implantation region skin histology PRF absorbing state and have or not lymphocytic infiltration.
1.6 lyophilizing allogene PRF mouse subcutaneous transplanting experiment
With 3% pentobarbital sodium 1ml/kg, the intraperitoneal injection of anesthesia mice, sterilization mouse back skin at spinal column two lateral incision 10mm otch, moves under water that it is subcutaneous to separate, and the heavily about 50mg of 10X10mm lyophilizing PRF(respectively) transplant in mouse back subcutaneous, sew up wound.Postoperative is conventional raises, and observes to change substantially in 1 week, 2 weeks, the activity of 3 all mices, skin of back.Respectively at 1 week of postoperative, 2 weeks, 3 weeks, draw materials conventional fixing, paraffin embedding, section (thick 5 μ m) HE dyeing, om observation PRF absorbing state and have or not lymphocytic infiltration at PRF graft area skin.And all do contrast with the normal mouse skin histology.
1.7 statistical analysis
Data with ± the SD form is represented, is that statistical tool carries out data analysis with the SPSS16.0 statistical software.Statistical analysis is carried out in lymphocyte CD 4 and CD8 subgroup, CD4/CD8 ratio and plasma IL-2, the variance analysis of IL-4 horizontal application repeated measure, and inspection level α value is got bilateral 0.05.
2 results
2.1 rabbit gross examination of skeletal muscle result
Lyophilizing PRF implants the back and observed for 3 weeks continuously, and two experimental grouies there is no skin infection, redness, fester and the appearance of downright bad symptom, and showing does not have tangible immunological rejection to occur.
2.2 the level of rabbit lymphocyte CD 4 and CD8 subgroup
2.2.1CD4 the subgroup testing result shows: two groups of laboratory animals do not have statistical significance (P〉0.05) with the embedding CD4 testing result difference of different time points afterwards embedding before, can think and implant the variation zero difference that does not have influence, different time points CD4 value from the detection of body lyophilizing PRF and the CD4 value of implantation allosome lyophilizing PRF, as shown in table 3.
The variation of table 3 liang experimental group different time points CD4
P〉different time points is relatively between 0.05, two group
2.2.2CD8 the subgroup testing result shows: two groups of laboratory animals do not have statistical significance (p〉0.05) with the embedding CD8 testing result difference of different time points afterwards embedding before, can think implant from body lyophilizing PRF with implant allosome lyophilizing PRF after the variation zero difference of CD8 detected value zero difference, different time points CD8 value, as shown in table 4.
The variation of table 4 liang experimental group different time points CD8
3.2.3CD4/CD8 ratio calculation result shows: two groups of laboratory animals do not have statistical significance (P〉0.05) with the embedding CD4/CD8 ratio difference of different time points afterwards embedding before, can think implant from body lyophilizing PRF with implant allosome lyophilizing PRF after the variation zero difference of CD4/CD8 ratio zero difference, different time points CD4/CD8 value, Non Apparent Abnormality immunne response phenomenon is described, as shown in table 5.
The variation of table 5 liang experimental group different time points CD4/CD8 (
± S)
P〉different time points is relatively between 0.05, two group
2.3 plasma in rabbit IL-2 and IL-4 level
2.3.1 plasma IL-2 horizontal detection result shows: shown in Fig. 5 and table 6, the IL-2 testing result difference not statistically significant of two groups of laboratory animals different time points before embedding and after embedding (P〉0.05), can think and Non Apparent Abnormality immunne response phenomenon is described the variation zero difference of implanting IL-2 value zero difference, different time points IL-2 value behind body lyophilizing PRF and implantation allosome lyophilizing PRF.
The variation (Pg/ml) of table 6 liang experimental group different time points IL-2 level
P〉different time points is relatively between 0.05, two group
2.3.2 plasma IL-4 horizontal detection result shows: shown in Fig. 6 and table 7, two groups of laboratory animals before transplanting with the IL-4 testing result difference not statistically significant of transplanting the back different time points (P〉0.05), can think and Non Apparent Abnormality immunne response phenomenon is described the variation zero difference of implanting IL-4 value zero difference, different time points IL-4 value behind body lyophilizing PRF and implantation allosome lyophilizing PRF.
The variation of different time points IL-4 before and after table 7 liang group laboratory animal is transplanted
2.4 rabbit PRF graft area tectology characteristics
Allosome lyophilizing PRF group: implant back 1w at PRF, as seen section still has PRF to exist around skin histology, and the organizational structure of show color densification does not have lymphocytic infiltration on every side under the light microscopic; Behind the 2w, PRF is absorbed gradually, and smaller volume merges with surrounding tissue, and surrounding tissue does not have lymphocytic infiltration; Behind the 3w, skin histology presents normal organizational structure, and PRF is absorbed fully, and is consistent with blank group structure.Show same result as shown in Figure 7 from body lyophilizing PRF group.The transplanting that shows allosome lyophilizing PRF can not cause tangible immunological rejection.
2.5 mice PRF graft area tectology characteristics
It is all normal to transplant 1 week behind the allogene lyophilizing PRF, 2 weeks, 3 all mice activities, and skin wound healing is all good; Tangible inflammatory reaction is not seen in postoperative 1 all skin and subcutaneous tissue HE dyeing, only the visible PRF that does not absorb fully in the part visual field; Inflammatory reaction is not seen in skin and subcutaneous tissue HE dyeing after 2 weeks, and lyophilizing PRF is degraded and absorbed; Skin and subcutaneous tissue HE dyeing and normal skin section zero difference after 3 weeks, as shown in Figure 8.
To sum up, the transplanting of lyophilizing PRF can not cause can detected tangible immunological rejection, can be used as a kind of safe biomaterial and uses.
Experiment three, lyophilization PRF repair the experimentation of rabbit ear cartilage defect
This experiment purpose is intended to verify that lyophilization PRF repairs the damaged repairing effect of the credulous bone holostrome of rabbit.Cartilage injury and damaged reparation are compared with soft tissue and osseous tissue, still have more difficulty clinically.If lyophilizing PRF has the cartilage injury of promotion and damaged repair, will produce positive meaning for other tissue injurys and damaged reparation so.
1 materials and methods
1.1 20 of laboratory animal healthy rabbits, body weight 2--3Kg purchases the animal center in The Fourth Military Medical University, raises the Experimental Animal Center barrier environment in Stomatological Hospital, Military Surgeon Univ. No. 4.
1.2 main agents and instrument and equipment DMEM/F12 culture medium (Hyclone, the U.S.), green grass or young crops-streptomycin (Hyclone, the U.S.), I Collagen Type VI enzyme (sigma, the U.S.), calf hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company, Hangzhou), pancreatin cell dissociation buffer (green skies biotechnology research institute, Chinese Shanghai), alizarin red (Shanghai Suo Laibao bio tech ltd, Chinese Shanghai), oil red O(Sigma, the U.S.), FITC labelling goat anti-mouse monoclonal antibody (Abcam, the U.S.), mouse anti rabbit CD31 and CD29 monoclonal antibody (Abcam, the U.S.), flow cytometer (BD company, the U.S.), cell culture incubator (Thermo, the U.S.), inverted microscope and biological microscope (Nikon, Japan).
1.3 experimental technique
1.3.1 rabbit fat stem cell (ADSCs) is cultivated and is identified
Get healthy 6 the monthly age rabbit, the about 3～4kg of body weight, male and female are not limit, 3% pentobarbital sodium is pressed 1ml/kg anesthesia, gets omoplate district, back fatty tissue 10ml, adopts the tissue digestion method to carry out the stem cell cultivation, with cell inoculation in 25cm
2Culture bottle in, volume fraction is 5%CO
2, 37 ℃ of saturated humidities are cultivated.3d changes liquid, and flush away is attached cell not, changes liquid 1 time in per 3～4 days according to the cell growing state later on, when cell grow about 90% the time, in the inoculation of going down to posterity of 1:3 ratio.Living cells and HE dyeing are observed under the inverted microscope and are taken a picture.
1.3.1.1ADSCs one-tenth fat inducing culture: get the 3rd generation ADSCs, with 1*10
5/ cm
2Be inoculated in 1.3*1.3cm
2Coverslip adds into the fat inducing culture after fusion reaches 90%, changes liquid once in per 3 days, and after 3 weeks, oil red O stain is identified.
1.3.1.2ADSCs osteogenic induction cultivate: get the 3rd generation ADSCs, with 1*10
5/ cm
2Be inoculated in 1.3*1.3cm
2Coverslip, fusion reach 80% back and add the osteogenic induction culture medium, change liquid once in per 3 days, and after 3 weeks, the calcium tuberosity is identified in alizarin red dyeing.
1.3.1.3 Flow cytometry stem cell surface CD molecule
Collect the 3-5 rabbit fat fat stem cell in generation, and to adjust cell density be 106/ml, at 1000r/min, under the 5min condition, PBS centrifuge washing 2 times, 100 μ l PBS re-suspended cells, add mouse-anti rabbit monoclonal antibodies primary antibodie CD29 and CD31 respectively, place 4 ℃ to hatch 30min, the PBS rinsing, the antibody two that adds FITC labelling goat-anti mice is anti-, 4 ℃ of lucifuges are hatched 30min, the centrifugal rinsing of PBS, 1000r/min, 5min, machine testing on the fluidic cell detector.
1.3.2 the preparation of lyophilizing PRF
With experiment one.
1.3.3 the experiment that the rabbit cartilage defect of lyophilizing PRF is repaired
Get 10 of healthy rabbits, in tame rabbit ear medium-sized artery mid point both sides difference separate skin holostrome, expose cartilage, with knife blade the cartilage holostrome is excised, it is damaged that every ear of rabbit is done two places, is divided into four groups of A, B, C, D at random, the damaged square area that is length of side 5mm in every place, each organizes the defective region implant and consumption is: A group, blank; The B group is from body ADSCs(1.0x10
7); The C group, lyophilizing PRF(0.05g); The D group is from body ADSCs(1.0x10
7)+lyophilizing PRF(0.05g); Every group of 5 samples.After the implantation, skin suture is closed otch.Every the conventional injection of rabbit penicillin infection, continuous three days, the conventional raising.Repeated trials 2 times.
1.3.4 laboratory animal is drawn materials and is detected
After transplanting back 1 month, 2 months, 3 months, laboratory animal auricular vein injection air under the general anesthesia state is put to death, separate top layer skin, expose cartilage defect reparation district, cut and respectively organize specimen, the repairing effect of gross examination of skeletal muscle cartilage.Subsequently specimen is placed fixedly 24h of 4% formalin, dehydration, transparent, paraffin embedding, the section of making the 5um bed thickness.A Erxinlan dyeing is done in section.Use the IPP6.0 image analysis software that newborn cartilage amount is carried out semi-quantitative analysis.
1.4 statistical analysis
Data are used
Form is represented, is that statistical tool carries out data analysis with the SPSS16.0 statistical software.Inspection level α value is got bilateral 0.05.
2 results
2.1 rabbit ADSCs morphologic observation and evaluation
2.1.1 rabbit ADSCs morphology characteristics
The ADSCs in former generation is spindle shape, the growth of colony sample.Inoculation back is about two weeks, about 80% converge.More unified through the ADSCs form that goes down to posterity after cultivating, be spindle shape fibroblast sample, the cell integral body that covers with is swirl shape, radial arrangement.ADSCs is after HE dyeing, and as seen typically endochylema is red dyes, and nucleus indigo plant is dyed, and cellular morphology is consistent with living cells form under the mirror, as shown in Figure 9.
2.1.2 rabbit ADSCs becomes the ability of fat and skeletonization
(1) ADSCs becomes fat to induce: after adding into the fat induced liquid, visible cell gradually becomes polygon, occur little fat behind the 3d in the cell and drip, one week back fat drip to merge and become big, along with the prolongation fat of incubation time drips more and morely, oil red O stain is positive, as shown in figure 10.
(2) ADSCs osteogenic induction: the ADSCs through osteogenic induction grows in an overlapping, form becomes short fusiformis, polygon etc. by spindle shape, form the nodal-like structure about about 10d, 3 weeks dyeed by alizarin red, as seen the calcium deposition place is red lumps of dying, prompting calcium tuberosity forms, and shows that ASC is divided into osteoblast
As shown in figure 11.
2.1.3ADSCs the expression of surface C D molecule
Flow cytometry result shows that ADSCs cell CD29 The positive expression rate is that 84.28 ± 2.85%, CD31 The positive expression rate is 1.65 ± 0.31%, meets the feature of adipose-derived stem cell.
2.2 the repair of the rabbit ear cartilage defect of lyophilizing PRF
The blank group: transplant back 1m, 2m, 3m, can see that the cartilage defect district broken ends of fractured bone is neat, the cell engrain does not have any cambium and occurs, as shown in figure 12.
Organize from body ADSCs: transplant back 1m and do not see tangible newborn cartilage behind the 2m, the cartilage defect edge begins to occur a small amount of newborn cartilage behind the 3m; The coloration result of drawing materials in March all shows have a small amount of light light blue newborn cartilaginous tissue that dyes to occur at the ripe cartilage broken ends of fractured bone of the navy blue place of engrain, and is mutually continuous with ripe cartilaginous tissue, to the center growth, but quantity seldom, two groups of repair of cartilage effect unanimities, all relatively poor, as shown in figure 12.
Lyophilizing PRF group: transplant 1m cartilage defect edge, back and begin to occur a small amount of newborn cartilage, continuity along with the time, nattier blue newborn cartilaginous tissue appears in the defective region edge, by the damaged center growth of the broken ends of fractured bone two side direction, and its newborn cartilage amount obviously wants many than the ADSCs group, this group has certain repairing effect to cartilage defect, but desirable not enough, as shown in figure 12.
Organize from body ADSCs+ lyophilizing PRF: after body ADSCs+ lyophilizing PRF complex is implanted 1m, begin to occur more newborn cartilage at the cartilage defect edge, account for defective region 1/3rd; Along with time lengthening, newborn cartilage increases gradually, transplants to reach about 2/3rds behind the 2m, transplant 3m after visible cartilage defect district almost all covered by nattier blue newborn cartilaginous tissue, some tissue color is deepened, becomes ripe newborn cartilaginous tissue.High power lens is observed and see have a large amount of light blue inmature chondrocytes to surround around the ripe cartilaginous tissue of engrain, is indicated as newborn cartilaginous tissue, as Figure 12 and shown in Figure 13.
Newborn cartilage amount is carried out semi-quantitative analysis in the tissue slice of use IPP6.0, represents repair of cartilage effect (cartilage defect repair rate=newborn cartilage Mian Ji cartilage defect district gross area * 100%) with the cartilage defect repair rate, and the result is as shown in table 8.
Each group of table 8 is transplanted back different time points cartilage defect repair rate (%)
Compare with matched group: a.P＜0.05, b.P＜0.01, c.P＜0.001
As can be seen from Table 1: transplant ADSCs cartilage defect repair rate merely and only have an appointment 15.4%; The simple lyophilizing PRF that implants, the cartilage defect repair rate rises to about 32.8%(P＜0.01); With ADSCs and lyophilizing PRF use in conjunction, it is about 87.36% that the cartilage defect repair rate significantly is elevated to, and repairing effect is far superior to other each group (P＜0.001).
3 discuss
Test a result and show, that lyophilizing PRF has is loose, loose structure and can discharge biotic factor VEGF for a long time; Test two results and show, there is not immunological rejection in lyophilizing PRF in the transplantation experiments in allogeneic or heterogenous animal body, can be used as a kind of safe biomaterial.The result of study of this experiment three shows that lyophilizing PRF can significantly promote the damaged reparation of the credulous bone holostrome of rabbit, is a kind of biomaterial safely and effectively.
We observe rabbit ear cartilage defect district because providing of somatomedin is provided, and add that cartilaginous tissue self repair ability is very limited, therefore almost do not have newborn cartilaginous tissue to occur.Implant ADSCs in rabbit ear cartilage defect district, because cell does not have the anchorage that adheres to, injection cell runs off rapidly to the interior back of body, and is difficult for surviving, and lacks somatomedin, so only there is micro-cartilaginous tissue to form.Implant lyophilizing PRF in rabbit ear cartilage defect district, some more newborn cartilages appearred in 3 months in postoperative.As everyone knows, Freeze Drying Technique can be preserved the cytokine vigor among the PRF, can not destroy its biological activity, and, reduce immunogenicity widely.After implanting lyophilizing PRF in the rabbit body, PRF is rehydration gradually, slowly release its contained multiple somatomedin, for example, PDGF, VEGF, TGF-β, FGF, IGF etc., might the contiguous cartilaginous tissue of chemotactic under the effect of relevant somatomedin in chondroblast arrival cartilage defect district form newborn cartilage.This research is with lyophilizing PRF and ADSCs Application of composite, and postoperative 3 months promotes the holostrome reparation of rabbit ear cartilage defect significantly.The loose structure that shows lyophilizing PRF is conducive to adhering to of ADSCs stem cell and grows, and, can also the slow-release bio factor, promoted ADSCs propagation and differentiation, particularly stem cell differentiation to chondroblast in the cartilage defect district makes the defective region cartilaginous tissue be able to new life and recovery.Bibliographical information, Makoto etc. have carried out effective for repairing as a kind of biomaterial to the synovial membrane wound of Mus by using cryodesiccated platelet rich plasma (PRP) composite collagen sponge.PRF possesses the advantage of not adding ectogenic anticoagulant as the substitute products of PRP, and its quality is more tough, can be shaped, and compared with the character of the approximate liquid state of PRP remarkable advantages is arranged, and in addition, the interpolation of exogenous anticoagulant makes its safety in utilization reduce.
In sum, lyophilizing PRF demonstrates cartilage defect repair preferably in this experiment, the preparation that shows lyophilizing PRF has kept the active component among the PRF, and its repair in trauma effect to the soft or hard tissue is still effective, and the state of its lyophilizing makes it more convenient aspect preservation and transportation.Lyophilizing PRF is expected to become a kind of commercially produced product that is similar to biological product such as bone meal, is widely used in medical fields such as soft or hard tissue injury and defect repair.
Claims (10)
1. new fibrinous preparation method of lyophilizing rich platelet, it is characterized in that, from the centrifugal acquisition gel of fresh blood rich platelet fibrin (PRF), at 400Pa, lyophilization is 24 hours in-45 to-4 ℃ the vacuum low-temperature environment, obtains the lyophilizing rich platelet fibrin (PRF) of biologically active.
2. according to the described preparation method of claim 1, it is characterized in that described centrifugation step comprises:
Extract blood with asepsis injector from human or animal's vein, add the sterilization centrifuge tube rapidly, 3000r/min is centrifugal 10 minutes immediately, left standstill 30 minutes at 4 ℃ of refrigerators, after treating blood coagulation, the blood coagulation thing is taken out, with the blood clot excision of bottom, keep light yellow gel shape material, i.e. rich platelet fibrin (PRF).
3. according to the described preparation method of claim 1, it is characterized in that, described gel rich platelet fibrin (PRF) is directly handled through lyophilization made loose porous lyophilizing rich platelet fibrin (PRF).
4. according to the described preparation method of claim 1, it is characterized in that, described gel rich platelet fibrin (PRF) is pushed with sterile gauze parcel back, liquid is wherein extruded, form a membrane-like object, handle through lyophilization afterwards and make chaffy lyophilizing rich platelet fibrin (PRF)
5. according to the described preparation method of claim 1, it is characterized in that, described gel rich platelet fibrin (PRF) is cut into microgranule, handle through lyophilization afterwards and make particulate lyophilizing rich platelet fibrin (PRF).
6. according to the described preparation method of claim 5, it is characterized in that, described particulate lyophilizing rich platelet fibrin (PRF) is made pulverous lyophilizing rich platelet fibrin (PRF) through grinding.
7. a new lyophilizing rich platelet fibrin is characterized in that, is prepared by above-mentioned preparation method.
8. according to the described new lyophilizing rich platelet fibrin of claim 7, it is characterized in that, in described lyophilizing rich platelet fibrin (PRF), sneak into from body ADSCs.
9. described new lyophilizing rich platelet fibrin according to Claim 8 is characterized in that, sneaks into 1.0x10 in every 500mg lyophilizing rich platelet fibrin (PRF)
7From body ADSCs.
10. according to the purposes of the described new lyophilizing rich platelet fibrin of claim 7 for the preparation of wound adjuvant or tissue engineering bracket material.
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