CN1846789A - A kind of injection type bone repair material and its preparation and application - Google Patents
A kind of injection type bone repair material and its preparation and application Download PDFInfo
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- CN1846789A CN1846789A CNA2005100341191A CN200510034119A CN1846789A CN 1846789 A CN1846789 A CN 1846789A CN A2005100341191 A CNA2005100341191 A CN A2005100341191A CN 200510034119 A CN200510034119 A CN 200510034119A CN 1846789 A CN1846789 A CN 1846789A
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Abstract
The present invention discloses one kind of injected bone repairing material and its preparation and application. The injected bone repairing material consists of solution I and solution II, the solution I is the mixture of platelet rich plasma (PRP) and 80-100 mg/ml concentration fibrinogen solution in the ratio of 1 to 3-6; and the solution II is solution mixture of calcium chloride in 40 mmol/ml concentration and thrombin in 400-1000 U/ml concentration. When the injected bone repairing material is used, solution I and solution II in the volume ratio of 1-9 are sucked with two syringes and injected into body to form gel with high adhesive force, with PRP accounting for 10-30 vol%. The injected bone repairing material is used in preparing implant for treating bone defect and bone nonunion and promoting fracture healing.
Description
Technical field
The invention belongs in the biomedical engineering and prepare the artificial organ technical field, specifically relate to a kind of injected bone repairing material and preparation thereof and application with Method of Tissue Engineering.
Background technology
At present, bone renovating material mostly is the subpattern of timbering material load cell growth factor greatly.The selection of timbering material and somatomedin is the key content of tissue engineering research.Timbering material has the effect of load cell and slow release somatomedin as the seed cell carrier, and timbering material commonly used at present mostly is the solid kind material, and it has obtained the effect that attracts people's attention in the preparation of bone renovating material.But, also exist to some extent to be difficult to moulding, growth factor-loaded and seed cell complicated operation, load factor are not high, the easy defective such as loss of cell.Therefore, injection-type or hydrogel class material are the focuses of present bone renovating material research.The cell growth factor that is usually used in the bone renovating material field has BMP, TGF-β, bFGF, VEGF etc., is extrinsic protein (xenogenesis or the foreign protein factor).From the preparation method analysis of these somatomedin, comprise two kinds of allograph bone extraction and gene recombinaton, it prepares complexity, costs an arm and a leg.For example, application number be 02114509.1 Chinese patent application " is the injection type multiple biological factor composite bone repair materials of carrier with the animal fibrin " to disclose a kind of be the injection type multiple biological factor composite bone repair materials of carrier with the animal fibrin, what it adopted is exactly recombinant human bone morphogenetic protein, fibroblast growth factor and β transforming growth factor.From the immunology angle, all kinds of somatomedin commonly used at present are exogenous (non-from body) albumen or polypeptide class, have immunological rejection in various degree.Research and development preparations is simple, cheap, no immunologic rejection be used for the preparation of bone renovating material from the bulk-growth factor, be the major issue that the further clinical practice of bone renovating material needs to be resolved hurrily.
Platelet rich plasma (Plate Rich Plasma, PRP) be from whole blood, separate contain the hematoblastic blood plasma of high concentration.Discovering in recent years, the somatomedin that helps promoting osteanagenesis that contains multiple high concentration from autologous vein blood in the platelet rich plasma that extracts (PRP) is as platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-β), insulin like growth factor (IGF), vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF) etc.When PRP with after calcium chloride and thrombin mix, can impelling wherein, platelet α particle release goes out these somatomedin.Damaged healing has obvious facilitation to these somatomedin to bone.The interior normal concentration of various somatomedin concentration ratio and body is in similar proportion among the PRP, and synergy is arranged between the somatomedin.But use PRP merely in the part, these somatomedin can only work easily because of blood circulation runs off within a short period of time.At present, existing with PRP and solid material such as the compound report that is used for the Oral and Maxillofacial Surgery repairing bone defect such as TCP or homogeneous allogenic bone.But PRP and the compound bone reparation aspect that is used for of injection-type material then are not reported.
Summary of the invention
At above-mentioned prior art situation, be difficult to moulding and complicated operation and expensive deficiencies such as exogenous cytokine in order to overcome existing bone renovating material, the invention provides a kind of injected bone repairing material, this bone renovating material is that injectable shape material load constitutes from the compound again autologous bone marrow stromal cell of body PRP, can be moulding arbitrarily and compound cells is easy, and use the autogenous cell somatomedin, do not have the immunologic rejection problem.
A kind of injected bone repairing material of the present invention is made up of solution one and solution two, and wherein, solution one is to be that PRP and concentration are that the fibrinogen solution of 80-100mg/ml is with 1: 3～6 mixed from the body platelet rich plasma; Solution two is mixed solutions of calcium chloride and thrombin, and wherein, calcium chloride concentration is 40mmol/ml, and concentration of thrombin is 400～1000U/ml; During use, extracting solution one and solution two respectively with the duplex syringe, is 9: 1～1: 1 mixed injection with volume ratio, forms the gel bone renovating material, and wherein the shared volume ratio of PRP is 10～30%.
Fibrinogen solution in the described solution one is former the obtaining of aprotinin solution fibrin of 2000～6000U/ml with concentration.
Described Fibrinogen is a commercial prod, can be available from Guangzhou Beixiu Biological Technology Co., Ltd or similar company.
Each component concentrations of injected bone repairing material of the present invention and ratio all are the optimums of groping to obtain through experiment, so can form the gel bone renovating material of biologically active after in injecting body at 20-30 in second rapidly.
Described PRP preferably takes from autologous vein blood, the platelet rich plasma that gets with the extraction of secondary centrifuging method.
Described extraction may further comprise the steps from the secondary centrifuging method of body RPR: with anticoagulation in 20 ℃ with 3000r/min centrifugal 10 minutes for the first time, draw 3mm composition under supernatant and the tunica albuginea layer; With 3600r/min centrifugal 15 minutes for the second time, abandon upper strata 3/4 supernatant under the same temperature, remainder is PRP.
The seed cell that adds certain cell density before the hybrid injection in solution one promptly obtains corresponding injection type tissue engineering bone by injected bone repairing material compound seed cell.
Another object of the present invention provides a kind of preparation method of injected bone repairing material.
The preparation method of injected bone repairing material of the present invention may further comprise the steps:
A. material is prepared:
(1) from body RPR: take from vena systemica blood, extracting platelet rich plasma with the secondary centrifuging method is PRP, puts-70 ℃ of refrigerators and preserves standby; Former with aprotinin solution solution fibrin, make into solution state, preparation calcium chloride-thrombin mixed solution, wherein, calcium chloride concentration is 40mmol/ml, concentration of thrombin is 400～1000I U/ml;
Described extraction may further comprise the steps from the secondary centrifuging method of body RPR: with anticoagulation in 20 ℃ with 3000r/min centrifugal 10 minutes for the first time, draw supernatant and 3mm composition under rete; With 3600r/min centrifugal 15 minutes for the second time, abandon upper strata 3/4 supernatant under the same temperature, remainder is PRP.
(2) fibrinogen solution: former with the aprotinin solution fibrin, make it become solution state;
(3) calcium chloride-thrombin mixed solution: configuration calcium chloride-thrombin mixed solution, wherein, calcium chloride concentration is 40mmol/ml, concentration of thrombin is 400IU/ml;
B. will be from body RPR and fibrinogen solution with 1: 3～6 mixed, sonic oscillation makes mix homogeneously, obtains containing the fibrinogen solution of PRP;
C. with the duplex syringe fibrinogen solution that contains PRP and the calcium chloride-thrombin mixed solution that obtain of extraction step B respectively, by mixing needle injection, the fibrinogen solution and the calcium chloride-thrombin solution ratio that contain PRP during injection are 9: 1～1: 1, form the stronger gel of adhesion strength, wherein the shared volume ratio of PRP is 10～30%, promptly obtains described injected bone repairing material.
In the fibrinogen solution that contains PRP that step B obtains, add a certain amount of seed cell, go in the body according to step C hybrid injection again, obtain injection type tissue engineering bone by injected bone repairing material compound seed cell.
Another object of the present invention is to provide the purposes of above-mentioned injected bone repairing material.
Injected bone repairing material of the present invention, the graft that can be used for preparing the damaged and bone does not connect of treatment bone and promote union of fracture comprises direct injection affected part, and compound seed cell construction tissue engineered bone.
The present invention forms fibrin gel (fibringlue after utilizing fibrinogen solution to meet calcium chloride-thrombin mixed solution, FG) principle, to add calcium chloride-thrombin mixed solution again behind PRP and the high concentration fibrinogen solution mixing, the fibrin gel that forms not only has multiple functions such as wound closure, hemostasis, promotion healing, bioadhesive, also can be used as medicine or growth factor slow-release carrier because of its distinctive fibrin network structure makes it.Like this, in the process that fibrin gel forms, PRP also discharges various somatomedin.These somatomedin are present in the fleece trellis gel, discharge gradually along with fibrinous degraded, can keep longer effective somatomedin concentration in the part.The Fibrinogen of solution state also makes the structure of tissue engineered bone more simple, and the gel of its formation has stronger tackability, make seed cell be difficult for running off, the Fibrin Glue injectable uses, and can be used for Minimally Invasive Surgery or non-operative treatment bone damaged and bone does not connect and promotion union of fracture.
The designed technical scheme of the present invention is that Fibrinogen and catalyst materials such as (calcium chloride, thrombins) are separated packing, earlier PRP or MSCs are mixed with fibrinogen solution before the art, this process operation is simple, when making up tissue engineered bone, seed cell and PRP liquid material are easy to compound, and are evenly distributed; Again this mixed liquor and catalyst are injected in the body with the bigeminy syringe when using in the art; Form gel FG in vivo, its cohesiveness is strong, makes seed cell and PRP be difficult for running off.
In the designed technical scheme of the present invention, add a certain amount of aprotinin in fibrinogen solution, its degraded that mainly act as regulation and control FG is to strengthen the stability of FG.Find in the research that FG injects that the back formed fruit jelly shape gel in about 20 seconds in the body in the art, postoperative 4 all fashion have small part FG residual, postoperative during 8 weeks FG degrade fully.The postoperative animal does not have whole body performances such as heating, suppurating sore, and implant site is organized and also do not found obvious edema and inflammatory.This shows nontoxic, the no obvious antigenicity of this composite.
In sum, from extract PRP from the body whole blood, its preparation is easy, cheap.In body PRP, be rich in multiple somatomedin such as the PDGF that bone is repaired that help, TGF-β, VEGF etc., be from body and originate, can not produce immunological rejection and pathophoresis, prepared injected bone repairing material has the injection-type material of flexible Application concurrently and from the two advantage of the bulk-growth factor, simple to operate, easy to use, adhesion strength is stronger, plasticity is good, can be in vivo arbitrarily plastotype become the shape of damage location, make new bone shape revert to original physiological status fully, the skeletonization effect is clear and definite, and is damaged applicable to various irregular bones, the bone does not connect treatment, and can use at local injection, also can be used for non-operative treatment treatment fracture, have broad clinical application prospect.
Description of drawings
Fig. 1 is the sem photograph of injected bone repairing material of the present invention.
Fig. 2 is the radius normotopia X sheet of postoperative during 4 weeks, is respectively A, B, C group from left to right.
Fig. 3 is the radius normotopia X sheet of postoperative during 8 weeks, is respectively A, B, C group from left to right.
Fig. 4 is the radius normotopia X sheet of postoperative during 12 weeks, is respectively A, B, C group from left to right.
Fig. 5 is the D group radius normotopia X sheet of postoperative during 16 weeks.
Fig. 6 is A group (experimental group) radius Histological section of postoperative during 12 weeks;
Fig. 7 is C group (matched group) radius Histological section of postoperative during 12 weeks.
The specific embodiment
Injected bone repairing material of the present invention, the graft that can be used for preparing the damaged and bone does not connect of treatment bone and promote union of fracture comprises direct injection affected part, and compound seed cell construction tissue engineered bone.Be the embodiment that the foundation load seed cell makes up tissue engineered bone with injected bone repairing material of the present invention below, come technical scheme of the present invention and implementation result are described further.
Embodiment one: seed cell---the cultivation of bone marrow stroma stem cell and induce differentiation.
The seed cell that this experiment is adopted is a bone marrow stroma stem cell, makes up tissue engineered bone jointly with injected bone repairing material of the present invention.
New zealand white rabbit is provided by Nanfang Medical Univ (former No.1 Military Medical Univ.) Nanfang Hospital animal, adopts speed sleep new, ketamine and the intramuscular anesthesia of atropine complex liquid.Bilateral ilium place extracts red bone marrow 3ml, and the DMEM culture medium is sneaked in the heparin solution anticoagulant, and the centrifugal 10min of 1000r/min behind the mixing inhales and abandons suspension adipose cell and part supernatant, crosses 100 mesh filter screens, adds DMEM complete medium (containing 15% hyclone) inoculation.37 ℃, 5%CO
2Incubator is cultivated, and full dose is changed liquid after 3 days, and amount was changed liquid 1 time in later 2～3 days half, treat cell be paved with bottle at the bottom of 80% back with 0.25% trypsinization, the cultivation of going down to posterity (contains 10% hyclone, 50ug/ml ascorbic acid, 10 with the DMEM conditioned medium
-8The mol/L dexamethasone, 10
-3The mol/L sodium) induces to osteoblast differentiation, changed liquid 1 time in conventional 3 days.(cell density reaches 10 to get biography 4 generation inner cell
8The order of magnitude) standby.Described complete medium contains 15% hyclone, and conditioned medium contains 10% hyclone, 50ug/ml ascorbic acid, 10
-8The mol/L dexamethasone, 10
-3The mol/L sodium.
Embodiment two: the extraction of PRP.
Aseptic condition extracts rabbit ear back of the body central artery blood 5ml down, with 10% sodium citrate 0.5ml anticoagulant.With centrifugal 10 minutes for the first time, draw 3mm composition under supernatant and the tunica albuginea layer under 20 ℃ of temperature with 3000r/min.Centrifugal 15 minutes of centrifugal 3600r/min for the second time under the same temperature abandons upper strata 3/4 supernatant, and remainder is PRP.The light shake of vortex oscillator makes mixing.High power microscope is counted its platelet content down and is compared with whole blood.Platelet counts has improved more than 4 times than whole blood among the PRP of this law preparation.Every 5ml whole blood is made PRP0.6ml approximately, and-70 ℃ of refrigerators are preserved standby.
Embodiment three: the preparation of injected bone repairing material.
Injected bone repairing material is made up of solution A and solution B.Solution A be with aprotinin solution solution fibrin former and with PRP with 3～6: 1 mixed.Aprotinin concentration is 2000～6000U/ml.Solution B is calcium chloride and thrombin solution, and its calcium chloride concentration is 40mmol/ml, and concentration of thrombin is 400U/ml.Extract solution A and solution B respectively with the duplex syringe during use.With solution A, 9: 1～1: 1 hybrid injection of B ratio.Form fruit jelly shape gel in second at 20-30 when injecting in the body, at the external gel that also is shaped as.
When being used to make up tissue engineered bone, before solution A, B hybrid injection, in solution A, add and press the bone marrow stroma stem cell of embodiment one described method through external evoked differentiation and proliferation, its cell density is 5 * 10
6～10 * 10
7/ ml.
As shown in Figure 1, the scanning electron microscopic observation gel is a fleece trellis structure, visible more marrow stromal cell and platelet in it.Platelet is from PRP (platelet rich plasma).
Embodiment four: the damaged model of preparation radius bone
Get healthy new zealand white rabbit, at 6 monthly ages, body weight 2-3kg adopts speed sleep new, ketamine and atropine complex liquid intramuscular injection anesthesia.Under the aseptic condition, operation appears radius stage casing, right side, together with periosteum excision radius 1.5cm, causes the damaged model of segmental bone.
Embodiment five: animal is implanted experiment
Experimental design:
36 healthy new zealand white rabbits are provided by Nanfang Medical Univ (former No.1 Military Medical Univ.) Nanfang Hospital animal, press the damaged model of embodiment four preparation radius bones, are divided into three groups of A, B, C at random, 12 every group.All animal arts the last weeks by embodiment two described methods extract hard of hearing central artery blood extract from the body platelet rich plasma (Plate Rich Plasma, PRP).A group Rhizoma Atractylodis Macrocephalae prepares bone marrow stroma stem cell (MSCs) by embodiment one described method preceding 1～February, the MSCs adding embodiment three described methods that art will digest behind the counting in preceding 30 minutes are prepared into injected bone repairing material, implantation is damaged from body radius 1.5cm segmental bone, is experimental group.It is matched group in the damaged place of same bone that B, C implant PRP+FG, FG respectively for two groups.(fibrin glue is that Fibrinogen is mixed a kind of gelling material that the back forms with calcium chloride, thrombin FG) to Fibrin Glue.A, B, three groups of layer-by-layer suture otch of C, three days 400,000 u/ days of intramuscular injection penicillin of postoperative.Other gets 4 same positions of radius and damaged spacious the putting as blank group of big ossiculum, i.e. D group.
Postoperative is taken radius normotopia X-ray film during 4,8,12 weeks.Compare the damaged reparation situation of bone with irradiation image scoring and graphical analysis X line resistance projection.
Detection method:
(1) gross examination of skeletal muscle: the ordinary circumstance of tightly observing the new zealand white rabbit postoperative.Postoperative 4,8,12 is put to death 2 animals and is drawn materials for all every group, observes surface variation, callus formation, bone defect repair situation and the surrounding tissue reaction of bone renovating material.
(2) radiological examination: A, B, C three treated animals are in all take the photograph right radius normotopia x mating plate (throw range from 1m, throw according to condition 46KV, 50mA, time of exposure 0.14s) in 4,8,12 weeks of postoperative, and D is organized in 12,16 weeks of postoperative and takes the photograph sheet, observes the bone defect healing situation.All photos are according to (Yang CY such as Yang, Simmons DJ, Lozano R.The healing of graftscombining freeze-dried and demineralized allogeneic bone in rabbits.Clin Orthop.1994, standards of grading 298:289) are carried out the radiology scoring.Density measurement reference literature (Yang Fuchun, Yang Zhiming etc., the radiology assessment that preparation of rhesus monkey tissue through engineering approaches bone and reparation allograph bone thereof are damaged, Chinese clinical rehabilitation 2004 are penetrated in the resistance of bone defect x line; 8 (2) 223-225).
The result:
(1) gross examination of skeletal muscle: all rabbit otch of postoperative are swollen, oozing of blood of show and dense sexual secretion not, the equal first phase healing of otch.4 weeks of postoperative, A group, the damaged place of B group bone are that a large amount of light red chromaticness hard fibres are organized filling, cutting fibrous tissue sees and still has a small amount of jelly in it, the far and near broken ends of fractured bone of radius is seen a large amount of fiber callus, A group callus is organized more than B, and C organizes damaged place and caves in slightly, is matter soft fibre tissue growth, the defective region central part still has part FG residual, does not see obvious callus.Each is organized the damaged muscle on every side of bone and there is no edema.In 8 weeks of postoperative, A, B group bone defective region is all seen a large amount of seriality callus, is expansive growth, and its diameter surpasses normal radius; Both do not have obvious difference, and material absorbs fully; The C group is damaged to be the fibrous tissue filling still, sees to be dispersed in growth of spur on a small quantity.Each organizes the reaction of surrounding tissue no abnormality seen.12 weeks of postoperative, all healings fully of two damaged places of Os Leporis seu Oryctolagi of A group, local for the periosteum bone tissue is normally arranged, moulding good, be difficult to distinguish the broken ends of fractured bone.Cleft lip of B group decreases healing fully, and moulding good, another cleft lip damage place is newborn callus filling, and the broken ends of fractured bone is fuzzy.The C group sees that far and near two broken ends of fractured bone respectively have the part growth of spur, and the damaged pars intermedia of bone still is a fibrous tissue.Surrounding tissue is normal.16 weeks of D group postoperative are observed the damaged size of bone when putting to death the same substantially, the sclerosis of two epiphysis, and the medullary cavity sealing, fibrous tissue filling pulp cavity does not have obvious growth of spur.
(2) X sheet observation: to shown in Figure 4, the damaged place of A group postoperative 4 all bones is patch shape resistance projection as Fig. 2, and the broken ends of fractured bone is still clear, and two broken ends of fractured bone portion density are high slightly.Postoperative 8 during week two broken ends of fractured bone fuzzy, the damaged place of bone is a seriality high density resistance projection, the outside callus of radius is clear, the inboard ulna cortical density in damaged place increases.The damaged place of bone is uniformity high density shadow during 12 weeks, has 7 rabbit radius bilateral cortical bone continuous, and pulp cavity connects, and postoperative damaged healing fully of bone during 12 weeks is described.The damaged place of B group postoperative 4 all bones is the cotton-shaped resistance projection of seriality, and the broken ends of fractured bone is clear.Postoperative 8 all bone defective regions are fuzzy, are seriality high density resistance projection, and yield of callus increases.The damaged disappearance of bone during 12 weeks, 5 cortical bone are continuous, and are moulding good, and pulp cavity is logical again.The big portion of C group postoperative 4 all defective regions is the printing opacity shadow, only sees cotton-shaped resistance projection around the broken ends of fractured bone.See that the broken ends of fractured bone is clear during 8 weeks, defective region has strip resistance projection.See that defective region is still clear in 12 weeks of postoperative, there is a small amount of growth of spur at rarely seen two ends.As shown in Figure 5, D group postoperative 16 all bone defective regions still are radiolucent shadow, and bone stump sclerosis, fuzzy illustrate and blankly organizes that postoperative 16 all bones are damaged all not to have a healing.
(3) X-ray film iconography appraisal result: A, B, the scoring of C group normotopia photo bone defect repair can be found out from table 1, A group and B group are all learned scoring than the irradiation image of C group in 4,8,12 weeks and are excellent (P＜0.05), the no significant difference (P all＞0.05) but A group and B group are compared.Positive bit slice bone defect resistance is penetrated density measurement and can be found out from table 2, and A, B, the C group is penetrated density measurement in the damaged resistance of bone in 4,8,12 weeks that significant difference (P＜0.05) is arranged, aspect what and density of newborn callus formation, and A＞B＞C.
Table 1 about the scoring of the normotopia photo of bone defect repair (X ± s, n=8)
| Group | Time (week) | ||
| 4 | 8 | 12 | |
| A | 5.333±0.512 | 9.156±0.408 | 10.683±0.546 |
| B | 5.060±0.726 | 8.833±0.516 | 10.030±0.804 |
| C | 2.310±0.535 | 4.267±0.547 | 6.083±0.841 |
Table 2 about bone defect resistance penetrate density measurement (X ± s, n=8)
| Group | Time (week) | ||
| 4 | 8 | 12 | |
| A | 58.501±2.725 | 73.638±1.265 | 87.683±2.101 |
| B | 52.663±2.541 | 65.375±0.992 | 76.582±1.017 |
| C | 25.578±2.948 | 34.203±1.556 | 46.585±1.975 |
Experimental result shows that A, B group is penetrated in YangShi radiology scoring and the resistance of defective region callus and all is better than simple FG group aspect the density measure, though and in the radiology scoring, do not have significant difference between two groups of A, B, at callus formation qualitatively, the A group is better than the B group.From this interpretation of result, PRP has the effect of tangible promotion bone defect repair, and the interpolation of seed cell plays an important role to promoting callus formation.
As shown in Figure 6 and Figure 7, Histological section observes and sees that experimental group is the normal bone tissues structure, and has pulp cavity to form, and matched group is a structure of fibrous tissue, does not have obvious skeletonization.
Conclusion: PRP has obvious facilitation to bone defect healing, and the injection-type material of compound PRP and MSCs prepare bone renovating material can to repair rabbit radius segmental bone damaged.
In the normal healing process of fracture, the blood clot at impaction fracture broken ends of fractured bone position plays an important role, and wherein the various somatomedin that discharge in fibrin in the blood clot and the platelet aggregation process play a leading role.PRP among the present invention is to extract from the body whole blood in art the last week, prepare through the secondary centrifuging method, its PC than whole blood improved 4 times surplus.Along with the degraded of FG, these somatomedin discharge gradually and can keep longer-term valid density in the part.People's such as Obarrio research (Obarrio J J, Arauz-Dutari J I, Chamberlain T M, etal.The use ofautologous growth factors in periodontal surgical l therapy:Plateletgelbiotechnology-casereports.IntJPeriodo ntics Restorative Dent, 2000; 486) and people's such as Soory research (Soory M 20 (5):, Virdi H.Implications of minocycline, platelet-derivedgrowth factor, and transforming growthfactor-bet on infiamma-tory repair potentialin the periodontium.J Periodontol, 1999; 70 (10): 1136) find, on bone marrow stroma stem cell and osteoprogenitor cells film, have PDGF, TGF-β 1 and TGF-beta 2 receptor.PDGF among the PRP and TGF-β may by with target cell (MSCs and osteoprogenitor cells) film on receptors bind play a role, promote MSCs and osteoprogenitor cells to osteoblast differentiation, thereby strengthen new osteanagenesis, the reparation that accelerated bone is damaged.And the VEGF among the PRP has and promotes that new vessels formation effect, the vascularization of bone renovating material are the bases that promotes new osteanagenesis, and the seed cell that forms of a large amount of new vesselses provides abundant nutrition, also can promote the reparation that bone is damaged.
Based on above-mentioned analysis, the graft that injected bone repairing material of the present invention can be used for preparing the damaged and bone does not connect of treatment bone and promotes union of fracture.
Claims (8)
1, a kind of injected bone repairing material is characterized in that: be made up of solution one and solution two, wherein
Solution one is to be that PRP and concentration are that the fibrinogen solution of 80-100mg/ml is with 1: 3～6 mixed from the body platelet rich plasma;
Solution two is mixed solutions of calcium chloride and thrombin, and wherein, calcium chloride concentration is 40mmol/ml, and concentration of thrombin is 400～1000U/ml;
During use, extracting solution one and solution two respectively with the duplex syringe, is 9: 1～1: 1 mixed injection with volume ratio, forms the gel bone renovating material, and wherein the shared volume ratio of PRP is 10～30%.
2, injected bone repairing material according to claim 1 is characterized in that: the fibrinogen solution in the described solution one is former the obtaining of aprotinin solution fibrin of 2000～6000U/ml with concentration.
3, bone renovating material according to claim 1 is characterized in that: described PRP takes from autologous vein blood, the platelet rich plasma that gets with the extraction of secondary centrifuging method.
4, tissue engineered bone according to claim 3 is characterized in that: described extraction may further comprise the steps from the secondary centrifuging method of body RPR: with anticoagulation in 20 ℃ with 3000r/min centrifugal 10 minutes for the first time, draw 3mm composition under supernatant and the tunica albuginea layer; With 3600r/min centrifugal 15 minutes for the second time, abandon upper strata 3/4 supernatant under the same temperature, remainder is PRP.
5, according to claim 1 or 2 or 3 described injected bone repairing materials, it is characterized in that: in solution one, add the seed cell of certain cell density before the hybrid injection, promptly obtain corresponding injection type tissue engineering bone by injected bone repairing material compound seed cell.
6, the preparation method of injected bone repairing material as claimed in claim 1 is characterized in that, may further comprise the steps:
A. material is prepared:
(1) from body RPR: take from vena systemica blood, extracting platelet rich plasma with the secondary centrifuging method is PRP, puts-70 ℃ of refrigerators and preserves standby; Former with aprotinin solution solution fibrin, make into solution state, preparation calcium chloride-thrombin mixed solution, wherein, calcium chloride concentration is 40mmol/ml, concentration of thrombin is 400～1000IU/ml;
(2) fibrinogen solution: former with the aprotinin solution fibrin, make it become solution state;
(3) calcium chloride-thrombin mixed solution: configuration calcium chloride-thrombin mixed solution, wherein, calcium chloride concentration is 40mmol/ml, concentration of thrombin is 400IU/ml;
B. will be from body RPR and fibrinogen solution with 1: 3～6 mixed, sonic oscillation makes mix homogeneously, obtains containing the fibrinogen solution of PRP;
C. with the duplex syringe fibrinogen solution that contains PRP and the calcium chloride-thrombin mixed solution that obtain of extraction step B respectively, by mixing needle injection, the fibrinogen solution and the calcium chloride-thrombin solution ratio that contain PRP during injection are 9: 1～1: 1, form the stronger gel of adhesion strength, wherein the shared volume ratio of PRP is 10～30%, promptly obtains described injected bone repairing material.
7, the preparation method of injected bone repairing material according to claim 6, it is characterized in that: in the fibrinogen solution that contains PRP that step B obtains, add a certain amount of seed cell, go in the body according to step C hybrid injection again, obtain injection type tissue engineering bone by injected bone repairing material compound seed cell.
8, the purposes that is used to prepare the damaged and bone does not connect of treatment bone and promotes the graft of union of fracture as claim 1 or 2 or 3 described injected bone repairing materials.
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