CN100406071C

CN100406071C - Method for preparing HAP/beta-TCP structured tissue engineering bone

Info

Publication number: CN100406071C
Application number: CNB2006100906717A
Authority: CN
Inventors: 陈大福; 田伟
Original assignee: TRAUMA ORTHOPAEDICS INST BEJING
Current assignee: TRAUMA ORTHOPAEDICS INST BEJING
Priority date: 2006-07-06
Filing date: 2006-07-06
Publication date: 2008-07-30
Anticipated expiration: 2026-07-06
Also published as: CN1883719A

Abstract

The present invention discloses a method for preparing HAP/beta-TCP tissue engineered bones with biological structure, which comprises: firstly, spongy bones at the lower ends of cattle femurs are used as raw material, and after marrow, fat and protein are removed, the spongy bones are rinsed, dried and treated at high temperature so that HAP is obtained; after HAP reacts with NH4H2PO4, products are heated again at high temperature to obtain an HAP/beta-TCP material support with biological structure; secondly, after being separated in vitro, bone marrow stem cells are inoculated in a culture bottle, DMEM culture solution is added into an incubator to be cultured at 37 DEG C and under 5% of CO2, and culture solution is exchanged every two to three days; when growing to 90%, the cells are digested by trypsinase for subculture, and after reaching 10<6> to 10<7>/ml, bone marrow stem cells are reserved; the HAP/beta-TCP material support with biological structure is cleaned by ultrasonic waves, subcultured cells are uniformly dripped on the support for induction culture, and after inner pores of the support material is overgrown with the cells, an HAP/beta-TCP tissue engineered bone with biological structure is formed. The microstructure of the HAP/beta-TCP tissue engineered bone is completely similar to that of a spongy bone, which is favorable for bone formation in vivo and degradation of support material, the degrading speed can be controlled, and bone defect repair is speeded up. As a result, the HAP/beta-TCP tissue engineered bone has a wide application prospect in the field of bone transplantation.

Description

The preparation method of biological structure HAP/ β-TCP tissue-engineered bone

Affiliated technical field

The present invention relates to medical science and biomedical engineering field, specially refer to the preparation method of a kind of biological structure HAP/ β-TCP tissue-engineered bone.

Background technology

The especially big damaged reparation of the bone defect repair that causes after a variety of causes such as complicated injuries, the bone tumor curettage, it is still problem to be solved for the bone source.Because limited, and there is graft donor site to cause the possibility of complication such as hematoma, infection, pain from body bone bone amount; There are potential dangers such as immune inflammation reacts, spreads disease in allograph bone.Therefore, research organization's through engineering approaches bone is significant as bone impairment renovation material.Organizational project relates to timbering material, seed cell and induces or somatomedin, and wherein timbering material and seed cell are the keys of organizational project.

The biologic inorganic material is the important research object of bone tissue engineering scaffold, hydroxyapatite (Hydroxyapatite, HAP) be the natural inorganic composition of bone, biological characteristics such as have excellent biological compatibility and can combine with the bone key, therefore, the HAP that develops varied various trait and structure with distinct methods has been used for repairing bone defect.Be difficult to degrade but HAP is stable in vivo and limited its application clinically.(it is fast than HAP to degrade for β-Tri-calcium Phosphate, β-TCP) have excellent biological compatibility equally, but the degradation speed instability is difficult to control for bata-tricalcium phosphate.Have and studies show that transplanting HAP and the composite of β-TCP are more conducive to the reparation of defective bone than their homogenous materials wherein.

HAP/ β-TCP the composite of synthetic is often because of the pore morphology inequality, be spindle, aperture, Kong Yukong junction is less, and the size distribution in aperture differs greatly, all have to 700 microns from several 10 microns, even have bulla to form, and can not reach connection fully between the hole, the part hole is poor efficiency or invalid hole.Therefore, synthetic porous HA/ β-TCP has very big influence to the autism growth of nutrient, metabolite exchange and the bone of repopulating cell.

The hole of the no machine support after the calcination of xenogenesis spongy bone and spongy bone structural similarity, the xenogenesis spongy bone is the main HAP that obtains after the physics and chemistry method is dispeled lipid, albumen and collagen, HAP handles through superphosphate again, part changes into β-TCP, thereby obtaining HAP/ β-TCP composite, this composite is more suitable for being used for the bone defect repair than synthetic HAP/ β-TCP on the structure of material.At present, from HAP/ β-TCP composite material and preparation method thereof that the calcination of xenogenesis spongy bone obtains, generally adopted the technology of boiling 5-10 hour, calcium ion and phosphate radical are lost, main is the micro structure distortion that causes spongy bone, subside, the girder fracture, the micro structure and the spongy bone architectural difference of the final HAP/ β-TCP composite that obtains are very big, therefore, be necessary existing method is improved to obtain form, the on all four biological structure HAP/ β of micro structure and spongy bone-TCP composite satisfies the structural requirement of bone tissue engineer seed cell to timbering material better.

At senile osteoporosis, tumor, bone after patients such as the diabetes femur head necrosis fracture is damaged, its reparation is slower, one of major reason is that whole body and/or the damaged local stem cell population to osteoblast differentiation as new bone formation source significantly reduce, treat the relatively poor or healing time prolongation of the simple timbering material effect of the damaged usefulness of this type of bone, be still a difficult problem that needs solution, therefore, need development to plant bone marrow stroma stem cell and give the tissue-engineered bone that the skeletonization directional induction makes up with the inorganic timbering material of biological structure HAP/ β-TCP, not only for new bone formation provides support but also seed cell is provided, to accelerate the damaged reparation of bone.

Summary of the invention

The purpose of this invention is to provide a kind ofly be convenient to vivo degradation, degradation speed is controlled, form is similar fully to spongy bone with micro structure, is more conducive to the preparation method of the biological structure HAP/ β-TCP tissue-engineered bone of bone defect repair.

In order to achieve the above object, the present invention has adopted technical scheme described as follows: the preparation method of a kind of biological structure HAP/ β-TCP tissue-engineered bone, comprise HAP/ β-TCP composite material bracket that preparation has biological structure, the stroma stem cell in-vitro separation, the amplification and to the osteoblast directional induction, make up tissue-engineered bone, be characterized in: the preparation of a. biological structure HAP/ β-TCP composite material bracket, vertebral body with the cattle that grows up, femoral head or distal part of femur spongy bone are raw material, dispel lipid through repeatedly cleaning the assorted chemical method that reaches of dispelling, behind the albumen, again through repeatedly rinsing, the dehydration after drying, carry out the high-temperature process first time, a plurality of temperature program(me) sections are set in the temperature interval from low to high, to obtain to keep the HAP of spongy bone trabecularism, with the NH of HAP and 0.5-2.0M concentration ₄H ₂PO ₄React after 1-8 hour, through the high-temperature process second time, acquisition HAP is the biological structure HAP/ β-TCP composite material bracket of 80-30 than 20-70 with the weight ratio of β-TCP; B. bone marrow stroma stem cell (MSC) in-vitro separation, amplification, conventional local anesthesia in being extracted animal body, the posterior superior iliac spine puncture, extract the about 2-6ml of bone marrow fluid, anticoagulant heparin, PBS dilution, add Ficoll separating medium gradient centrifugation, with the sucking-off of MSC layer, add PBS, centrifugal, be inoculated in culture bottle, add high sugared DMEM culture fluid, go into 37 ℃ of incubators, volume ratio is that 5%CO2 cultivates, and changes liquid according to cell growth condition, equilibrated ph value, cultivations of going down to posterity that cell growth reaches at 90% o'clock, trypsin use in passage digestion, go down to posterity cultivate several after bone marrow stroma stem cell quantity reach 10 ⁶-10 ⁷Collect standby behind individual/ml; C. make up tissue-engineered bone,, material is soaked in a small amount of culture medium and in super-clean bench, dry with preceding, 10 with above-mentioned biological structure HAP/ β-TCP stock support ultrasonic cleaning in high purity water, drying, encapsulation, sterilization ⁶-10 ⁷/ ml cell evenly drops on the block timbering material, 37 ℃ of lower volume are than hatching 2-5 hour for 5%CO2, add culture medium again, the DMEM culture medium changes inducing culture into after 24 hours, changed one time inducing culture in every 2-3 days, 12-16 days cells cover with the tissue-engineered bone that is built into the HAP/ β-TCP with biological structure behind the timbering material internal void.

Described biological structure HAP/ β-TCP composite material bracket be shaped as the square body bulk, or with the square body bulk through graininess broken, screening back formation 0.5mm-2mm.

In described biological structure HAP/ β-TCP composite material bracket, the weight ratio of preparation HAP and β-TCP is respectively 80/20,65/35, the NH of biological structure HAP/ β-TCP composite of 50/50,30/70 ₄H ₂PO ₄Concentration is respectively 0.50～0.9M, 0.95～1.30M, 1.35～1.70M, 1.75～2.0M, and the response time is 1～8 hour.

In the preparation of a. biological structure HAP/ β-TCP composite material bracket described in the above technical scheme, the high-temperature process temperature is provided with 12-18 temperature program(me) section from 100 ℃ to 1000 ℃ for the first time, the fastest heating rate 15-30 ℃/minute, the slowest heating rate 2-5 ℃/minute, cooling naturally; High-temperature process was held warm 3-5 hour for 800-1000 ℃ for the second time, and heating rate 5-12 ℃/minute, cooling naturally.

In b. marrow stroma stem cell in-vitro separation described in the above technical scheme, the amplification, the prescription of high sugared DMEM culture fluid is:

The high sugared DMEM stock solution of preparation 1000ml

The a small amount of high purity deionized water of one bag of usefulness of DMEM dry powder is earlier molten

Folic acid 6mg

Arginine-hydrochloric acid 116mg

Glutamine 216mg

Aspartic acid 36mg

Heper?acid 4.766g

Glucose 4g

Sodium bicarbonate-after add 2g

The gentamycin 1ml of 40,000 units

Preparation institute water is a high purity deionized water

And trypsin cell dissociation formula of liquid is:

Claiming 0.25g trypsin dry powder to be dissolved in 100ml does not have in calcium magnesium Hank ' the s liquid, and fully the aseptic filtration packing is standby behind the mixing;

The preparation of no calcium, magnesium Hank ' s liquid

4 ℃ of preservations of pH=7.2

Sodium chloride 8g

Potassium chloride 0.4g

Sodium hydrogen phosphate 0.7g

Potassium dihydrogen phosphate 0.06g

Glucose 1ml

Distilled water 1000ml

1% phenol red 2ml.

Described c. makes up in the tissue-engineered bone, and inducing culture is to contain sodium 10 ^-3Mol/L, dexamethasone 10 ^-8Mol/L, vitamin C 50mg/L, BMP ₂The high sugared DMEM culture medium of 100ng/ml.

Because HAP/ β-TCP composite that the present invention has adopted biological method to prepare has biological structure, the bone marrow stroma stem cell in-vitro separation, the amplification and to osteoblast directional induction bone marrow stroma stem cell, and be configured to tissue-engineered bone as support plantation bone marrow stroma stem cell with the HAP/ β-TCP composite of biological structure, because the form of this tissue-engineered bone, micro structure is similar fully to spongy bone, HAP/ β-TCP composite material bracket with biological structure is convenient to degraded in vivo, degradation speed can be controlled, and plant bone marrow stroma stem cell on the HAP/ of biological structure β-TCP composite inorganic material support and give the skeletonization directional induction, not only support was provided but also seed cell was provided for new bone formation, so accelerated the damaged reparation of bone greatly, thereby make the HAP/ β-TCP tissue-engineered bone with biological structure have broad application prospects in the bone fields of implantation, the scope that biological structure HAP/ β-TCP tissue-engineered bone is used is:

1. repair because of the bone after wound, tumor and the excision of congenital pseudarthrosis damaged;

2. the bone bridge in fusion between vertebral body, the other fusion of vertebra, the enlargement of cervical canal is implanted;

3. the damaged filling of bone that forms that resets after tibial plateau, calcaneus compression fracture and the Colles fracture is rebuild;

4. the damaged filling of femur head necrosis focus cleaning postoperative bone is rebuild;

5. the damaged filling of Periprosthetic bone was rebuild when bone dissolved focus processing or overhaul technology behind the artificial joint replacement;

6. jaw defect reparation, alveolar ridge increase;

7. supply the damaged backfill of bone district's bone after taking from the body bone.

Description of drawings:

Fig. 1 biological structure HAP/ β of the present invention-TCP tissue-engineered bone structure chart

The micro structure figure of Fig. 2 biological structure HAP/ β-TCP timbering material

Fig. 3 60 all HAP/ β-TCP tissue-engineered bone is repaired the damaged figure of 1.5cm rabbit radius segmental

Simple HAP/ β of 60 weeks of Fig. 4-TCP stock support is repaired the damaged figure of 1.5cm rabbit radius segmental

The specific embodiment

Embodiment

Make up the biological structure HAP/ β-TCP tissue-engineered bone and the application of rabbit bone marrow stroma stem cell

One, the preparation of biological structure HAP/ β-TCP timbering material:

1. the source of spongy bone: get 3 years old adult bulls bone lower end spongy bone, cut 30mm * 20mm * 15mm piece material, (also desirable cattle vertebral body or bulls bone replace bulls bone lower end);

2. raw material high pressure flowing water flushing, warm water soaking, rinsed with deionized water is removed impurity, bone marrow and part fat for each 2-5 time;

3.1-2M KOH or NaOH soaked rinsed with deionized water 2-4 time 24-48 hour;

4.2%-20%H ₂O ₂Soaked 24-48 hour, rinsed with deionized water 2-4 time is dried;

5.75%-100% series dehydration of alcohol 24-48 hour dries;

6.60 ℃-100 ℃ dry 4-24 hour;

7. high-temperature process for the first time is provided with 12-18 temperature program(me) section from 100 ℃ to 1000 ℃, and the fastest heating rate 15-30 ℃/minute, the slowest heating rate 2-5 ℃/minute, the temperature program(me) section specifically is allocated as follows:

1) room temperature rise to 100-130 ℃ 10-20 minute, held warm 10-20 minute;

2) 100-130 ℃ rise to 200-220 ℃ 10-30 minute, held warm 60-90 minute;

3) 200-220 ℃ rise to 250-260 ℃ 15-25 minute, held warm 20-40 minute;

4) 250-260 ℃ rise to 280-310 ℃ 5-15 minute, held warm 10-20 minute;

5) 280-310 ℃ rise to 320-330 ℃ 8-15 minute, held warm 20-60 minute;

6) 320-330 ℃ rise to 360-400 ℃ 5-10 minute, held warm 20-35 minute;

7) 360-400 ℃ rise to 450-500 ℃ 10-20 minute, held warm 10-30 minute;

8) 450-500 ℃ rise to 540-560 ℃ 5-20 minute, held warm 10-20 minute;

9) 540-560 ℃ rise to 580-610 ℃ 15-25 minute, held warm 30-60 minute;

10) 580-610 ℃ rise to 660-680 ℃ 6-15 minute, held warm 10-20 minute;

11) 660-680 ℃ rise to 750-800 ℃ 10-15 minute, held warm 15-20 minute;

12) 750-800 ℃ rise to 880-910 ℃ 15-25 minute, held warm 15-30 minute;

13) 880-910 ℃ rise to 940-960 ℃ 10-20 minute, held warm 90-180 minute;

14) be cooled to room temperature naturally;

8. material ultrasonic cleaning 3-5 time, 80-100 ℃ dry 4-8 hour;

9. material is dipped in the NH that concentration is 0.95-1.50M ₄H ₂PO ₄In 3-6 hour, 80 ℃ of dryings 4 hours;

10. high-temperature process was held warm 3-5 hour for 800-1000 ℃ for the second time, and heating rate 5-12 ℃/minute, cooling naturally;

11. material ultrasonic cleaning 1-3 time, 80-100 ℃ dry 4-8 hour, obtain HAP and β-TCP ratio and be biological structure HAP/ β-TCP composite material bracket of 65/35;

12. double-deck encapsulation, cobalt ₆₀Sterilization.

The micro structure of its HAP/ β-TCP timbering material is seen Fig. 2.

Two, the bone marrow stroma stem cell in-vitro separation, increase, induce:

Specimen is obtained and cell separation

Conventional local anesthesia, the posterior superior iliac spine puncture is extracted the about 4ml of bone marrow fluid, anticoagulant heparin.

1 takes out two monthly age rabbit bone marrow 2ml, 2 times of PBS dilutions, and mixing is put into the 20ml centrifuge tube;

2 prepare 2 of 10ml centrifuge tubes, and each adds lymphocyte separation medium 3ml, extract the bone marrow diluent with the 5ml syringe, slowly inject along the centrifugal tube wall of 10ml, make single cell suspension;

3 centrifugal 2000rpm, 15min;

4 with the sucking-off of MSC layer (white opacity shape), puts into the another one centrifuge tube, adds PBS, centrifugal 1500rpm, 7-8min;

5 repeat to wash once, abandon PBS, 3 * 10 ⁵The concentration of/ml is inoculated in 25cm ²Tissue Culture Flask in, add high sugared DMEM (12%FBS, the 5ml of gentamycin 40,000 units/100ml), 37 ℃ of 5%CO ₂Cultivate.

The cell culture amplification

Former foster 36 hour cells of being commissioned to train are adherent, wash suspension cell off, all change liquid.

Go down to posterity to cultivate and change liquid according to cell growth condition, the cell growth reaches at 90% o'clock and goes down to posterity:

1 sucking-off culture fluid adds 2ml PBS, rocks gently, abandons PBS;

225cm ²Culture bottle adds pancreatin 3ml, 37 ℃ of 3min, and microscope observing cell all suspends, and high sugared DMEM equivalent adds, and stops digestion;

3 draw cell suspension puts into centrifuge tube, 1500rpm, 3min;

4 centrifugal after, abandon PBS, add PBS 3-5ml according to cell number, counting, centrifugal 1500rpm, 3min;

5 abandon PBS, add culture medium, about 2000cm ²Inoculation, 37 ℃ of 5%CO ₂Cultivate.

Osteogenic induction

1) abductive approach: well-grown MSC, press 2000/cm ²Plant in 6 orifice plates that preset the IWAKI slide.After 24 hours, use the osteogenic induction system instead, changed liquid once in every 2-3 days, the alkaline phosphatase staining of 12 days histochemical methods (calcium-cobalt method), scarlet I, the III Collagen Type VI of dying of picric acid sky wolf;

2) osteoblast inducing culture system: contain sodium 10 ^-3Mol/L, dexamethasone 10 ^-8Mol/L, vitamin C 50mg/L, BMP ₂The high sugared DMEM culture medium of 100ng/ml;

Wherein, rabbit bone marrow stroma stem cell (MSC) cultivation is high sugared DMEM stock solution preparation (1000ml) with liquid:

Folic acid 6mg

Arginine-hydrochloric acid 116mg

Glutamine 216mg

Aspartic acid 36mg

Heper?acid 4.766g

Glucose 4g

Sodium bicarbonate-after add 2g

The gentamycin 1ml of 40,000 units

Preparation institute water is a high purity deionized water;

The trypsin preparation

Claiming 0.25g trypsin dry powder to be dissolved in 100ml does not have in calcium magnesium Hank ' the s liquid, and fully the aseptic filtration packing is standby behind the mixing.

The preparation of no calcium, magnesium Hank ' s liquid

PH=7.24 ℃ of preservation

Sodium chloride 8g

Potassium chloride 0.4g

Sodium hydrogen phosphate 0.7g

Potassium dihydrogen phosphate 0.06g

Glucose 1ml

Distilled water 1000ml

1% phenol red 2ml.

Serum be in 56 ℃ of water-baths of hyclone deactivation 30min to destroy complement and some infectious virus.

Lymphocyte separation medium (Ficoll), density 1.077g/ml is used for the mononuclear cell of gradient separations bone marrow.

Three, biological structure HAP/ β-TCP kind is planted the preparation that the rabbit bone marrow stroma stem cell makes up tissue-engineered bone

1. with biological structure HAP/ β-TCP support ultrasonic cleaning in high purity water, drying, encapsulation, sterilization;

2. material is soaked in the sugared DMEM culture medium of a small amount of height and in super-clean bench, dry;

3. with 0.2-0.5ml 10 ⁶-10 ⁷/ ml MSC cell evenly drops in the block support material of 0.4-1ml

On the material, 37 ℃ of 5%CO ₂Hatched 2-5 hour, and added high sugared DMEM culture medium again;

4.24-48 high sugared DMEM culture medium changes the MSC inducing culture into after hour, changed a subculture in every 2-3 days, first changes liquid with the MSC inducing culture for the second time, uses high sugared DMEM culture medium later on, and 14 days cells cover with the timbering material internal void and are built into tissue-engineered bone.

The engineered bone structure of biological structure HAP/ β-TCP is seen Fig. 1.

Four, biological structure HAP/ β-TCP kind is planted the tissue-engineered bone repairing bone defect animal experiment that the rabbit bone marrow stroma stem cell makes up

1. the adult new zealand rabbit of usefulness body weight 3.0-3.4kg is 8, the new 0.25mlkg of speed dormancy ^-1Aseptic operation is implemented in intramuscular anesthesia, and it is damaged to make the 1.5cm segmental with abrasive drilling in bilateral radius, constantly washes with normal saline during brill.Both sides are column biological structure HAP/ β-TCP tissue-engineered bone, the biological structure HAP/ β-TCP of the long 1.5mm of implanted diameter 3mm respectively, postoperative gentamycin 40,000 a units/intramuscular injection three days, single cage raising;

2.60 week is put to death animal, and get the bilateral radius and ulna and go into 10% formalin fixed, gradient ethanol dehydration, PMMA embedding, serial section (Leica SM2500E), Von kossa Ponceaux is dyed.

3. the histology shows 60 all that biological structure HAP/ β-the TCP tissue-engineered bone has been repaired 1.5cm radius segmental is damaged, sees Fig. 3, the most of degraded of timbering material, and the skeletonization effect is better than simple biological structure HAP/ β-TCP material, sees Fig. 4.

More than be to make up the biological structure HAP/ β-TCP tissue-engineered bone of rabbit bone marrow stroma stem cell and the specific embodiment of application, if can also be built into the biological structure HAP/ β-TCP tissue-engineered bone of human bone marrow stroma stem cell according to this preparation method, the application that the bone that is used for human body is damaged, bone is transplanted.

Claims

1. the preparation method of biological structure HAP/ β-TCP tissue-engineered bone, comprise HAP/ β-TCP composite material bracket that preparation has biological structure, bone marrow stroma stem cell in-vitro separation, amplification and to the osteoblast directional induction make up tissue-engineered bone, it is characterized in that:

A. the preparation of biological structure HAP/ β-TCP composite material bracket, vertebral body, femoral head or distal part of femur spongy bone with the cattle that grows up are raw material, dispelling assorted and after chemical method dispels lipid, albumen through repeatedly cleaning, again through repeatedly rinsing, dehydration after drying, carry out the high-temperature process first time, a plurality of temperature program(me) sections are set, to obtain to keep the HAP of spongy bone trabecularism, with the NH of HAP and 0.5-2.0M concentration in the temperature interval from low to high ₄H ₂PO ₄React after 1-8 hour, through the high-temperature process second time, acquisition HAP is the biological structure HAP/ β-TCP composite material bracket of 80-30 than 20-70 with the weight ratio of β-TCP;

B. bone marrow stroma stem cell in-vitro separation, amplification, conventional local anesthesia in being extracted animal body, posterior superior iliac spine puncture, extract bone marrow fluid 2-10ml, anticoagulant heparin, the PBS dilution adds lymphocyte separation medium separating medium gradient centrifugation, with the sucking-off of MSC layer, add PBS, centrifugal, be inoculated in culture bottle, add high sugared DMEM culture fluid, go into 37 ℃ of incubators, volume ratio is 5%CO ₂Cultivate, change liquid according to cell growth condition, the balance pH value, cultivations of going down to posterity that the cell growth reaches at 90% o'clock, trypsin is used in passage digestion, go down to posterity cultivate for several times after bone marrow stroma stem cell quantity reach 10 ⁶-10 ⁷Collect standby behind individual/ml;

C. make up tissue-engineered bone,, material is soaked in a small amount of culture medium and in super-clean bench, dry with preceding, 10 with above-mentioned biological structure HAP/ β-TCP stock support ultrasonic cleaning in high purity water, drying, encapsulation, sterilization ⁶-10 ⁷/ ml cell evenly drops on the block timbering material, 37 ℃ of 5%CO ₂Hatched 2-5 hour, add culture medium again, high sugared DMEM culture medium changes inducing culture into after 24 hours, changes one time inducing culture in every 2-3 days, and 12-16 days cells cover with the tissue-engineered bone that is built into the HAP/ β-TCP with biological structure behind the timbering material internal void.

2. according to the preparation method of the described tissue-engineered bone of claim 1, it is characterized in that: in the preparation of described a. biological structure HAP/ β-TCP composite material bracket, high-temperature process temperature program(me) section is that 12-18 temperature program(me) section is set in the temperature range from 100 ℃ to 1000 ℃ for the first time, the fastest heating rate 15-30 ℃/minute, the slowest heating rate 2-5 ℃/minute, cooling naturally; High-temperature process was held warm 3-5 hour for 800-1000 ℃ for the second time, and heating rate 5-12 ℃/minute, cooling naturally.

3. according to the preparation method of the described tissue-engineered bone of claim 1, it is characterized in that: described a. biological structure HAP/ β-TCP composite material bracket be shaped as the square body bulk, or through graininess broken, screening back formation 0.5mm-2mm.

4. according to the preparation method of the described tissue-engineered bone of claim 1, it is characterized in that: in described a. biological structure HAP/ β-TCP composite material bracket, the weight ratio of preparation HAP and β-TCP is respectively 80/20,65/35, the NH of biological structure HAP/ β-TCP composite of 50/50,30/70 ₄H ₂PO ₄Concentration is respectively 0.50～0.9M, 0.95～1.30M, 1.35～1.70M, 1.75～2.0M, and the response time is 1～8 hour.

5. according to the preparation method of the described tissue-engineered bone of claim 1, it is characterized in that: in described b. marrow stroma stem cell in-vitro separation, the amplification, the prescription of high sugared DMEM culture fluid is:

The high sugared DMEM stock solution of preparation 1000ml

Folic acid 6mg

Arginine-hydrochloric acid 116mg

Glutamine 216mg

Aspartic acid 36mg

Heper?acid 4.766g

Glucose 4g

Sodium bicarbonate-after add 2g

The gentamycin 1ml of 40,000 units

Preparation institute water is a high purity deionized water

The tryptic digestive juice prescription is:

The preparation of no calcium, magnesium Hank ' s liquid

4 ℃ of preservations of pH=7.2

Sodium chloride 8g

Potassium chloride 0.4g

Sodium hydrogen phosphate 0.7g

Potassium dihydrogen phosphate 0.06g

Glucose 1ml

Distilled water 1000ml

1% phenol red 2ml.

6. according to the preparation method of the described tissue-engineered bone of claim 1, it is characterized in that: described c. makes up in the tissue-engineered bone, and inducing culture is to contain sodium 10 ^-3Mol/L, dexamethasone 10 ^-8Mol/L, vitamin C 50mg/L, BMP ₂The high sugared DMEM culture medium of 100ng/ml.