CN104147641B

CN104147641B - A kind of for personalized bone renovating material and its preparation method

Info

Publication number: CN104147641B
Application number: CN201410330587.2A
Authority: CN
Inventors: 张丽君; 张英; 张登央; 王飞; 王妍
Original assignee: Shenzhen Polytechnic
Current assignee: Shenzhen Polytechnic
Priority date: 2014-07-11
Filing date: 2014-07-11
Publication date: 2016-08-31
Anticipated expiration: 2034-07-11
Also published as: CN104147641A

Abstract

The present invention relates to a kind of for personalized bone renovating material and preparation method thereof, bone renovating material includes organic material and the inorganic material that mass ratio is 0.1 0.6:1, and has wrapped up the lipid microsphere powder of protein.This bone renovating material can be used for the Bone Defect Repari of personalization, and it has biocompatibility and degradable, has the safety required for biological bone material, space network, the feature of balance degraded.

Description

A kind of for personalized bone renovating material and its preparation method

Technical field

The present invention relates to bioengineering field, be specifically related to a kind of bioprosthetic material.

The invention still further relates to the preparation method of described bioprosthetic material.

Background technology

Osseous tissue damage is a great problem that human health faces.Along with medical science applied development, artificial bone graft's technology It is applied to clinic the most relatively broadly, but artificial bone graft still suffers from some problems, the most seriously osseous tissue so far Shortage, donor is not enough, and meanwhile, artificial bone graft's therapeutic scheme is the most still difficult to repair the osseous tissue sustained damage or be allowed to function Recovered for a long time.Organism manufacturing is a kind of important potential replacement therapy measure, is expected to finally solve this problem.When When individual osseous tissue damage or disappearance, Biotechnology uses the mode implanting organ succedaneum to carry out personalized treatment, this Kind of mode can have a repertoire of tissue or organ at the implantation initial stage, and progressively has during growing in vivo The tissue needed or the function of organ.Additionally osseous tissue is a three-dimensional tissue in human body, and inside has blood vessel, nerve, bone thin The biotic component of the Various Complex such as born of the same parents, bone marrow, solid and its internal passage of manufacture that design is three-dimensional are also to need to solve Problem.

The purpose of tissue engineering bracket material is for building the cell of tissue offer one three-dimensional rack, beneficially cell Stick, breed or even break up, provide suitable external environment for cell growth.Biodegradable material rises in Tissue Engineering Study Very important effect, it is the key that organizational project realizes industrialization.

In addition to meeting the requirement of general biomedical material, the ideal biomaterial needed for tissue engineering also needs to meet Following requirement: possess good biocompatibility, neoblastic function will not be affected because of the rejection of adjacent tissue；Have Degradability and suitable degradation rate, when the cell or tissue transplanted is survived in receptor, timbering material can be degraded voluntarily； Have and meet cell, the biomechanical strength of histoorgan requirement；There is good Cellular interfaces relation, can interact to protect Deposit and promote cell function；Be prone to plastotype, it is simple to be processed into preferably two dimension or three dimensional structure, and be transplanted to internal after can protect Hold original shape；Can be combined with other bioactive molecules, promote propagation and the differentiation of seed cell；Easily sterilizability.

The addition of computer aided technique at present, the progress of Medical Imaging Technology and material fabrication and processing technology is again for tissue The personalized treatment of engineering creates new opportunity.As by non-intruding computed tomography (CT) and NMR (Nuclear Magnetic Resonance)-imaging (MRI) carry out three-dimensional reconstruction, instruct tissue typing or wound to differentiate.At present computer-aided design and manufacture (CAD/CAM) with And rapid shaping technique (RP) makes sclerous tissues's model, organization bracket and Custom Prosthesis prosthese, the application of 3D printing technique nearly Step has promoted cad technique, can set up personalization damage or the data base of alternate sites by medical imaging data, obtain accurately Volume and the osseous tissue individuation module such as size and internal voids.

The final purpose of organizational project is to allow cell grow in vitro, be divided into functional organization and organ.The source of cell Can be roughly divided into cell and the stem cell of maturation, ripe cell can be divided into again autologous with two different sources of allosome.Autologous Cell derived should be optimal cell derived.Then step to be passed through consists essentially of collects a small amount of autogenous cell Cultivate propagation in vitro.These tissues may be used for organizational project or are directly used in autologous treatment afterwards.It is now known that, Not only present in myeloid tissue, interstital stem cell (MSCs) under certain condition, can be thin to osteoblast, chondrocyte, one-tenth flesh Born of the same parents, adipose cell, fibroblast etc. break up, and there is also stem cell at its hetero-organization such as skin, liver, nerve etc., these Stem cell there is also the phenomenon of mutually conversion.Research shows, tissue-specific stem cells is careful possible as the kind of organizational project Born of the same parents originate, and the most autologous MSCs and timbering material are combined the case report of the existing clinical practice of tissue engineered bone of structure, display The good prospect of clinical practice.MSCs is also easy to realize the importing of exogenous gene and expression, has started application for gene therapy Prospect.Human marrow mesenchymal stem cell (human marrow mesenchymal stem cells, hMSCs) is owing to obtaining way Footpath is more convenient, and amplification phenotype is stable, avoids Medical Ethics dispute simultaneously, thus it is careful to become preferable tissue engineered bone kind Born of the same parents, for using hMSCs to build cartilage to chondroblast direction induction to build the researchs such as tissue engineered bone .Tali, external Inoculation bioactive materials is also implanted in nude mouse, and it becomes cartilage satisfactory for result.

Mature cell In vitro culture remains the problem being worth research, because how to be effectively improved cell in vitro Organizational project will be had a direct impact by multiplication capacity, and particularly patient is badly in need of this cell or the tissue of autogenous cell formation or device Official.Bioreactor is to be mounted with nutrient, gas (such as oxygen and carbon dioxide) and control of waste in right quantity level Agitator and the culturing room of sensor.Along with the growth being organized in bioreactor so that it is mechanical property reaches most preferably to Extremely crucial problem because many is organized in by extension, pulls or can make a response during compression, be reconstructed or Change their population structure.

Summary of the invention

It is an object of the present invention to provide a kind of bone renovating material, this bone renovating material can be used for the bone of personalization and repaiies Multiple, it has biocompatibility and degradable, has the safety required for biological bone material, space network, balance degraded Feature.

It is also another object of the present invention to provide the preparation method of described bone renovating material, by by bio-compatible degradable Organic material, after suitable solvent dissolves, after mixing with inorganic phosphate calcium salt, adds the lipid microsphere being enclosed with protein Powder mixes, and according to the 3D model data program of the design data that CT scan obtains, prints the bone of applicable specific individuals treatment Repairing 3D material, material removes remaining solvent through vacuum drying, inoculates bone marrow interstital stem cell, in the life of Automated condtrol Thing reactor carries out stereoscopic culture, it is thus achieved that personalized bone renovating material, finally implant pathological changes position to be repaired into the human body.

According to an aspect of the present invention, a kind of bone renovating material for personalization, including

Mass ratio is organic material and the inorganic material of 0.1-0.6:1, and

Wrap up the lipid microsphere powder of protein；Wherein:

Described organic material is selected from PLGA, PLA and PGA, PLA:PGA=5%-95%:95%-5% in described PLGA, divides Sub-weight range is 8000-250000；Described PLA molecular weight ranges is 8000-250000,；Described PGA molecular weight ranges is 8000-250000；

Described inorganic material is the one in bata-tricalcium phosphate, type alpha tricalcium phosphate, hydroxyapatite and tetracalcium phosphate Or multiple phosphoric acid class calcium salt；

The described protein being wrapped in lipid microsphere is selected from NFG, insulin like growth factor, weight Group human beta interferon, erythropoietin, Transforming growth factor-β3, one or several of bone morphogenetic protein, every part of fat In matter microsphere powder, the content of protein is；NFG 50～200ng/ part, insulin like growth factor 50～ 200ng/ part, erythropoietin 5～20IU/ part, recombinant human interferon alpha 2 50～200ng/ part, Transforming growth factor-β3 50- 120ng/ part, bone morphogenic protein BMP-2-250-100ng/ part, bone morphogenic protein BMP-2-7 is 50-100ng/ part；

Preferably protein is recombinant human interferon alpha 2 IFN-β, and the content of IFN-β is 100ng/ml；Preferred protein Combination for IFN-β Yu TGF-β 3.

According to a further aspect in the invention, the method for the bone renovating material that preparation is personalized, comprise the steps:

1) mix described organic material and inorganic material according to predetermined ratio with organic solvent, add and wrapped up protein Lyophilizing lipid microsphere powder, prepare bone renovating material；

2) by step 1) bone renovating material proceed to 3D printer, become with nuclear magnetic resonance, NMR with non-intruding computed tomography As carrying out individuation bone injury site three dimensional data collection, the faultage image collected section is converted into 3 D-printing model, Rapid shaping technique is utilized to make osseous tissue model and organization bracket；

3) by step 2) 3D print preparation tissue scaffold design be placed in cell bottle inoculation osteoblast, cell implantation concentrations To 1 × 10⁵/ mL, cultivates and transfers to carry out in bioreactor stereoscopic culture to cell attachment or formation cell monolayer；

4) the personalized bone renovating material of implantable bone damage location is obtained.

Preparation method of the present invention, wherein material low temperature state to be in print procedure, temperature range is-30 DEG C～-10 DEG C, and the material printed persistently to give cold wind and make it fixed-type；The tissue scaffold design printed to be carried out very Vacuum freecing-dry.

Described tissue scaffold design needs to carry out aseptic process before inoculating cell, can irradiate with gamma-rays, or epoxy Ethane is fumigated.And described support is soaked through culture fluid.

Inoculating cell, to biological support, is cultivated the cell attachment treated on biological bone support for 2 days in culture bottle or is formed single Shifting tissue scaffold design after Ceng in bioreactor, stereoscopic culture, wherein the speed of fed-batch medium is 5ml/min-15ml/ Min, the composition of culture medium adjusts according to incubation time and growing state.Osteoblast (hMSC) complete medium includes becoming as follows Point: low sugar DMEM88% (v/v), hyclone 10% (v/v), Pidolidone 1% (v/v), green grass or young crops/streptomycin 1% (v/v). HMSC becomes cartilage differentiation culture medium to include following composition: DMEM in high glucose 96.3% (v/v), Pidolidone 1% (v/v), green grass or young crops/strepto- Element 1% (v/v), 10mmol/L ascorbic acid 0.5% (v/v), 1mmol/L dexamethasone 0.01% (v/v), 40 μ g/L L-dried meat Propylhomoserin 0.1% (v/v), 1000 μ g/L Sodium Pyruvates 0.1% (v/v), ITS+Premix1% (v/v), 10ng/ml TGF-β 3.

The osseous tissue material of the present invention, in individualized therapy, can keep degradation speed and the area of new bone group of biomaterial The balance of the speed of growth knitted, present invention employs cytokine and the parcel of bone protein interventional technique, especially lipid microsphere Technology, makes the growth in vitro of personalized osseous tissue and in the tumor growth phase, and more preferable diversification stimulates surrounding tissue and osseous tissue The growth of the various tissue systems being associated of itself.

Present invention finds IFN-β possibly as having the ability promoting that hMSCs becomes cartilage differentiation, and IFN-β can generation Acting on for TGF-β 3, the two is used in combination and can more promote into cartilage differentiation.

The present invention uses organic-biological material, and application advanced manufacturing technology and 3D printing technique, in conjunction with modern individuals image Learn a skill, make the treatment of personalized bone more conform to three-dimensional size, it is ensured that cell passage osseous tissue within, after implantation faster with Autologous tissue merges, and reduces allosome rejection.

The osseous tissue that the bone renovating material of the personalization that the present invention provides and its preparation method obtain, has biological bone material Required safety, space network, the feature of balance degraded.

Accompanying drawing explanation

Fig. 1 is for adding the alcian blue coloration result of different proteins (cytokine)；

Fig. 2 is for adding the GAG testing result of each protein (cytokine)；

Fig. 3 is to add IFN-β to promote that hMSCs becomes the GAG testing result of cartilage differentiation；

Fig. 4 is to add IFN-β to promote that hMSCs becomes the qRT-PCR result of cartilage differentiation, is shown as cartilage differentiation the 14th day each Group CollagenII expression；

Fig. 5 is to add IFN-β to promote that hMSCs becomes the qRT-PCR result of cartilage differentiation, is shown as cartilage differentiation the 14th day each Group SOX9 expression；

Fig. 6 is to add IFN-β to promote that hMSCs becomes the WESTERN BLOT result of cartilage differentiation；

Fig. 7 is that variable concentrations IFN-β induces into cell GAG total content variation tendency after cartilage differentiation；

Fig. 8 is the scanning electron microscope examination result of hMSCs Osteoblast Differentiation on PLGA/TCP material；

Fig. 9 be the ALP activity ratio of the hMSC cell of Osteoblast Differentiation on PLGA/TCP material relatively；

Figure 10 is the bioreactor construction schematic diagram for stereoscopic culture.

Detailed description of the invention

Material source:

In addition to indicating especially, all material is commercially available purchase；

HMSC cell derived: the hMSC cell strain in the 5th generation is purchased from Guangzhou Sai Ye company

Prepared by the lipid microsphere of parcel protein:

It is as follows that film evaporation method prepares lipid microsphere step: 1. film forming: film material dispensing: by injection lecithin, cholesterol Weigh by charge ratio 3～7:1～3:1 with 18-amine. (or vitamin E), be then dissolved in the dichloro of final concentration of 0.5～1.5% In methane or chloroform, being placed in by this liquid in the eggplant type bottle of Rotary Evaporators, 60 DEG C of water-baths make as evaporating temperature, rotation Solvent evaporates, and final stage nitrogen dries up solvent, makes liposome film forming in eggplant type bottle.2. aquation: add factor-containing (such as: recombined human IFN β 1a (final concentration of 100ng/ml) and bone morphogenic protein BMP-2-2 (final concentration of 80ng/ml)) Aqueous phase solution about 50ml (film material is 1% in the concentration of solution), adds several little beades, acutely shakes, treat that thin film is from bottle wall On completely fall off after, water bath sonicator about 30min, in bottle, liquid is light cloud.3. high-pressure homogenization: by rough lipoid microsphere high pressure The temperature that refiner exports, in pressure is 15Kpa～30Kpa pressure limit, by condensed water, is controlled at 30 DEG C by refiner ～37 DEG C, homogenizing to 200～the particle diameter of 400nm.

Freeze-drying (FDM) combines and prepares lipid microsphere, increases following steps the most on the basis of the above: 4. freezing dry Dry: by the liposomal mixtures after homogenate, concussion, to add appropriate trehalose and make the most final concentration of 1.5%, by liquid in pressure It is the leaf filter of 0.45um by aperture under power, carries out aseptic process, also the pore size control of liposome can be existed simultaneously Below 450nm, it is also possible to the particle diameter further making liposome is the most homogeneous, takes the sample after filtration and aseptically divides Dress, 2ml/ bottle, it is divided in cillin bottle；5. freezing, it is dried.Lipid microsphere albumen lyophilizing sample is in use, certain body can be added Long-pending PBS or normal saline (2ml), concussion to light cloud.

Instrument and equipment:

3D printing device, CT and MRI are common apparatus；

Bioreactor for stereoscopic culture: structure sees Figure 10, and bioreactor 100, including tank body 1, difference position In the first cover 2A and the second cover 2B at tank body two ends, the first cover is provided with the first hold assembly 3A stretching to tank interior With at least one the first conduit 4A passed in and out for material, the second cover is provided with the second hold assembly 3B stretching to tank interior With at least one the second conduit 4B passed in and out for material, formed between the first cover 2A and the second cover 2B and tank body 1 and seal. By commercialization bioreactor, being injected by the culture fluid conduit from this bioreactor lower end, culture fluid is full of tank body 1, from The conduit on tank body top flows out, and is back to commercialization bioreactor.Gas is automatically controlled by described commercialization bioreactor Body, the dissolved oxygen of liquid, pH, pressure, temperature and the interpolation of additional material, anti-to the biology of stereoscopic culture cultivating different times Answer device to supplement different nutritional labelings, it is achieved tank body 1 inner tissue's block and the solid of biomaterial, continuously, close to the cultivation of body fluid.

Below in conjunction with specific embodiment, the present invention is described in detail:

The personalized osseous tissue material mixture ratio of embodiment 1.

Weigh 1g hydroxyapatite or type alpha tricalcium phosphate or bata-tricalcium phosphate or tetracalcium phosphate is placed in the container that one end is airtight In, after adding 2ml acetone, high-ranking military officer one end is airtight, and vibration makes acetone infiltrate hydroxyapatite.

Weigh 0.2g, mean molecule quantity be 100,000 PLGA (poly-(D, Pfansteihl-co-glycolic), PLA:PGA=75: 25) or PGA (PGA) or PLA (PLLA), being placed in the container that one end is airtight, adding volume is the third of 0.2ml After ketone the other end is airtight, make PLGA be completely dissolved.

Organic material after dissolving is completely added in inorganic material, and vibration stirs, airtight.

Take a deal is enclosed with lyophilizing lipid microsphere powder, contains NFG in this powder simultaneously (NGF) being 100ng/ part, recombinant human interferon alpha 2 (IFN-β) is 120ng/ part, and bone protein (BMP-2) is 100ng/ part.Added Enter in the above-mentioned marking liquid prepared, mix rapidly.

Prepared by the personalized osseous tissue material of embodiment 2

Individuation bone injury site is carried out three-dimensional by non-intruding computed tomography (CT) and NMR (Nuclear Magnetic Resonance)-imaging (MRI) Data acquisition, the faultage image collected section uses mimic or 3dMed software processes, is converted into the space vector number of three-dimensional According to, then by Three-dimensional Design Software Autodesk3ds Max, it is converted into 3 D-printing model, it is compiled as 3D printer identification literary composition Part form such as stl file.

The print parameters of cubic materials support, between layers material square crossing stacking are set, use flat in every layer Line is placed, and the gap between every is 1mm, the movement that step height is 0.1mm, X-axis and Y direction that Z-direction is each Speed is 2mm/s, the pore size control of discharging opening between 0.5mm-0.1mm, the Stress control of discharging 20psi-60psi it Between.

Weighing 1g type alpha tricalcium phosphate to be placed in the container that one end is airtight, after adding 2ml acetone, high-ranking military officer one end is airtight, and vibration makes Acetone infiltration hydroxyapatite.

Weighing 0.3g, mean molecule quantity is the PLA of 100,000, is placed in the container that one end is airtight, and adding volume is 0.2ml's After acetone the other end is airtight, make PLA be completely dissolved.

Take a deal is enclosed with lyophilizing lipid microsphere powder, and this part of powder includes insulin like growth factor (IGF- I) being 100ng/ part, erythropoietin (EPO) is 20IU/ part, and recombinant human interferon alpha 2 (IFN β) is 80ng/ part, bone protein (BMP-7) it is 100ng/ part.It is added in the above-mentioned marking liquid prepared, mixes rapidly.

The material mixed is joined in the raw material storage room of 3D printer, run program, according to the program set Print designed cubic materials support.Print procedure maintains rack platform and environment temperature to be-20 DEG C, and persistently gives Cold wind makes Solid-Liquid Separation molding.

After support prints, support need to carry out vacuum lyophilization process, first paragraph temperature is set to-50 DEG C, and vacuum sets For 20pa, run 4h；Second segment temperature controls at 28～32 DEG C, 42 hours persistent period.

Embodiment 3 personalized osseous tissue material (adding cytokine IFN-β) induction hMSC cell becomes cartilage differentiation

Weighing 1g bata-tricalcium phosphate to be placed in the container that one end is airtight, after adding 2ml acetone, high-ranking military officer one end is airtight, and vibration makes Acetone infiltration hydroxyapatite.

Weighing 0.3g, mean molecule quantity is the PGA of 120,000, is placed in the container that one end is airtight, and adding volume is 0.2ml's After acetone the other end is airtight, make PGA be completely dissolved.

Take a deal is enclosed with lyophilizing lipid microsphere powder, and wherein (IFN-β) Han recombinant human interferon alpha 2 is 120ng/ part. It is added in the above-mentioned marking liquid prepared, mixes rapidly.

Three-dimensional rack ray is done aseptic process, aseptically, the hMSC cell of logarithm division stage will be in, dense Degree is 5 × 10⁵Cell/ml, takes 0.5ml respectively and is inoculated in three-dimensional rack and 6 porocyte plates (negative control), quiescent culture 20 My god, within the most every 4 days, change a cell culture complete medium.HMSC complete medium includes following composition: low sugar DMEM88% (v/ V), hyclone 10% (v/v), Pidolidone 1% (v/v), green grass or young crops/streptomycin 1% (v/v).HMSC becomes cartilage differentiation culture medium Including following composition: DMEM in high glucose 96.3% (v/v), Pidolidone 1% (v/v), green grass or young crops/streptomycin 1% (v/v), 10mmol/L Ascorbic acid 0.5% (v/v), 1mmol/L dexamethasone 0.01% (v/v), 40 μ g/L L-PROLINEs 0.1% (v/v), 1000 μ G/L Sodium Pyruvate 0.1% (v/v), ITS+Premix1% (v/v), 10ng/ml TGF-β 3.

Collect experimental group cell and cellular control unit in the 20th day, carry out biochemical analysis detection.

Glycosaminoglycan (GAG) total content is the important symbol that cell becomes cartilage differentiation level, and GAG content detection result shows Cartilage experimental group is become to have significant difference, GAG total content the most constantly to increase with blank group after cultivating 4d. whole In experimental period, the cell GAG total content of experimental group has significant difference compared with cellular control unit.Show prepared by the method Three-dimensional rack likely promotes the one-tenth cartilage differentiation of cell.

Embodiment 4. is added the osseous tissue material of different cytokines and is promoted that hMSCs becomes cartilage differentiation

Experimental technique:

Add different cytokines IGF-1, NGF, EPO and the group technology of IFN-β:

Take the logarithm the 8th generation hMSCs of trophophase, resuspended with complete medium, adjusts cell concentration to 1 × 10⁵/ mL.6 hole 200 μ L cell suspension are inoculated in the every hole of plate, and supplementing culture medium to 2mL. cell is divided into blank group, become cartilage matched group, IFN-β Group, IGF-1 group, NGF group and EPO group, complete medium during 4d, become cartilage differentiation culture medium, containing 100ng/ml IFN-β Complete medium, containing the complete medium of 100ng/ml IGF-1, complete medium containing 100ng/ml NGF and containing 10IU/ml The complete medium correspondence of EPO changes liquid, and every 4d changes liquid afterwards.

Alcian blue dyeing detection method:

After liquid is changed in packet, 14d takes out 6 orifice plates, carefully pumps the culture medium in hole, adds cleaning liquid and cleans, and Acidic Liquid is hatched Cleaning 2 times after 3min, dyeing liquor processes 30min, avoids illumination, cleans 2 times, and core fast red is redyed to observe under rear inverted microscope and clapped According to, the often multiple hole of group 3.

Experimental result

(1) the alcian blue coloration result of different cytokines is added

Alcian blue coloration result shows (Fig. 1) blank group, IGF-1 group, shuttle-type after NGF group and the dyeing of EPO group Cellular morphology is the most high-visible, and cell peripheral also has no that blue glycoprotein occurs, forming cartilage matched group has obvious cell Metamorphosis, cell peripheral has a large amount of glycoprotein to assemble, the trend of the oriented Chondrocyte Differentiation of IFN-β group cellular morphology, but only There is a small amount of glycoprotein visible.

(2) the GAG testing result of each cytokine is added

Glycosaminoglycan total content is the important symbol that cell becomes cartilage differentiation level, and GAG total content testing result shows (figure 2), becoming cartilage matched group 4d after using into cartilage differentiation culture medium instead to have significant difference (p < 0.01) with other groups, GAG is total Content the most constantly increase .IFN-β group cell GAG total content 4d, 8d and blank group have significant difference (p < 0.05), but the later stage both is close, does not has significant difference, and remaining respectively organizes GAG total content and blank group does not has significance poor Different (p > 0.05), experiment shows that IFN-β may promote that hMSCs becomes cartilage differentiation.

Embodiment 5. is added IFN-β and is promoted that hMSCs becomes cartilage differentiation

Experimental technique:

Inspection IFN-β facilitates the group technology of cartilage differentiation effect:

Take the logarithm the 8th generation hMSCs of trophophase, resuspended with complete medium, adjusts cell concentration to 1 × 10⁵/ mL.6 hole 200 μ L cell suspension are inoculated in the every hole of plate, and supplementing culture medium to 2mL. cell is divided into blank group, become cartilage matched group, IFN-β Group and IFN-β+TGF-β 3 (Transforming growth factor-β3) group, during 4d with complete medium, become cartilage differentiation culture medium, contain The one-tenth cartilage differentiation culture medium (without TGF-β 3) of 100ng/ml IFN-β is trained with the cartilage differentiation that becomes containing 100ng/ml IFN-β Supporting base correspondence and change liquid, every 4d changes liquid afterwards.

Experimental result:

(1) add IFN-β and promote that hMSCs becomes the GAG testing result of cartilage differentiation

GAG total content testing result shows (Fig. 3), becomes cartilage matched group, IFN-β group and IFN-β+TGF-β 3 groups with blank Matched group has significant difference (p < 0.01), and GAG total content the most constantly increases..IFN-β group cell GAG always contains Amount is slightly above into cartilage matched group, 4d and 11d has a significant difference (p<0.01), but 7d and 14d do not have significant difference (p> 0.05), 3 groups of cell GAG total contents of IFN-β+TGF-β are significantly larger than into cartilage matched group and IFN-β group, and become cartilage matched group Having significant difference (p < 0.01), experiment to show that IFN-β can substitute for TGF-β 3 and acts on, the two is used in combination and can more promote Become cartilage differentiation.

(2) add IFN-β and promote that hMSCs becomes the qRT-PCR result of cartilage differentiation

QRT-PCR result shows (Fig. 4,5), IFN-β group cell Collagen II and SOX9 gene relative expression during 14d Rate blank to be significantly larger than group (p < 0.01), and becomes cartilage matched group to be closer to, and 3 groups of cells of IFN-β+TGF-β Collagen II and SOX9 gene relative expression leads then apparently higher than other each group (p < 0.01).

(3) add IFN-β and promote that hMSCs becomes the WESTERN BLOT result of cartilage differentiation

WESTERN BLOT result shows (Fig. 6), and 14d respectively organizes all has Collagen II and SOX9 band to occur, wherein 3 groups of bands of IFN-β+TGF-β are the brightest.

Gray analysis value (table 1) proves that IFN-β+TGF-β 3 groups of Collagen II and SOX9 relative expression quantity is each compared with other Group has notable difference (p < 0.01).

Table 1 WESTERN BLOT sxemiquantitative gray analysis

Embodiment 6. is added variable concentrations IFN-β and is promoted that hMSCs becomes cartilage differentiation

Experimental technique:

The group technology of variable concentrations IFN-β:

Take the logarithm the 8th generation hMSCs of trophophase, resuspended with complete medium, adjusts cell concentration to 1 × 10⁵/ mL.6 hole 200 μ L cell suspension are inoculated in the every hole of plate, and supplementing culture medium to 2mL. cell is divided into into cartilage matched group, IFN-β 50 groups, IFN-β 100 groups, IFN-β 200 groups and IFN-β 400 groups, with becoming cartilage differentiation culture medium and respectively containing 50,100,200,400ng/ during 4d The one-tenth cartilage differentiation culture medium correspondence of ml IFN-β changes liquid, and every 4d changes liquid afterwards.

Experimental result:

GAG total content testing result shows (Fig. 7), IFN-β 100 groups, IFN-β 200 groups and IFN-β 400 groups with become cartilage Matched group has a significant difference (p < 0.01), and 50 groups of GAG total content levels of IFN-β are with to become cartilage matched group close, only exist 7d and 11d has 100 groups of cell GAG total contents of significant difference (p < 0.05) .IFN-β to show to add apparently higher than other groups, experiment Adding the IFN-β of 100ng/ml, to promote into cartilage differentiation effect the most notable.

Embodiment 7 human marrow mesenchymal stem cell and personalized osseous tissue material biocompatibility in vitro

(1) detection that hMSC cell is bred on PLGA/TCP (tricalcium phosphate) material

HMSCs growth rate on PLGA/TCP material is very fast, and cultivation effect becomes apparent from. propagation 1d and 3d, material Peace is in the face of being closer to according to growth rate, and both OD values do not have significant difference (P > 0.05), 5d and 7d, hMSC on material Growth rate compares apparently higher than plane, and both OD values have significant difference (P < 0.01). and we analyze the upper growth of plane comparison HMSC between 3d to 5d, be in exponential phase, enter at 7d subsequently and grow relatively slow plateau, and on material The hMSC of growth still has higher growth rate at 7d, traces it to its cause, should have with the loose structure of PLGA/TCP material The biggest surface area/volume ratio is relevant, and cell has more space can to adhere to and grow.

(2) hMSCs Osteoblast Differentiation effect on PLGA/TCP material

Under scanning electron microscope, observation Osteoblast Differentiation culture medium changes 7d after liquid, and cellular matrix layer has covered material surface area More than 60%, may be seen indistinctly calcium tuberosity (Fig. 8)..

ALP enzyme (alkali phosphatase) Activity determination result shows, the hMSC cell of Osteoblast Differentiation on PLGA/TCP material Its ALP enzymatic activity is higher than plane comparison, it means that material can promote the Osteoblast Differentiation of cell. differentiation 1d, material and plane Comparison ALP enzymatic activity is closer to, and both do not have significant difference (P > 0.05), 3d, and on material, the ALP enzymatic activity of hMSC is the highest Compareing in plane, both have significant difference (P < 0.05), 5d and 7d difference the most notable (P < 0.01) is shown in Fig. 9.

The MTT testing result (n=6) that table 2 hMSCs breeds on PLGA/TCP material

*: compare with matched group, P ＜ 0.01

Safety evaluation in the personalized osseous tissue animal body of embodiment 8.

Experimental technique: the relevant marks such as the BiologicalEvaluationofMedicalDevice standard promulgated by existing national Bureau of Drugs Supervision and pharmacopeia Accurate pyrogen, haemolysis, systemic acute toxi-city, Intradermal zest to subject material, the content such as it is subcutaneously implanted and tests.

Result: the test of the pyrogen test of subject material, hemolytic test, systemic acute toxi-city, Intradermal irritation test, subcutaneous The contents such as Implantation Test all meet BiologicalEvaluationofMedicalDevice standard.

1 experiment material

1.1 test medicine: subject material normal saline lixiviating solution, subject material Oleum Gossypii semen lixiviating solution, subject material block 0.5cm × 0.5cm × 0.5cm, lot number: 20131001, provided by Shenzhen Polytechnic, the preparation method of subject material Such as embodiment 1,2 and 3.

1.2 laboratory animals: health large ear rabbit, body weight 1.8～2.0kg, male and female do not limit, by Changchun Biological Products Institute Co., Ltd provides；Kunming mouse, body weight 20 22g, male and female half and half, by by the limited duty of Changchun Biological Products Institute Ren company provides, ticket number: SCXK (lucky)-2012-0001.

1.3 instrument

Enlightening auspicious CS-600B automatic clinical chemistry analyzer

Shenzhen semi-automatic cellanalyzer of Pu Kang PE-6800

1.4 reagent

ALT test kit, lot number 140113028, valid till 2014-09；

AST test kit, lot number 140213010, valid till 2014-03；

γ-GT test kit, lot number 140413006, valid till 2014-04；

ALP test kit, lot number 140313006, valid till 2014-04；

PT test kit, lot number 140813005, valid till 2014-02；

BUN test kit, lot number 141313018, valid till 2014-06；

UA test kit, lot number 141213006, valid till 2014-04；

Above test kit is provided by Mairui Biological Medical Electronic Co., Ltd., Shenzhen.

CHE test kit, lot number 20130801, valid till 20140825, Hui Qiang biotech inc of Jilin Province.

CRP test kit, lot number 130944, valid till 201409, Shanghai Jiemen Bio-Tech Co., Ltd..

2 methods and result

2.1 pyrogen testing

Screen qualified rabbit: taking 8 health large ear rabbits, body weight 1.8～2.0kg, male and female do not limit.

Rabbit anus temperature is measured with temperature probe, the degree of depth about 6cm that thermometer inserts, measures 1 time every 30min, surveys 8 altogether Secondary, select 8 body temperature all in the range of 38.0～39.6 DEG C, and the highest and minimum body temperature differs 3 families less than 0.4 DEG C Rabbit is used for testing.Should select in 3～7 days before test sample inspection.

Table 2.1.1 screens qualified rabbit result

Number

Weight kg

1 time DEG C

2 times DEG C

3 times DEG C

4 times DEG C

5 times DEG C

6 times DEG C

7 times DEG C

8 times DEG C

1♂	2.0	40.1	39.8	39.6	39.4	39.4	39.3	39.2	39.1
										2♂	2.2	40.0	39.8	39.3	39.5	39.5	39.5	39.3	39.3
3♂	1.7	39.4	39.7	39.5	39.5	39.6	39.6	39.8	39.6
										√4♂	2.0	39.6	39.6	39.5	39.5	39.4	39.4	39.4	39.5
5♀	1.6	38.8	38.6	38.4	38.3	38.6	38.6	38.5	38.5
										√6♀	2.1	39.5	39.4	39.4	39.3	39.4	39.3	39.4	39.3
7♀	2.2	39.9	39.6	39.5	39.4	39.3	39.3	39.2	39.3
										√8♀	2.6	39.6	39.5	39.5	39.4	39.5	39.6	39.5	39.4

Select 8 anus Wen Jun in the range of 38.0～39.6 DEG C, and the highest with the minimum temperature difference less than 0.4 DEG C 3 Rabbit is used for testing, and 4,6, No. 8 rabbit meet above-mentioned requirements as a result.

Test before prepare: test front 1～2 day, for try out rabbit should be in as far as possible in same temperature environment, laboratory with Receptacle temperature difference cannot be greater than 5 DEG C, laboratory temperature should 17～25 DEG C test all processes in it should be noted that room temperature become Change and cannot be greater than 3 DEG C, it is to avoid noise jamming.Within before rabbit test at least 1 hour, start fasting to be placed in suitable device, until Test complete, the degree of depth about 6cm that thermometer inserts, 3 rabbits being used for test are placed in holder, inject pre-test starting 2 body temperature, every minor tick 30min, it is averaged the body temperature basal body temperature as this rabbit, the difference of twice body temperature must not exceed 0.2 DEG C, And regular using warming therapy difference must not exceed 1 DEG C between each rabbit.

Pyrogen is tested: be slowly injected into the subject material physiology salt being pre-heated to 38 DEG C by 10mL/kg dosage through auricular vein Water extract, it is ensured that inject complete in 15min.Measure a body temperature every 30min after injection, survey 6 times altogether, take anus temperature Peak deducts basal body temperature and is body temperature lift-off value.

Table 2.1.2 pyrogen test result:

Result judges: in 3 rabbit of preliminary examination, and body temperature raises and is below 0.6 DEG C, and 3 rabbit body temperatures raise total With less than 1.4 DEG C, it is believed that the heat source check of test sample meets regulation.

2.2 hemolytic test

Take healthy rabbits through Culling heart blood 8ml, collect to potassium oxalate anticoagulant tube, use 10ml normal saline dilution subsequently. 10ml subject material normal saline lixiviating solution is as test subject material, and 10ml distilled water is as positive control, 10ml physiology salt Water is as negative control, and often group operation repetitive 3 is managed.After all test tube puts people's water bath with thermostatic control 37 DEG C insulation 30min, every test tube adds Enter and O.2ml dilute rabbit whole blood, mix follow-up continuation of insurance temperature 60min.Incubation terminates to pour out liquid in pipe and is centrifuged 5min with 800g, inhales Take appropriate supernatant to be transferred in spectrophotometer measurement cup, at 545nm, measure its optical density (OD).It is calculated as follows haemolysis Percentage rate: percentage of hemolysis=(OD sample one OD negative control)/(OD positive control one OD negative control) × 100%.

Table 2.2 hemolytic test result

Result judges: percentage of hemolysis is 0.0528%, and hemolysis rate < 1%, meet the hemolytic test requirement of medical material.

2.3 acute systemic toxicity

Take 40 healthy kunming mices, body weight 18 22g, male and female half and half, be randomly divided into intravenous contrast group, the tested material of vein Material group, abdominal cavity matched group, abdominal cavity subject material group, often group 10, male and female divide cage to feed.The initial of every mice is recorded before test Body weight, injects according to 50ml/kg dosage, intravenous contrast group by tail vein injection saline, vein subject material group by The normal saline lixiviating solution of tail vein injection subject material, abdominal cavity matched group are by lumbar injection Oleum Gossypii semen, abdominal cavity subject material group By lumbar injection subject material Oleum Gossypii semen lixiviating solution.In observing Mice Body after injection immediately, whether side effect occurs, and in injection Rear 4,24,48,72h continues to observe the ordinary circumstances such as the feed of mice, activity, death, and records body weight change.

Result: subject material compares by group mice with matched group, activity, hair color, feed, two the most normal, none is dead, body Weight compares no significant difference with matched group, shows that subject material disposably contacts without overt toxicity, meets the whole body of medical material The requirement of acute toxicity test.

Table 2.3 acute systemic toxicity Mouse Weight (g) (N=10)

Group	Before administration	4h after administration	24h after administration	48h after administration	72h after administration
						Intravenous contrast group	22.40±2.06	22.13±2.03	23.26±2.25	24.66±2.54	26.45±2.75
Tested group of vein	23.54±1.47	23.38±1.42	23.78±1.36	25.29±1.90	26.75±1.79
						Abdominal cavity matched group	22.37±2.28	22.29±2.24	22.65±2.26	23.98±2.72	25.78±1.96
Tested group of abdominal cavity	21.95±1.56	21.81±1.50	22.73±1.49	23.81±1.99	25.21±1.97

2.4 Intradermal irritant reaction tests

Taking the new zealand white rabbit grown up at the beginning of 4 health, male and female do not limit, and body weight is not less than 2kg, divide equally 2 groups: polar solvent Group, non-polar solven group.Before test, 24h thoroughly shaves except tested rabbit back spinal column both sides are by hair, it is to avoid injured skin.75% The skin that (volume fraction) ethanol disinfection exposes.5 points, every some interval 2cm is selected every rabbit spinal column both sides.Polar solvent Every some intradermal injection 0.2mL subject material normal saline lixiviating solution on the left of group rabbit, every, right side injection 0.2mL normal saline is made Negative control；Every some intradermal injection 0.2mL subject material Oleum Gossypii semen lixiviating solution on the left of non-polar solven group rabbit, every, right side is noted Penetrate 0.2mL Oleum Gossypii semen and make negative control；After injection at once and 24,48,72h observe each injection site and surrounding skin tissue instead Should, according to dermoreaction marking system, erythema and the edematous tissue reaction of each injection site are marked, calculate animal pair The final mean score of every kind of material lixiviating solution.

Table 2.4.1 Intradermal irritant reaction test erythema and eschar form score-sheet

Group	Injection is at once	24h after injection	48h after injection	72h after injection
					Polarity control side	0.0±0.0	0.0±0.0	0.0±0.0	0.0±0.0
Polarity tested material side	0.0±0.0	0.0±0.0	0.0±0.0	0.0±0.0
					Nonpolar control sides	0.0±0.0	0.0±0.0	0.0±0.0	0.0±0.0
Nonpolar tested material side	0.0±0.0	0.0±0.0	0.0±0.0	0.0±0.0

Table 2.4.2 Intradermal irritant reaction test edema forms score-sheet

As a result, subject material normal saline lixiviating solution and Oleum Gossypii semen lixiviating solution intradermal injection, in the 0-72h observation period, equal nothing Erythema, eschar, edema etc. stimulate phenomenon, meet the irritant test requirement of medical material.

2.5 subcutaneous implant test

Taking healthy new zealand white rabbit 3, male and female do not limit, and blunt solution, by hair, is used in removing rabbit back test position after sterilization Method of cuing open is preparing a subcutaneous capsule at other 2cm skin incision position, distance spinal column both sides, the bottom of capsule away from skin incision should be 1cm with On, subject material block to be implanted subcutaneous, by skin closure, raises 12 weeks, period notes observing and recording rabbit general status, work The situation such as dynamic, diet, drinking-water, body weight growth, before being subcutaneously implanted, implant after within 4 weeks, 8 weeks, 12 weeks, weigh, ear edge is quiet Arteries and veins is taken a blood sample, and surveys routine blood indexes and Biochemical Indices In Serum.

After implanting, within 12 weeks, it is individually separated spinal column both sides subcutaneous, exposes and implant subcutaneous material, observe and implant polylactic acid Size, form, surrounding tissue is with or without suppuration and secretions situation etc., and takes the surrounding tissue position specimen comprising material, uses 10% formalin is fixed, is dehydrated transparent, paraffin embedding, serial section, and thickness is 5 μm, and (HE contaminates hematoxylin-eosin staining Color), basis of microscopic observation histopathology situation.

Table 2.5.1 rabbit body weight growth pattern (kg)

Number

Before implantation

After implantation 4 weeks

After implantation 8 weeks

After implantation 12 weeks

1	2.0	2.5	2.8	3.0
					2	1.9	2.1	2.4	2.5
3	2.1	2.6	2.9	3.2

Table 2.5.2 on the impact of routine blood indexes (N=3)

Routine blood indexes	Before implantation	After implantation 4 weeks	After implantation 8 weeks	After implantation 12 weeks
					WBC(10⁹/L)	8.40±0.70	8.30±1.04	7.77±1.03	8.07±1.15
Lymph#(10⁹/L)	2.13±0.31	2.00±0.17	2.00±0.17	2.23±0.45
					Mid#(10⁹/L)	0.70±0.10	0.67±0.12	0.63±0.06	0.60±0.10
Gran#(10⁹/L)	5.57±0.45	5.63±0.84	5.13±0.85	5.23±0.60
					Lymph% (%)	25.07±3.13	24.10±2.00	25.57±1.96	27.63±1.40
Mid% (%)	8.63±0.46	8.23±0.42	8.13±0.25	7.67±0.21
					Gran% (%)	66.30±2.79	67.47±1.99	66.37±1.62	64.70±1.31
RBC(10¹²/L)	5.17±0.07	5.24±0.04	5.34±0.09	5.27±0.11
					HGB(g/L)	129.3±1.53	129.7±2.52	131.3±3.06	129.3±1.53
HCT (%)	35.13±5.52	38.33±0.67	38.53±0.98	38.13±0.25
					MCV(fL)	74.27±3.00	73.10±1.35	72.03±1.27	72.33±1.66
MCH(pg)	24.93±0.61	24.70±0.35	24.53±0.31	27.87±5.40
					MCHC(g/L)	336.0±9.00	337.7±4.04	340.3±2.89	338.7±3.21
RDW-CV (%)	14.23±0.06	14.10±0.17	14.13±0.06	14.23±0.06
					RDW-SD(fL)	36.23±0.71	36.60±0.35	36.80±0.53	36.37±1.34
PLT(10⁹/L)	212.0±5.29	224.3±2.08	345.0±10.44	234.3±5.77
					MPV(fL)	7.63±0.31	7.57±0.35	7.77±0.15	7.73±0.25

PDW	14.33±0.25	14.20±0.26	14.40±0.10	14.33±0.25
					PCT (%)	0.16±0.01	0.17±0.01	0.19±0.01	0.18±0.004

Table 2.5.3 on the impact of blood biochemistry index (N=3)

Blood biochemistry index	Before implantation	After implantation 4 weeks	After implantation 8 weeks	After implantation 12 weeks
					TP(g/L)	64.60±5.89	64.67±4.61	65.37±3.31	65.30±5.37
ALB(g/L)	40.67±1.17	41.37±1.08	41.97±0.83	43.20±1.48
					GLB(g/L)	23.97±4.99	23.30±3.90	23.40±3.32	22.10±3.90
A/G	1.77±0.38	1.83±0.32	1.83±0.25	2.00±0.35
					ALT(U/L)	51.07±8.13	40.43±6.37	50.77±9.12	52.00±9.03
AST(U/L)	22.50±2.29	22.47±1.78	23.60±2.61	25.33±1.32
					AST/ALT	0.43±0.06	0.43±0.06	0.43±0.06	0.50±0.10
r-GT(U/L)	11.37±1.16	9.87±0.59	9.43±1.53	9.40±0.26
					CHE(U/L)	4062.3±78.75	4086.3±44.61	4180.3±71.93	4160.7±35.84
ALP(U/L)	149.4±44.33	144.0±45.14	133.5±39.12	135.2±47.66
					BUN(mmol/L)	5.90±1.22	6.01±0.86	5.34±0.58	5.59±0.54
CREA(μmol/L)	75.60±9.84	73.63±10.29	69.03±9.35	68.17±5.86
					UA(μmol/L)	7.17±3.76	7.10±4.78	7.13±3.18	7.33±1.64
CRP(mg/L)	3.13±0.21	3.70±0.26	4.17±0.06	3.13±0.25

As a result, rabbit is subcutaneously implanted after subject material during 12 weeks, and its general status, activity, diet, drinking-water, body weight increase The situations such as length are the most without exception；Its routine blood indexes compares Non Apparent Abnormality before implanting；Its Biochemical Indices In Serum compares before implanting More also Non Apparent Abnormality.

After implanting 12 weeks, the subject material block of implantation has been degraded and has absorbed, and at implantation, surrounding tissue is without suppurating, also without dividing Secreting, the surrounding tissue position pathology detection result of material shows and is normal structure.

Claims

1. for a personalized bone renovating material, including

Mass ratio is organic material and the inorganic material of 0.1-0.6:1, and

Wrap up the lipid microsphere powder of protein；Wherein:

Described organic material is selected from PLGA, PLA and PGA, PLA:PGA=5%-95%:95%-5% in described PLGA, molecular weight Scope is 8000-250000；Described PLA molecular weight ranges is 8000-250000；Described PGA molecular weight ranges is 8000- 250000；

Described inorganic material is the one in bata-tricalcium phosphate, type alpha tricalcium phosphate, hydroxyapatite and tetracalcium phosphate or many The phosphoric acid class calcium salt planted；

The described protein being wrapped in lipid microsphere is restructuring human beta interferon, recombined human β interference in every part of lipid microsphere powder The content of element is；50～200ng/ parts.

2. the personalized bone renovating material described in claim 1, being wherein wrapped in the protein in lipid microsphere is recombined human Interferon IFN-β, the content of IFN-β is 100ng/ml.

3. the personalized bone renovating material described in claim 1 or 2, the protein being wherein wrapped in lipid microsphere is restructuring Interferon-beta and the combination of Transforming growth factor-β3.

4. the method for the personalized bone renovating material described in preparation claim 1, comprises the steps:

1) mix described organic material and inorganic material according to predetermined ratio with organic solvent, add and wrapped up freezing of protein Dry lipid microsphere powder, prepares bone renovating material；

2) by step 1) bone renovating material proceed to 3D printer, enter with non-intruding computed tomography and NMR (Nuclear Magnetic Resonance)-imaging Row individuation bone injury site three dimensional data collection, is converted into 3 D-printing model by the faultage image collected section, utilizes Rapid shaping technique makes osseous tissue model and organization bracket；

3) by step 2) 3D prints the tissue scaffold design of preparation and is placed in cell bottle inoculation osteoblast, cultivate to cell attachment or Transfer to bioreactor is carried out stereoscopic culture after forming cell monolayer；

5. the preparation method described in claim 4, wherein step 2) in described print procedure material temperature scope be-30 DEG C～- 10 DEG C, and the material printed persistently gives cold wind and makes it fixed-type；The tissue scaffold design printed carries out vacuum freezing to be done Dry.

6. the preparation method described in claim 4, wherein said tissue scaffold design carry out before inoculating cell gamma-rays irradiation or The aseptic process of epoxyethane fumigation, and described support is soaked through culture fluid.

7. the preparation method described in claim 4, wherein step 3) in, inoculating cell to tissue scaffold design, train in culture bottle Support 2 days and shift tissue scaffold design after monolayer in bioreactor until the cell attachment on tissue scaffold design or formed, three-dimensional training Supporting, wherein the speed of fed-batch medium is 5ml/min-15ml/min, and osteoblast complete medium includes following composition:

Low sugar DMEM 88% (v/v), hyclone 10% (v/v), Pidolidone 1% (v/v), green grass or young crops/streptomycin 1% (v/v). HMSC becomes cartilage differentiation culture medium to include following composition: DMEM in high glucose 96.3% (v/v), Pidolidone 1% (v/v), green grass or young crops/chain Mycin 1% (v/v), 10mmol/L ascorbic acid 0.5% (v/v), 1mmol/L dexamethasone 0.01% (v/v), 40 μ g/L L- Proline 0.1% (v/v), 1000 μ g/L Sodium Pyruvates 0.1% (v/v), ITS+Premix1% (v/v), 10ng/ml TGF-β 3。