CN101032631A - Preparation method of xenogeneic bone graft material - Google Patents
Preparation method of xenogeneic bone graft material Download PDFInfo
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- CN101032631A CN101032631A CNA2007100488227A CN200710048822A CN101032631A CN 101032631 A CN101032631 A CN 101032631A CN A2007100488227 A CNA2007100488227 A CN A2007100488227A CN 200710048822 A CN200710048822 A CN 200710048822A CN 101032631 A CN101032631 A CN 101032631A
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Abstract
A method for preparing a heterogeneous bone graft material, which takes animal bones as raw materials, comprises the following steps: (1) removing bone marrow and blood cells, washing the animal bone obtained under aseptic condition with high-pressure pulse distilled water or normal saline under normal pressure, room temperature and aseptic condition for a limited time for removing bone and marrow and blood cells in marrow cavity: (2) degreasing and washing by using ether, namely placing the animal bone from which the bone marrow and blood cells are removed in the step (1) in ether with the mass concentration of 95-100% for degreasing at normal temperature and normal pressure, wherein the use amount of the ether is limited by submerging the treated animal bone, the degreasing time is limited by removing fat in the animal bone, and after degreasing, washing by using sterile water to remove the ether; (3) ultra-deep low-temperature freezing, namely freezing the animal bone treated in the step (2) under normal pressure by liquid nitrogen; (4) and (3) gamma-ray irradiation, namely irradiating the animal bone frozen at the ultra-deep low temperature in the step (3) with gamma-ray.
Description
Technical field
The invention belongs to the bone grafting material field, particularly the preparation method of heterotransplantatioof of bones.
Background technology
The bone that illness such as wound, tumor and artificial joint overhaul technology cause is damaged to be one of difficult problem of puzzlement orthopedist always, though at present have a lot of methods to be used for the damaged reconstruction of bone, the bone transplanting is considered to wherein one of effective method.Autologous bone transplanting is owing to have good osteogenic activity, osteoinductive and bone conductibility, the standard method of transplanting as bone always.Yet, because limited from body bone source, and the position of drawing materials complication such as local hemorrhage, the nerve injury etc. that may bring, limited clinical practice from the body bone.Homogeneous allogenic bone transplantation is an another kind of bone grafting mode commonly used clinically, but not only there is certain immunogenicity in allogenic bone transplantation, have the danger of propagating human diseases, and the same with other xenotransplant, face donor wretched insufficiency and medical ethics problem.Bone xenograft is considered to one of effective ways that can replace autologous bone transplanting and homogeneous allogenic bone transplantation, but owing to the immunology obstacle between the xenogenesis species, has hindered the progress of bone xenograft.
The patent No. provides a kind of preparation method that is used for the novel non-immunogenicity heterotransplantatioof of bones of human body for the patent application of WO9947080-A, and this kind method may further comprise the steps: a, get the part bone be used to prepare the xenogenesis bone material in animal body; B, water and alcohol wash; C, adopt following a kind of processing at least: ultraviolet radiation, alcohol-pickled, ozonidation, frozen-thaw process or sterilization, and cytoclasis; The cell surface carbohydrate is removed in d, glycosidase digestion.The problem that this kind method exists is: 1, do not mention α-Gal antigenic processing method of xenotransplantation in addition; 2, the purpose of water and alcohol wash only is to remove synovial fluid and solubility lipid pollutant, rather than removes the fat in the bone material.Therefore, it is former and fatty to remove the xenogenesis fracture fully with the heterotransplantatioof of bones of this kind method preparation, transplants if be used for bone, has the transplantation immunity rejection inevitably and bone defect healing is had considerable influence.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of improved heterotransplantatioof of bones preparation method is provided, this kind method can not only be removed the various antigens and the fat of heterotransplantatioof of bones better, and can keep required bone-inducting active and biomechanics characteristic.
Heteroantigen should be removed during bone xenograft, the heteroimmune rejection could be reduced.Heteroantigen mainly comprises α-galactosyl antigen (α-galactosyl residues, α-Gal) and major histocompatibility antigen (MHC).According to the 26S Proteasome Structure and Function difference, the major histocompatibility antigen relevant with graft-rejection can be divided into MHC-I class and MHC-II class antigen.The inventor of present patent application found through experiments, and α-Gal, MHC-I antigen wide expression are around the medullary cell and osteocyte, osteoblast after birth and Haversian canal of animal bone, and MHC-II antigen mainly is expressed in the medullary cell of animal bone.
Fat has a significant impact bone defect healing in bone is transplanted, there are some researches show [Chappard D.Fressonnet C.Genty C.etal.Fat in bone xenografts:importance of the purification procedures oncleanliness, wettability and biocompatibility.Biomaterials.14 (7): 507-12,1993 Jun.] bone material of not ungrease treatment is behind radiation gamma, peroxidization, toxigenicity can take place in fat.
The method of the invention is raw material with the animal bone, according to above-mentioned basic research, may further comprise the steps:
(1) removes bone marrow and hemocyte
The animal bone of obtaining under the aseptic condition is used high-voltage pulse distilled water or normal saline flushing under normal pressure, room temperature and aseptic condition, washing time exceeds with bone marrow and the hemocyte of removing in bone and the pulp cavity thereof;
(2) ether defatting and flushing
At normal temperatures and pressures step (1) is removed the ether defat that the animal bone of bone marrow and hemocyte places mass concentration 95%～100%, the consumption of ether exceeds to flood processed animal bone, the time of ungrease treatment exceeds with the fat of removing in the animal bone, after the ungrease treatment, remove ether with aseptic water washing;
(3) super deep-frozen
Will be through animal bone liquid nitrogen freezing (196 ℃ are freezing) under normal pressure of step (2) processing, cooling time was at least 1 month, to destroy osteocyte, reduced antigenicity;
(4) radiation gamma
With the animal bone behind the super deep-frozen of step (3) under normal pressure, room temperature with radiation gamma sterilization with further destroy osteocyte, promptly obtain to have removed the heterotransplantatioof of bones of heteroantigen.
Because the variation of thermograde has certain influence to the biomechanical property of bone material, can increase profound hypothermia and preserve step after above-mentioned super deep-frozen step, soon the animal bone behind the liquid nitrogen freezing is carried out radiation gamma at least again in normal pressure ,-60 ℃～-80 ℃ preservations after 1 month.
In the said method, the pressure of described high-voltage pulse distilled water of step (1) or normal saline is 350KPa～600KPa; Flushing sterilized water in the step (2) is distilled water or normal saline or distilled water; The dosage of radiation gamma is 25KGy～30KGy, and the time of radiation gamma is 7 hours～8 hours.
The described normal pressure of said method is 1 atmospheric pressure, and room temperature refers to indoor natural temperature.
The present invention has following beneficial effect:
1, the method for the invention is owing to containing removal bone marrow and hemocyte, ether defatting step, and the reasonable arrangement of each sequence of steps, not only can remove α-Gal, MHC-I and the MHC-II antigen and the fat of animal bone, and make prepared bone grafting material have required bone-inducting active and biomechanics characteristic.
2, because prepared bone grafting material has been removed α-Gal, MHC-I and MHC-II antigen, thereby the transplantation immunity rejection reduces greatly.
3, because prepared bone grafting material has been removed fat, in transplanting, bone helps symphysis, behind radiation gamma, and can the toxigenicity reaction.
4, after super deep-frozen step, increase profound hypothermia and preserve step, can reduce the influence of variations in temperature the bone grafting material biomechanics.
Description of drawings
Fig. 1 is pig cortical bone α-Gal Detection of antigen figure (* 400): osteocyte nuclear is indigo plant and dyes, brown yellow granule shape electrodeposition substance around cell membrane and the Haversian canal, and the dyeing that is positive, and do not see positive staining in the bone matrix of extracellular.
Fig. 2 is pig spongy bone α-Gal Detection of antigen figure (* 400): osteoblast is spindle shape around osteocyte ellipse in the bone trabecula, the bone trabecula, nucleus is indigo plant and dyes, brown yellow granule shape electrodeposition substance is arranged on osteocyte film and the osteoblast film, and positive staining is not seen in the dyeing that is positive in the bone matrix.
Fig. 3 is pig Grafting Cancellous Bone Bolt myelocyte and osteocyte, osteoblast after birth and Haversian canal week MHC-I antigen positive expression figure (* 200).
Fig. 4 is pig Grafting Cancellous Bone Bolt myelocyte MHC-II antigen positive expression figure (* 400), is pale brown color dyeing.
Fig. 5 is the antigen positive expression figure of the Os Sus domestica graft materials after the method for the invention is handled, and nucleus indigo plant is dyed, osteocyte surface only visible minute quantity α-Gal, MHC-I and MHC-II antigen presentation.
Fig. 6 is the sem photograph (* 25) of the Os Sus domestica graft materials after the method for the invention is handled, and finds out that from this figure the bone material hole wall is smooth.
Fig. 7 is the sem photograph (* 100) of the Os Sus domestica graft materials after the method for the invention is handled, and finds out that from this figure the bone material hole wall is smooth.
Fig. 8 is the rabbit periphery serum IL-2 testing result figure in 1,2,4,6 weeks of postoperative.
Fig. 9 is the rabbit periphery serum IL-4 testing result figure in 1,2,4,6 weeks of postoperative.
The specific embodiment
Embodiment 1: antigenic expression of α-Gal, MHC-I and MHC-II and distribution situation detect in the original Os Sus domestica
(1) main agents
M86: anti-α-Gal monoclonal antibody, Alexis company produces;
SLA-1: mouse-anti pig MHC-I monoclonal antibody, Serotec company produces;
SLA-DR: mouse-anti pig MHC-II monoclonal antibody, BD company produces.
(2) main material and equipment
Specimen is taken from June～8 monthly age fresh adult inbred line inland river pig femur condyle spongy bone and ribs, and the inland river pig is buied by preclinical medicine zoopery center, Sichuan University West China.LEICA2500E type microtome, 1600 type treating block machines are mounted the sheet instrument, and German LEICA produces.
(3) SABC detects α-Gal, MHC-I and MHC-II antigen
1. specimen is fixed 24 hours through 4% paraformaldehyde;
2. the EDTA of 0.5M is after decalcification February～March under 4 ℃ of conditions, series dehydration paraffin embedding;
3. paraffin section is after taking off paraffin, 3%H
2O
2Suppress endogenous enzyme, 0.1% tryptic digestive juice was repaired antigen 15 minutes for 37 ℃.0.01mol/L PBS washing 2 times, each 5 minutes.Added notmal horse sera (1: 200) sealing non-specific antibody 30 minutes;
4. experimental group drips anti-α-Gal monoclonal antibody (M86), anti-MHC-I and the antigenic monoclonal antibody of MHC-II (SLA-1 and SLA-DR) 50 μ l respectively, antibody dilution multiple 1: 200, and negative control group drips PBS, 4 ℃ of refrigerator overnight incubation;
5. 0.01mol/L PBS washing is 2 times, each 5 minutes;
6. it is anti-as two to drip biotinylated horse anti-mouse antibody (1: 200) 50 μ l, hatches 0.01mol/L 40 minutes for 37 ℃
PBS washing 2 times, each 5 minutes;
7. it is anti-as three to add SP (1: 200), hatches 40 minutes for 37 ℃;
8. 0.01mol/L PBS washing is 2 times, each 5 minutes;
9. the colour developing of DAB lucifuge stops after 2 minutes～3 minutes, and haematoxylin was redyed nucleus 1 minute.
10. 95%, 100% ethanol dewatered respectively 15 minutes, and dimethylbenzene is after transparent 30 minutes, the canada balsam mounting.
(4) SABC testing result
Experimental group osteocyte nuclear is indigo plant and dyes, brown yellow granule shape electrodeposition substance around cell membrane and the Haversian canal, dyeing is positive, and do not see positive staining (seeing Fig. 1, Fig. 2, Fig. 3, Fig. 4) in the bone matrix of extracellular, α-Gal, MHC-I antigen wide expression are around the medullary cell and osteocyte, osteoblast after birth and Haversian canal of Os Sus domestica, and MHC-II antigen mainly is expressed in the medullary cell of Os Sus domestica.
Embodiment 2: the preparation of Os Sus domestica graft materials
Present embodiment is a raw material with the rib of monthly age in June～8 fresh adult inbred line inland river pig, and the inland river pig is buied by preclinical medicine zoopery center, Sichuan University West China.Concrete preparation method is as follows:
(1) removes bone marrow and hemocyte
Under aseptic condition, remove pig rib surrounding tissue and periosteum, get the rib stage casing and keep a side cortex, make 1.5cm * 0.5cm * 0.4cm bone bar, is the high-voltage pulse distilled water flushing of 350Kpa with pressure with the pig rib of preparation under normal pressure, room temperature (20 ℃) and aseptic condition, washes can remove pig rib and interior bone marrow and the hemocyte of pulp cavity thereof in about 60 minutes;
(2) ether defatting and flushing
At normal temperatures and pressures step (1) is removed the ether defat that the pig rib of bone marrow and hemocyte places mass concentration 95%, the consumption of ether exceeds to flood processed pig rib, the time of ungrease treatment is about 24 hours, can remove the fat in the pig rib, after the ungrease treatment, remove ether with the distilled water flushing;
(3) super deep-frozen
The pig rib that to handle through step (2) liquid nitrogen freezing (196 ℃ freezing) 1 month under normal pressure to destroy osteocyte, reduces antigenicity;
(4) radiation gamma
Pig rib behind the super deep-frozen of step (3) is used gamma-rays (Co down in normal pressure, room temperature (25 ℃)
60) illumination-based disinfection and the further cell that destroys, the dosage of radiation gamma is 25KGy, the time of radiation gamma is 8 hours, promptly obtains Os Sus domestica graft materials of the present invention behind radiation gamma.
Embodiment 3: the preparation of Os Sus domestica graft materials
Present embodiment is a raw material with the condyle of femur of monthly age in June～8 fresh adult inbred line inland river pig, and the inland river pig is buied by preclinical medicine zoopery center, Sichuan University West China.Concrete preparation method is as follows:
(1) removes bone marrow and hemocyte
Under aseptic condition, remove pig condyle of femur surrounding tissue and periosteum, get the middle spongy bone of condyle of femur, make 0.5cm * 0.5cm * 0.5cm bone piece, is the high-voltage pulse normal saline flushing of 600KPa with pressure with the pig spongy bone piece of preparation under normal pressure, room temperature (10 ℃) and aseptic condition, washes can remove pig spongy bone and interior bone marrow and the hemocyte of pulp cavity thereof in about 30 minutes;
(2) ether defatting and flushing
The pig spongy bone of at normal temperatures and pressures step (1) being removed bone marrow and hemocyte places absolute ether (ether of mass concentration 100%) defat, the consumption of ether exceeds to flood processed pig spongy bone, the time of ungrease treatment is about 48 hours, can remove the fat in the pig spongy bone, after the ungrease treatment, remove ether with normal saline flushing;
(3) super deep-frozen
The pig spongy bone that to handle through step (2) liquid nitrogen freezing (196 ℃ freezing) 4 months under normal pressure to destroy osteocyte, reduces antigenicity;
(4) profound hypothermia is preserved
Pig spongy bone behind step (3) liquid nitrogen freezing was preserved 1 month normal pressure ,-60 ℃.
(5) radiation gamma
Pig spongy bone behind the super deep-frozen of step (3) is sterilized and the further cell that destroys with radiation gamma under normal pressure, room temperature (5 ℃), the dosage of radiation gamma is 30KGy, the time of radiation gamma is 7 hours, promptly obtains Os Sus domestica graft materials of the present invention behind radiation gamma.
Embodiment 4: the α-Gal of Os Sus domestica graft materials, MHC-I and MHC-II Detection of antigen
Os Sus domestica graft materials with embodiment 2 and embodiment 3 preparations detects its α-Gal, MHC-I and MHC-II antigen respectively with the SABC method.Detect step with embodiment 1.
Testing result: bone lacuna and Haversian canal (Harversian pipe) only have the small amount of bone cell to retain on every side, and nucleus indigo plant is dyed, and osteocyte surface only visible minute quantity α-Gal, MHC-I and MHC-II antigen positive are expressed (see figure 5).
Embodiment 5: the pore structure of Os Sus domestica graft materials detects
Os Sus domestica graft materials scanning electron microscopic observation with embodiment 2 and embodiment 3 preparations: color is faint yellow, and micro-pore wall is smooth, and the aperture is (239.83 ± 150.19) μ m, and voidage (36.36 ± 9.36) % is as Fig. 6, shown in Figure 7.
There are some researches show (Hulbert SF, Young FA, Mathews RS.Potential of ceramic materials aspermanently implantable skelet al prostheses.J Biomed Mater Res.1970,4 (3): 433-456.), the optimum aperture of bone grafting material is 100 μ m～500 μ m, each other the aperture of Lian Jieing should＞100 μ m, big relatively pore size material helps direct skeletonization, helps vascularization and oxygen and effect.
The Os Sus domestica graft materials of the method for the invention preparation, its pore size and voidage satisfy above-mentioned condition, and suitable osteoblast is grown into.
Embodiment 6: the biomechanical property of Os Sus domestica graft materials detects
Under the normal temperature and pressure, (Instron Co., USA) the Os Sus domestica graft materials to embodiment 2 preparations carries out axial compression, with the fresh pig rib of same area in contrast to adopt INSTRON 8874 biomechanics test macros.Compression experiment loading velocity 0.5mm/min, stress decrease 25%-70% stop to load.Each compression experiment of organizing material the results are shown in Table 1.
Table 1 is respectively organized compression experiment result (N=4, the x ± S) of material
| Group | Maximum load (N) | Maximum displacement (mm) | Maximum stress (MPa) | Maximum strain (%) | Elastic modelling quantity (GPa) |
| The pig rib graft materials of embodiment 2 preparations | 85.35±22.02 | 0.32±0.03 | 17.26±6.89 | 1.31±0.14 | 1.92±1.10 |
| Fresh pig rib matched group | 149.2±61.95 | 0.56±0.21 | 20.45±7.02 | 3.28±2.31 | 1.54±0.65 |
Compression experiment data in the table 1 show that the Os Sus domestica graft materials of embodiment 2 preparations is compared with fresh pig rib matched group, and anti-compression properties does not have obvious change.
Embodiment 7: zoopery
1. the experimentation of transplantation immunity in the xenogenesis bone material body
(1) materials and methods
Main agents comprises: the anti-rabbit CD8 antibody (available from U.S. Pharmingen company) of the anti-rabbit CD4 antibody of FITC labelling, purification, and the anti-Mus CD8 antibody of FITC labelling is as two anti-(China fir reagent company produces in Beijing); The ELISA test kit of anti-rabbit IL-2, IL-4 (available from BD company).Capital equipment comprises: flow cytometer, microplate reader etc.
The xenogenesis bone material is the pig spongy bone of embodiment 3 preparations.
(2) animal grouping and experiment
30 of new zealand white rabbits, 3 monthly ages, male and female half and half, about body weight 2.0Kg (the Huaxi Hospital Attached to Sichuan Univ animal center provides), be divided into 2 groups at random, one group is the Os Sus domestica graft materials experimental group of embodiment 3 preparations, a kind of is fresh pig spongy bone experimental group, 15 every group.
The anesthesia of new zealand white rabbit 10% water and chloral (pressing the 3ml/kg dosed administration) intraperitoneal, the ventricumbent position is fixed on the operating-table, back preserved skin, sterilization, shop aseptic towel.Get the back median incision, cut skin, subcutaneous tissue, with the 4th lumbar vertebra body level, with the xenogenesis bone material handled respectively embedding implant in the musculus sacrospinalis of both sides, about bone material respectively, stitching muscular fasciae, layer-by-layer suture wound.Postoperative intramuscular injection penicillin 400,000 units, for three days on end.
(3) postoperative detects index
1. lymphocyte subgroup analysis:
Respectively at 1,2,4 weeks of postoperative getting rabbit ear edge venous blood, the variation of CD4+, CD8+T cell subsets and CD4/CD8 ratio behind the flow cytometer detection bone graft.Concrete operation method is as follows:
A. anticoagulant: get rabbit ear edge venous blood 0.5ml, add the anticoagulant tube that contains EDTA immediately, shake up gently;
B. add fluorescent antibody: get above-mentioned anticoagulation 20 μ l, add the anti-rabbit CD4 monoclonal antibody 10 μ l of FITC labelling; Other gets above-mentioned anticoagulation 20 μ l, adds the anti-rabbit CD8 monoclonal antibody 10 μ l of purification; The abundant mixing of antibody and sample, lucifuge left standstill 20 minutes under the room temperature;
C. isolated lymphocytes: add hemolytic agent 10 μ l, mixing, lucifuge was hatched 20 minutes under the room temperature, and centrifugal 3 minutes of 1500r/min abandons supernatant.It is anti-as two to detect the anti-mice IgG of CD8 group adding FITC labelled goat;
D. washing: add 0.01M PBS mixing, centrifugal 5 minutes of 1500r/min abandons supernatant;
E. add 0.01M PBS mixing, flow cytometer detects CD4+, CD8+T lymphocyte percentage ratio respectively.
2. cytokine IL-2, IL-4 detect
Respectively at 1,2,4,6 weeks of postoperative getting rabbit ear edge venous blood 2ml, put into the glass blood taking tube, left standstill under the room temperature 2 hours, centrifugal 15 minutes of 1500r/min obtains serum.IL-2 and IL-4 level in the ELISA detection by quantitative serum.
The IL-2 detection method:
A. set up standard curve: establish gauge orifice 6 holes, every hole adds standard substance 25 μ l, standard substance concentration is followed successively by 0,62.5pg/ml, 125pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml, treats that respectively every hole adds test serum 25 μ l respectively in the gaging hole, and every part of serum specimen drips 3 multiple holes;
B. application of sample: every hole adds biotin labeled anti-rabbit IL-2 antibody 25 μ l, and mixing is 30 seconds gently, hatches under the room temperature 30 minutes;
C. wash plate: get rid of liquid in the most plate, every hole adds the washing of 350 μ l cleaning mixture, and blots the removal water droplet in thick folded absorbent paper, cyclic washing 3 times;
D. every hole adds 50 μ l horseradish peroxidases, and mixing is 30 seconds gently, hatches under the room temperature 30 minutes;
E. wash plate: method is with (c.);
F. every hole adds 50 μ l colour developing liquid, and mixing is 10 seconds gently, hatches under the room temperature 15 minutes;
G. every hole adds 50 μ l stop buffers, and mixing is 30 seconds gently, reads the OD value with microplate reader at the 450nm place at once;
H. result's judgement: with the OD value as vertical coordinate, standard substance concentration 0,62.5pg/ml, 125pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml are abscissa drawing standard curve, and OD value per sample calculates the IL-2 content of respective sample on standard curve.
IL-4 detection method: with the IL-2 detection method.
(4) result
1. the lymphocyte subgroup analysis result see Table 3, table 4, table 5:
Table 3 Flow cytometry CD4+ cell (x ± S)
| The postoperative time | The Os Sus domestica graft materials experimental group of embodiment 3 preparations | Fresh pig spongy bone experimental group |
| 1 week | 20.10±11.89 | 40.30±3.77 |
| 2 weeks | 30.63±5.05 | 69.17±18.60 |
| 4 weeks | 34.80±2.72 | 60.13±12.54 |
Table 4 Flow cytometry CD8+ cell (x ± S)
| The postoperative time | The Os Sus domestica graft materials experimental group of embodiment 3 preparations | Fresh pig spongy bone experimental group |
| 1 week | 11.37±3.78 | 18.60±13.79 |
| 2 weeks | 19.37±4.54 | 28.37±7.25 |
| 4 weeks | 23.57±10.29 | 32.80±9.64 |
Table 5 Flow cytometry CD4+/CD8+ cell (x ± S)
| The postoperative time | The Os Sus domestica graft materials experimental group of embodiment 3 preparations | Fresh pig spongy bone experimental group |
| 1 week | 2.26±2.21 | 3.09±1.94 |
| 2 weeks | 1.66±0.54 | 2.63±1.18 |
| 4 weeks | 1.66±0.63 | 1.92±0.54 |
From table 3, table 4, table 5 as can be seen, each all CD4+ in experimental group postoperative 1 week～2, CD8+T lymphocyte all raise gradually, and fresh pig spongy bone experimental group raises obviously.CD4/CD8 ratio is the highest in each group 1 week of postoperative, descends fresh pig spongy bone experimental group higher (P＜0.05) later on gradually.
2. serum il-2, IL-4 testing result:
Each is organized bone material and transplants respectively at 1,2,4,6 weeks of postoperative getting rabbit ear edge venous blood 2ml, centrifugal acquisition serum, and IL-2 and IL-4 level in the ELISA detection by quantitative serum, testing result is seen Fig. 8, Fig. 9.
As can be seen from Figure 8, IL-2 level in the periphery serum behind the bone grafting operation, the Os Sus domestica graft materials experimental group＜fresh pig spongy bone experimental group of embodiment 3 preparations, difference obvious (P＜0.05) between two groups.As can be seen from Figure 9, IL-4 level in the periphery serum behind the bone grafting operation, the Os Sus domestica graft materials experimental group＜fresh pig spongy bone experimental group of all embodiment 3 preparations of postoperative 1,2, difference obviously (P＜0.05) between two groups, postoperative 4,6 does not have significant difference (P＞0.05) between all two groups.
(5) conclusion:
Immunology detection result shows that the bone xenograft immunological rejection mainly occurs in 1 week～2 week of postoperative, fresh pig spongy bone experimental group immunologic rejection is apparently higher than the Os Sus domestica graft materials experimental group of embodiment 3 preparations, and the Os Sus domestica graft materials experimental group immunological rejection of embodiment 3 preparations obviously reduces.
2. xenogenesis bone material body is repaired the damaged experimentation of rabbit radius
(1) materials and methods
The xenogenesis bone material is the pig rib graft materials of embodiment 2 preparations
(2) animal grouping and experiment
30 of new zealand white rabbits, at 3 monthly ages, male and female half and half about body weight 2.0Kg (the Huaxi Hospital Attached to Sichuan Univ animal center provides), are divided into 2 groups at random, i.e. the Os Sus domestica graft materials experimental group and the fresh pig rib experimental group of embodiment 2 preparations, 15 every group.
The anesthesia of 10% water and chloral (pressing the 3ml/kg dosed administration) intraperitoneal, the ventricumbent position is fixed on the operating-table, back preserved skin, sterilization, shop aseptic towel.Get the bilateral radius middle incision, cut skin, subcutaneous tissue, enter the exposure radius from spatium intermusculare, under head of radius the 20mm place with the radius of a segment length 15mm together with periosteum amputation on every side, hemostasis also irrigates, by the requirement of dividing into groups respectively with the different damaged places of xenogenesis bone material implantable bone.Will not fix in the art, sew up muscular fasciae, the layer-by-layer suture wound.Raise in the conventional cage of postoperative, suffer from the not all right external fixation of limb, intramuscular injection penicillin 400,000 units, for three days on end.
(3) clinical follow index
6 weeks of postoperative, 12 weeks, dead 5 rabbits in the every component of 24 all different times other places carry out x-ray observation.
(4) result
Postoperative different time points X-ray film Lane-Sandhux mark [Lane JM, Sandhu HS.Current approaches toexperimental bone grafting.Orthop Clin North Am.1987 Apr; 18 (2): 213-25.] result all shows, the Os Sus domestica graft materials repair ability of embodiment 2 preparations is better than fresh Os Sus domestica, and significant difference (P＜0.05) is arranged.
The radiology Lane-Sandhux scoring of table 6 repairing bone defect (n=5, x ± S)
| Group | 6 weeks | 12 weeks | 24 weeks |
| The Os Sus domestica graft materials experimental group of embodiment 2 preparations | 2.20±0.84 | 4.00±1.00 | 4.60±1.52 |
| Fresh pig rib experimental group | 1.40±0.55 | 2.60±0.55 | 3.00±0.71 |
(5) conclusion:
The Os Sus domestica graft materials experimental group repair ability of embodiment 2 preparations is better than fresh pig rib experimental group.
Claims (9)
1, a kind of preparation method of heterotransplantatioof of bones is a raw material with the animal bone, it is characterized in that may further comprise the steps:
(1) removes bone marrow and hemocyte
The animal bone of obtaining under the aseptic condition is used high-voltage pulse distilled water or normal saline flushing under normal pressure, room temperature and aseptic condition, washing time exceeds with bone marrow and the hemocyte of removing in bone and the pulp cavity thereof;
(2) ether defatting and flushing
At normal temperatures and pressures step (1) is removed the ether defat that the animal bone of bone marrow and hemocyte places mass concentration 95%～100%, the consumption of ether exceeds to flood processed animal bone, the time of ungrease treatment exceeds with the fat of removing in the animal bone, after the ungrease treatment, remove ether with aseptic water washing;
(3) super deep-frozen
Will be through animal bone liquid nitrogen freezing under normal pressure of step (2) processing, cooling time was at least 1 month, to destroy osteocyte, reduced antigenicity;
(4) radiation gamma
With the animal bone behind the super deep-frozen of step (3) under normal pressure, room temperature with radiation gamma sterilization with further destroy osteocyte, promptly obtain to have removed the heterotransplantatioof of bones of heteroantigen.
2, the preparation method of heterotransplantatioof of bones according to claim 1, it is characterized in that also comprising profound hypothermia preservation step, this step is after super deep-frozen step, and soon the animal bone behind the liquid nitrogen freezing is carried out radiation gamma at least again in normal pressure ,-60 ℃～-80 ℃ preservations after 1 month.
3, the preparation method of heterotransplantatioof of bones according to claim 1 and 2 is characterized in that the pressure of described high-voltage pulse distilled water of step (1) or normal saline is 350KPa～600KPa.
4, the preparation method of heterotransplantatioof of bones according to claim 1 and 2 is characterized in that the flushing sterilized water in the step (2) is distilled water or normal saline or distilled water.
5, the preparation method of heterotransplantatioof of bones according to claim 3 is characterized in that the flushing sterilized water in the step (2) is distilled water or normal saline or distilled water.
6, the preparation method of heterotransplantatioof of bones according to claim 1 and 2, the dosage that it is characterized in that radiation gamma is 25KGy～30KGy, the time of radiation gamma is 7 hours～8 hours.
7, the preparation method of heterotransplantatioof of bones according to claim 3, the dosage that it is characterized in that radiation gamma is 25KGy～30KGy, the time of radiation gamma is 7 hours～8 hours.
8, the preparation method of heterotransplantatioof of bones according to claim 4, the dosage that it is characterized in that radiation gamma is 25KGy～30KGy, the time of radiation gamma is 7 hours～8 hours.
9, the preparation method of heterotransplantatioof of bones according to claim 5, the dosage that it is characterized in that radiation gamma is 25KGy～30KGy, the time of radiation gamma is 7 hours～8 hours.
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| CNA2007100488227A CN101032631A (en) | 2007-04-06 | 2007-04-06 | Preparation method of xenogeneic bone graft material |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102600509A (en) * | 2011-01-25 | 2012-07-25 | 上海交通大学医学院附属第九人民医院 | Osteoblast derived from cryopreserved bone, and application of osteoblast derived from cryopreserved bone in bone tissue regeneration |
| CN102727934A (en) * | 2012-06-20 | 2012-10-17 | 上海骁博科技发展有限公司 | Manufacturing method and application of inductive artificial periosteum |
| CN103958671A (en) * | 2011-10-17 | 2014-07-30 | 李喜永 | Bio-fat material eliminated immunity and method for manufacturing same |
| CN107583108A (en) * | 2017-10-31 | 2018-01-16 | 陕西爱骨医疗股份有限公司 | A kind of preparation method of bone grafting material |
| CN111386042A (en) * | 2017-12-04 | 2020-07-07 | 热生物制品有限公司 | Production of cell-based vaccines |
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- 2007-04-06 CN CNA2007100488227A patent/CN101032631A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102600509A (en) * | 2011-01-25 | 2012-07-25 | 上海交通大学医学院附属第九人民医院 | Osteoblast derived from cryopreserved bone, and application of osteoblast derived from cryopreserved bone in bone tissue regeneration |
| CN103958671A (en) * | 2011-10-17 | 2014-07-30 | 李喜永 | Bio-fat material eliminated immunity and method for manufacturing same |
| JP2014531964A (en) * | 2011-10-17 | 2014-12-04 | ヤング リー，ヒー | Raw fat material from which immunity has been removed and method for producing the same |
| CN102727934A (en) * | 2012-06-20 | 2012-10-17 | 上海骁博科技发展有限公司 | Manufacturing method and application of inductive artificial periosteum |
| CN102727934B (en) * | 2012-06-20 | 2014-10-22 | 上海骁博科技发展有限公司 | Manufacturing method and application of inductive artificial periosteum |
| CN107583108A (en) * | 2017-10-31 | 2018-01-16 | 陕西爱骨医疗股份有限公司 | A kind of preparation method of bone grafting material |
| CN111386042A (en) * | 2017-12-04 | 2020-07-07 | 热生物制品有限公司 | Production of cell-based vaccines |
| CN111386042B (en) * | 2017-12-04 | 2023-01-31 | 热生物制品有限公司 | Production of cell-based vaccines |
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