CN102727936A - Construction method of sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries - Google Patents
Construction method of sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries Download PDFInfo
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Abstract
The invention relates to a construction method of a sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries, and especially relates to a construction method and an application of a stem-cell-containing sustained-release NT-3 gelatin sponge cylindrical stent. During application, the sustained-release NT-3 gelatin sponge cylindrical stent with planted stem cells and differentiated cells thereof is transplanted to a place of a transected spinal cord injury, such that injured central nerve regeneration and function reparation can be promoted. According to the invention, on a biological tissue engineering level, injured neuron protection and injured neuron axon regeneration promotion after a transected spinal cord injury are enhanced. The life quality of the injured is improved, and social and family burdens are reduced. Therefore, the invention has great significance in promoting the social and economical development of our nation.
Description
Technical field
The present invention relates to a kind of slow release neurenergen 3 (neurotrophin-3 that is used to repair spinal cord injury; NT-3) construction method of gelatin sponge cylinder bracket, especially a kind of construction method and application thereof that contains the slow release NT-3 gelatin sponge cylinder bracket of stem cell.
Background technology
Central nervous system injury such as serious spinal cord injuries receptor mainly show as paraplegia and quadriplegia, also do not have effective method for treatment at present.Tradition is thought, is difficult to regeneration behind the neurologic defict.Think that now spinal nerves regeneration reason of difficulty mainly is to lack the neurotrophic factor that promotes neuranagenesis in their microenvironments of living in.Therefore, want to regenerate behind the neurologic defict, must carry out manual intervention.Present stage promotes the strategy that neurologic defict is repaired; Put forth effort on more and improve the nerve fiber regeneration microenvironment that neuron sends, let regenerated nerve fiber pass damage zone, get in the myeloid tissue of opposite side by the bridge support; Rebuild neural pathway connection, recover original function.
In the strategy of repair of spinal cord injury, the bioengineered tissue material is used to treat spinal cord injury and causes people's attention gradually as having neuroprotective unit with the active factors that promotes its axon regeneration effect or as the carrier of transplanting stem cell.Tissue engineering material mainly contains natural polymer biomaterial and degradable macromolecule synthetic material.The natural polymer biomaterial comprises gelatin, collagen, chitosan and alginate etc.Natural material has excellent biological compatibility, can promote sticking, breed and breaking up of cell external.Their convenient neurotrophic factors that carries, good with the conformability of myeloid tissue, can reduce inflammatory reaction and reduce the formed cicatrix of astrocyte hypertrophy, can promote the 26S Proteasome Structure and Function of damaged spinal cord to recover to a certain extent.Because there is processing in natural material, mechanical performance is relatively poor and degradation rate problem such as easy-regulating not, uses in vivo and receives certain limitation.Degradable high molecular synthetic material with PLLA (poly D, L-lactic acid, PLLA) and polylactic acid-polyglycolic acid copolymer (poly D; L-lactic-co-glycolic acid; PLGA) be representative, its biggest advantage is easy to be processed into the structure and the shape of various complicacies, and certain mechanical strength is arranged; Can play the effect of bridge joint tissue, catabolite is easy to be absorbed.But this material possibly exist catabolite to be tart problem in the external function that does not promote cell adhesion and growth after implanting.
The gel that natural biologic material is made, gelfoam and collagen sponge all can be used for cross-section property repair of spinal cord injury.Gel can have been located filling effect in the spinal cord injury cavity, promotes axon regeneration, reduces the cicatrix that astrocytosis forms.But it can not guide the regeneration aixs cylinder to pass through damage field preferably, especially lacks mechanical strength.Gelatin and collagen have the advantage of gel, and can conveniently carry active substance.Gelatin comes from collagen, but (de-nature collagen) do not had the antigenicity of collagen behind the former natural quality that removes photoresist, and contains similar arginine-glycine-aspartic acid (RGD) sequence, can promote sticking and moving of cell.In addition, the gelatin price comparison is cheap, but its mechanical strength is not high yet.
Gelfoam is applied to clinical many year history has been arranged, and this has benefited from its favorable tissue compatibility and cellular affinity.But there is case report to point out in recent years,, situation occurs after finally causing implanting nervus centralis the surrounding tissue extruding because gelfoam suction expands easily.In view of the situation, we design with a kind of relative expansion coefficient materials with smaller PLGA film as the conduit shell, and at central authorities filling gelfoam (Ceng Yuanshan, the Ceng Xiang of conduit.A kind of structure that is used for the gelatin sponge cylinder bracket of repairing nerve damage.Obtain People's Republic of China's patent of invention, patent of invention number: ZL 200910040176.9).This gelatin sponge cylinder bracket can keep the good material characteristic of gelfoam on the one hand as far as possible, can reduce the excessive expansion of gelfoam on the other hand.In addition, owing to the higher PLGA film of mechanical strength has been arranged as the conduit shell, the intermediary gelfoam of conduit is not easy to subside.These advantages all help us and carry out the inside and outside experimentation of body.
Fibroin albumen (silk fibroin) has been compared obvious superiority with other natural polymer: (1) is safe and reliable; Fibroin albumen is the natural high-purity protein that derives from silkworm, and is fewer to the pathophorous risk of the mankind, and has clear and definite primary structure, do not have potential hazardness.(2) can obtain membranaceous or liquid character through the different disposal method.(3) can change its surface property through the chemical modification of some amino group of amino acids and side chain, be easier to adherent cell.(4) in vivo, can slowly degrade, has certain slow-releasing outward.
Using neurotrophic factor treatment central nervous system injury is one of focus of research at present; But (neurotrophin-3 NT-3) waits neurotrophic factor for be mostly nerve growth factor (NGF), BDNF (BDNF) and the neurenergen 3 that are used.Proved that at present NGF mainly acts on sensory neuron, not obvious to the motor neuron effect, and the neuron type scope of BDNF effect is narrower.Many researchs think, NT-3 plays an important role to neuronic growth, differentiation and to the survival and the axon regeneration thereof of damaged axoneuron.Have research also to confirm, NT-3 has obvious facilitation to the regeneration of spinal cord injury place corticospinal tract nerve fiber, and this has also obtained checking in our previous research.
Neural regeneration after spinal cord injury is effectively solved as yet at present, and it possibly be one of factor of most critical that the injury region microenvironment lacks neurotrophic factor.Therefore, it is necessary giving exogenous neurotrophic factor.Recently think that the therapeutic alliance strategy is expected to make repair of spinal cord injury to reach better effect, like neurotrophic factor and biologic bracket material Combined application etc.Research shows that because exogenous administering mode can not satisfy the requirement in the spinal nerves regenerative process, the clinical effectiveness that route of administration such as conventional injection neurotrophic factor obtain is unsatisfactory, makes the extensive use of neurotrophic factor be restricted.Therefore, need badly and find a kind of ideal carrying neurotrophic factor, and can make it in spinal cord injury place microenvironment certain hour, keep the method for valid density.
The problem that exists in the comprehensive above-mentioned research; This invention patent is attempted through in the gelatin sponge cylinder bracket of natural biologic material----porous crack, adding the NT-3/ fibroin albumen; Utilize the fibroin albumen characteristic that NT-3 will be attached on the gelatin, make up a kind of slow release NT-3 gelatin sponge cylinder bracket that is used to repair spinal cord injury.Then, in this support, plant mesenchymal stem cells MSCs (bone marrow mesenchymal stem cells, MSCs).
Summary of the invention
At present, do not see the construction method that has documents and materials reports to be used to repair the slow release NT-3 gelatin sponge cylinder bracket of spinal cord injury at home, outward as yet, especially a kind of construction method and application thereof that contains the slow release NT-3 gelatin sponge cylinder bracket of stem cell.The objective of the invention is to want to overcome the existing deficiency on the employed method of spinal cord injury of treating clinically; Use the slow release NT-3 gelatin sponge cylinder bracket material treatment spinal cord injuries receptor property disease that we make up voluntarily, may promote regeneration of damaged spinal nerves and function reparation better.
Basic scheme of the present invention comprises:
Be the main body with porous crack gelfoam, add fibroin albumen and NT-3, outside Using P LGA film wrapped forms a kind of pattern as cylindrical timbering material.Plant mesenchymal stem cells MSCs etc. on this basis, be built into and promote the regenerated artificial nerve catheter of spinal nerves.During application, it is transplanted to spinal cord transection property injury region.
The present invention has its remarkable advantage; It is at our last patent of invention (a kind of structure that is used for the gelatin sponge cylinder bracket of repairing nerve damage; Patent of invention number: make up a kind of gelatin sponge cylinder bracket that can slow release NT-3 on basis ZL200910040176.9); Be used to promote the spinal nerves regeneration and the function reparation thereof of damaged; The bigger traumatic nervus centralis disease of harm people ' s health is carried out preclinical study, will effectively promote the development of whole traumatic nervus centralis diseases prevention and treatment research field.This improves and hinders patient's life quality prolonging human longevity, alleviates society and family burden, promotes that the Chinese society economic development is all significant.The present invention will make the traumatic nervus centralis diseases prevention and treatment level of China occupy the leading level in the world.
The specific embodiment
Through specific embodiment the used key instrument of the present invention, degradable natural biomaterial, degradable macromolecule synthetic material, silkworm Bombyx bombycis, neurotrophic factor, experimental cell, laboratory animal and reagent are done detailed description below:
1. key instrument
Superclean bench (Purifying Equipment Co., Ltd., Suzhou), generic centrifuge (Japanese Kubo field makes institute), low-temperature and high-speed centrifuge (U.S. Eppendorf company), 5%CO
2Incubator (U.S. Queue company), inverted fluorescence microscope (German Leica company), enzyme-linked immunosorbent assay instrument (U.S. Bio-Rad company), cryostat microtome (Britain Shandon company) and XL30 FEG type scanning electron microscope (German Philips company).
2. degradable natural biomaterial
Gelfoam is available from the product of Nanjing Jinting Pharmaceutical Co., Ltd..
3. degradable macromolecule synthetic material
PLGA (50: 50) thin film is available from the product of Mount Tai, Jinan handle of the Big Dipper biotechnology company.
4. silkworm Bombyx bombycis
The used raw material of preparation silk fibroin protein solution---silkworm Bombyx bombycis is the product of being given by the Silk Worm Inst Zhejiang Prov. Agriculture Science Academy.
5. neurotrophic factor
People NT-3 gene recombinant protein (neurotrophin-3, NT-3; Product available from Sigma company)
6. experimental cell and laboratory animal
Green fluorescence mesenchymal stem cells MSCs (MSCs, separation and Culture is obtained voluntarily) and gfp transgene SD (Sprague-Dawley) rat neonatal rat (Osaka University, Osaka, Japan).
7. main agents
DMEM-LG (Gibico), top grade hyclone (TBD), poly-D-lysine (Sigma); D-Hank ' s balance liquid (autogamy), trypsin Sigma), EDTA (Sangon); 0.01mol/l PBS (middle China fir Golden Bridge), MTT (Sigma), dimethyl sulfoxide (DMSO) is (Sangon); Hoechst33342 (Sigma), lowlenthal serum (middle China fir Golden Bridge) etc.
The detailed concrete operations technical descriptioon of the present invention is following:
1. the structure of the porous crack gelfoam PLGA cylinder bracket of slow release NT-3
The structure of the porous crack gelfoam PLGA cylinder bracket of described slow release NT-3 can be divided into 4 steps.The 1st step is the preparation rack shell: the shell thin-walled is around cylindrical polylactic acid-polyglycolic acid copolymer (poly D; L-lactic-co-glycolic acid; PLGA); It is made up of degradable high molecular synthetic material PLGA (polylactic acid and polyglycolic acid ratio are 50: 50, and molecular weight is 100 000).0.02mm thick PLGA thin film is available from Mount Tai, Jinan, Shandong handle of the Big Dipper biotech company, the construction method of material is following: get a certain amount of PLGA and be dissolved in the dichloromethane, be made into 5% solution; After treating that PLGA dissolves fully, cast and gathering in the tetrafluoro mould of level-off, room temperature (the control temperature is at 20 ℃); Volatilized 24 hours; Carefully took thin film off on the 2nd day, be inverted in mould 24 hours, cut out suitable big or small after drying and preserve.Thin film is surrounded a circle on the rustless steel cylinder grinding tool that diameter is 3mm, and the edge is pasted with acetone, and forming diameter is the cylindric PLGA shell of 3mm.During use the PLGA shell is cut into 2mm length, alcohol-pickled 15 minutes, uses aseptic D-Hank ' s balance liquid to embathe each 10 minutes 3 times subsequently.
The 2nd step is the preparation silk fibroin protein solution: (1) is with 20g Na
2CO
3Be dissolved in 4 premium on currency, be heated to 100 ℃, put into 75g silkworm Bombyx bombycis (giving), keep that solution is little to boil, and constantly stir by the Silk Worm Inst Zhejiang Prov. Agriculture Science Academy.After 1 hour, remove solution.Repeat said process again 1 time.To boil good Bombyx bombycis natural cooling, rinse well, be placed on baking oven interior 24 hours with deionized water, subsequent use after 50 ℃ of oven dry.(2) get CaCl
244.40g, ethanol 46.00ml, deionized water 57.60ml processes mixed solution, in this solution, puts into the Bombyx bombycis 15.00g of oven dry.After making the abundant submergence Bombyx bombycis of solution, 80 ℃ of heating in water bath, stirring and dissolving.After 1 hour, Bombyx bombycis all is dissolved as silk fibroin protein solution.Stop heating and stirring, the natural cooling silk fibroin protein solution is to room temperature.(3) remove the salt ion in the silk fibroin protein solution with bag filter (available from Guangzhou Qi Yun Bioisystech Co., Ltd) dialysis.Soaked the bag filter contain silk fibroin protein solution with tap water in preceding 2 days, using deionized water instead in back 1 day soaks, and dialyses altogether 3 days.Between dialysis period, every 1 tap water or deionized water of changing at a distance from 3 hours.(4) silk fibroin protein solution after will dialysing is poured in the graduated cylinder, leaves standstill 4 hours, removes the solid impurity in the solution.Collect the silk fibroin protein solution after leaving standstill with conical flask, 4 ℃ of refrigerators are preserved, and in 1 week, finish using.(5) silk fibroin protein solution of getting after about 10ml leaves standstill is poured in the small beaker, 60 ℃ of oven dry.The quality of the fibroin albumen after the weighing oven dry divided by solution quality, can obtain the concentration of silk fibroin protein solution.Computing formula is:
C is the concentration of silk fibroin protein solution; m
0Quality for small beaker; m
1For silk fibroin protein solution and beaker quality; m
2Dry back fibroin albumen and beaker quality and.We are about 6%～7% at the silk fibroin protein solution concentration of preparation.
The 3rd step is the gelfoam of preparation load NT-3/ fibroin albumen: (1) is got 0.2 μ g people NT-3 gene recombinant protein (NT-3 is available from Sigma company) and is dissolved in the 1ml silk fibroin protein solution mixing.The NT-3/ silk fibroin protein solution of this mixing is joined gelfoam (aseptic medical gelatin sponge is available from Nanjing, Nanjing Pharma Inc.) upward to saturated.In 4 ℃ of refrigerators, left standstill 4 hours again, then put into-80 ℃ of refrigerator freeze overnight.(2) gelfoam of freezing back loading NT-3/ fibroin albumen is put into freezer dryer ,-80 ℃ of vacuum lyophilization 12 hours.(3) gelfoam of the load NT-3/ fibroin albumen after the lyophilizing is put into the centrifuge tube of being with medicated cap ,-80 ℃ of refrigerators are preserved.Before the gelfoam of application load NT-3/ fibroin albumen with methanol treatment 5 minutes.
The 4th step is the porous crack gelfoam PLGA cylinder bracket of preparation slow release NT-3: the gelfoam with load NT-3/ fibroin albumen in superclean bench is cut into diameter 3mm, thickness 2mm size, and its pattern is cylinder.Again the cylinder gelfoam of load NT-3/ fibroin albumen is carefully filled in sharp mouth tweezer and disinfected in the PLGA shell.The aseptic kept dry of slow release NT-3 gelatin sponge cylinder bracket is for use.
2. the NT-3 of the gelfoam of load NT-3/ fibroin albumen discharges and detects
Get the gelfoam material (volume with quality substantially identical) of two load NT-3/ fibroin albumens, 75% alcohol disinfecting 10 minutes is positioned in 24 orifice plates after using D-Hank ' s balance liquid to embathe 3 times.Every hole adds L-DMEM culture fluid 300 μ l, in 37 ℃ of incubators, places.Regularly take out L-DMEM culture fluid (putting into-30 ℃ of refrigerators after the collection preserves) every day, replenish the new L-DMEM culture fluid of adding 300 μ l simultaneously.So repeat to till 28 days.Got 1 day, 3 days, 5 days, 7 days, 14 days, 21 days and the culture fluid of 28 days 7 time points, detect NT-3 concentration wherein, and draw NT-3 and discharge string diagram with the method for ELISA.
3. the pattern of the gelfoam of load NT-3/ fibroin albumen and pore size thereof detect
Get the gelfoam material of 1 load NT-3/ fibroin albumen, directly spraying plating is golden under dry status, and under scanning electron microscope, observes, takes pictures.Observe the surface topography of the gelfoam material of load NT-3/ fibroin albumen, and in material, get 5 visuals field, calculate the diameter and the scope of its hole.
4. the in-vitro separation of rat bone marrow mesenchymal stem cells, cultivation and evaluation
Mesenchymal stem cells MSCs (MSCs) in-vitro separation, cultivation and authentication method (Ceng Yuanshan, Ceng Xiang with reference to our last patent of invention.A kind of structure that is used for the gelatin sponge cylinder bracket of repairing nerve damage.Obtained People's Republic of China's patent of invention on June 8th, 2011, patent of invention number: ZL200910040176.9).
5.MSCs plant the process in slow release NT-3 gelatin sponge cylinder bracket material of planting
Slow release NT-3 gelatin sponge cylinder bracket material is soaked sterilization in 10 minutes in 75% ethanol, use D-Hank ' s balance liquid to embathe 3 times, residual alcoholic content is removed.Obtain P3-P5 for mesenchymal stem cells MSCs (MSCs), abandon and use D-Hank ' s balance liquid to embathe 1 time after losing culture fluid, with 0.25% pancreatin (0.02% EDTA) digestion 1 minute, the L-DMEM culture fluid that reuse contains 10%FBS stopped digestion.1000r/ minute, centrifugal 5 minutes.Abandon supernatant,, count with counting chamber with the L-DMEM culture fluid that the contains 10%FBS MSCs that suspends again.By each timbering material plantation 2～3 * 10
5Individual cell/20 μ l.After the volume calculated, it is centrifugal again to obtain corresponding cell concentration, after abandon supernatant.With the L-DMEM culture fluid that contains 10%FBS of the respective volume MSCs that suspends again.During plantation MSCs, add the MSCs suspension at the timbering material upper and lower faces.Around timbering material, add at last the L-DMEM culture fluid that contains 10%FBS on a small quantity and spend the night, let the MSCs adherent growth better of plantation in the timbering material.Changed culture fluid on the 2nd day again, its amount is advisable with the submergence timbering material.Changed liquid 1 time in later two days.In above-mentioned culture fluid, being routinely added to 1% pair resists.
6.MSCs the adhesion in slow release NT-3 gelatin sponge cylinder bracket and the detection that distributes thereof
Cultivate 7 days slow release NT-3 gelatin sponge cylinder bracket material after taking out plantation MSCs, fix 30 minutes, with 0.01M PBS rinsing timbering material 3 times with 4% paraformaldehyde.With the capable continuous frozen section of timbering material, the thick 25 μ m of sheet.The distribution of MSCs is observed in fetch bit down in cryptoscope in the transverse section of the timbering material outside and center respectively.
7.MSCs the growth vigor in slow release NT-3 gelatin sponge cylinder bracket detects
Detect the MSCs growth vigor that is planted in the slow release NT-3 gelatin sponge cylinder bracket with the MTT method.Cultivate after 1 day, 3 days, 5 days and 7 days, each time point is respectively got 4 timbering materials and is done the detection of MSCs growth vigor.Every group 4 multiple holes.Every hole adds MTT (5mg/ml, i.e. 0.5%MTT) 50 μ l and L-DMEM culture fluid 450 μ l, and 37 ℃ were continued to hatch 4 hours.Abandon and lose supernatant, every hole adds DMSO 350 μ l, acutely rocks on the decolorization swinging table 20 minutes.The DMSO solution in every hole is transferred to respectively in another 96 orifice plate, and each timbering material adds 3 multiple holes, every hole 100 μ l.On the enzyme linked immunological monitor, select wavelength 490nm, measure the OD value in each hole.Respectively to be grouped into abscissa, OD value (OD value) is a vertical coordinate, draws MSCs growth vigor string diagram.
8. the growth conditions of scanning electron microscopic observation MSCs on slow release NT-3 gelatin sponge cylinder bracket
The timbering material of getting the 1st day, 7 days is with 0.01M PBS flushing 5 minutes * 2, and the back is with fixing behind the 2.5% glutaraldehyde 0.1mol/L phosphate buffer, and general room temperature is about 30 minutes.Use gradient alcohol dehydration subsequently: 30% ethanol, 10 minutes; 50% ethanol, 10 minutes * 1; 75% ethanol, 10 minutes * 1; 95% ethanol, 10 minutes * 1; 100% ethanol, 15 minutes * 1.Then, timbering material is placed natural drying, each timbering material metal spraying 120 seconds.Back line scanning electron microscopic observation, and take pictures.
9. statistical analysis
The MTT method detects MSCs growth vigor data; With mean ± standard deviation
expression, utilization variance analysis (one-way ANOVA).P＜0.05 expression difference has the significance meaning.
Experimental result shows:
1. the gelfoam of Using P LGA film parcel load NT-3/ fibroin albumen is built into slow release NT-3 gelatin sponge cylinder bracket
The pattern that shows prepared slow release NT-3 gelatin sponge cylinder bracket like Fig. 1.A. silkworm Bombyx bombycis; The B.PLGA thin film; C. medical gelatin sponge; D. a left side is the gelfoam of load NT-3/ fibroin albumen, and the right side is the medical gelatin sponge; The E.PLGA thin film encloses the hollow circular cylinder that is rolled into; F. slow release NT-3 gelatin sponge cylinder bracket.Fig. 6 A shows the low power cardinal principle pattern of slow release NT-3 gelatin sponge cylinder bracket; Fig. 6 B shows the high power appearance structure of internal stent;
2. the NT-3 release in vitro curve of the gelfoam of load NT-3/ fibroin albumen
Show that like Fig. 2 the NT-3 burst size of the gelfoam of two load NT-3/ fibroin albumens is the highest at the 1st day, along with the prolongation of In vitro culture time, the burst size of every day is downward trend gradually.In the time of the 28th day, still have the release of NT-3.This shows in 28 days observation period, all have every day NT-3 from the gelfoam of load NT-3/ fibroin albumen, to slowly release.
3. the pattern of slow release NT-3 gelatin sponge cylinder bracket and pore size thereof
Show that like Fig. 6 B the gelfoam of slow release NT-3 gelatin sponge cylinder bracket is a kind of irregular, multi-pore structure material.Get 5 high power fields at random and observe, the size of its hole is most of between 30 μ m～300 μ m.
4.MSCs In vitro culture and purification
In the former process of supporting of being commissioned to train of MSCs, separate and purification MSCs according to its adherent characteristic.After the 4th day, the cell in the cell clone is constantly bred, and number increases, and cellular morphology becomes spindle shape, fibroblast appearance or polygon, is radial, forms microcolony.When cell culture to approaching the fusion, the cell that is climbed out of by adjacent colony merges mutually, becomes radial or the swirl shape arrangement, the cell spindle shape, and born of the same parents circle are clear, and karyon is full.Along with going down to posterity of cell, cell also is tending towards purification gradually.When pass to P4 for the time, cellular morphology is homogeneous relatively, is mostly spindle shape or star (Fig. 3).
5.MSCs evaluation
See MSCs qualification result (Ceng Yuanshan, the Ceng Xiang of our last patent of invention.A kind of structure that is used for the gelatin sponge cylinder bracket of repairing nerve damage.Obtained People's Republic of China's patent of invention on June 8th, 2011, patent of invention number: ZL200910040176.9).
6.MSCs adhesion and distribution thereof in slow release NT-3 gelatin sponge cylinder bracket
Plant after 7 days, MSCs distributes even relatively in support.MSCs (Fig. 4 A) is evenly distributed than the MSCs (Fig. 4 B) of the transverse section at middle part on the transverse section of the support outside.MSCs is in the quantity of support different aspects, and outside aspect is more than the middle part aspect.
7.MSCs the vigor of in slow release NT-3 gelatin sponge cylinder bracket, growing
Use the MTT method and detect demonstration, MSCs has growth vigor (Fig. 5) preferably in slow release NT-3 gelatin sponge cylinder bracket.MSCs growth vigor string diagram shows that the MSCs growth vigor progressively increases with the prolongation of incubation time, and the 3rd born long-term job power begins obviously to increase, and increases to the 7th day always.
8. scanning electron microscope shows the form that MSCs grows in slow release NT-3 gelatin sponge cylinder bracket
Scanning electron microscope shows that the MSCs kind plants in the slow release NT-3 gelatin sponge cylinder bracket growthform of 1 day and 7 days.Shown in Fig. 6 C, at the 1st day, the timbering material surface had MSCs to adhere to, and cell is fusiformis more, had short projection to stretch out.Projection between some MSCs is in contact with one another.Fig. 6 D shows that the 7th day timbering material surface has many MSCs to adhere to, the cell uniform distribution, and majority is a fusiformis.They stretch out long projection, and come in contact each other.
Description of drawings
Fig. 1. the pattern A. silkworm Bombyx bombycis of prepared slow release NT-3 gelatin sponge cylinder bracket; The B.PLGA thin film; C. medical gelatin sponge; D. a left side is the gelfoam of load NT-3/ fibroin albumen, and the right side is the medical gelatin sponge; The E.PLGA thin film encloses the hollow circular cylinder that is rolled into; F. slow release NT-3 gelatin sponge cylinder bracket.
Fig. 2. show the NT-3 release in vitro curve of the gelfoam of two load NT-3/ fibroin albumens.
Fig. 3. the MSCs of In vitro culture and purification.The P4 that white arrow shows cultivation is for MSCs (green fluorescence), and they are the cell space of spindle shape or star, wherein contain the nucleus (Hoechst33342 labelling) of blue-fluorescence.Fluorescence microscope, scale=40 μ m.
Fig. 4. plant adhesion and the distribution of MSCs in slow release NT-3 gelatin sponge cylinder bracket after 7 days.A. white arrow shows the distribution of MSCs on the transverse section of the support outside; B. white arrow shows the distribution of MSCs on the transverse section of middle part.Scale=320 μ m
Fig. 5 .MTT method detects the growth vigor of MSCs in slow release NT-3 gelatin sponge cylinder bracket.
Fig. 6. scanning electron microscope shows adhesion and the distribution of MSCs in slow release NT-3 gelatin sponge cylinder bracket.A. the low power cardinal principle pattern that shows timbering material; B. show the high power appearance structure that timbering material is inner; C. red arrow is shown in the MSCs that cultivated in the support 1 day; D. red arrow is shown in the MSCs that cultivated in the support 7 days.
Claims (7)
1. slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury; Its shape characteristic is: the cylinder bracket surface is wrapped in by polylactic acid-polyglycolic acid copolymer (poly D; L-lactic-co-glycolic acid; PLGA) thin-walled of thin film formation, the gelfoam of load NT-3/ fibroin albumen is being filled by cylinder central authorities; Gelfoam through load NT-3/ fibroin albumen adsorbs the stem cell of plantation and the cell of differentiation thereof, is built into the slow release NT-3 gelatin sponge cylinder bracket that helps regeneration of damaged spinal nerves and function reparation thereof.
2. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 1, its architectural feature is: described cylinder bracket is that the gelfoam by the PLGA tube wall of appearance thin layer and inner load NT-3/ fibroin albumen constitutes.
3. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 2, its compositional characteristic is: polylactic acid and polyglycolic acid ratio are 50: 50 among the PLGA, molecular weight is 100 000.
4. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 3 is characterized in that: the pipe thickness that PLGA constitutes is 0.02mm, and cross-sectional diameter is 3mm, and length is 2mm.
5. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 2, it is characterized in that: the gelfoam of load NT-3/ fibroin albumen is a multi-pore structure.
6. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 5; Its architectural feature is: the gelfoam of load NT-3/ fibroin albumen is a cylinder; Cross-sectional diameter 3mm, length is 2mm.
7. the slow release NT-3 gelatin sponge cylinder bracket material that contains stem cell that is used to repair cross-section property spinal cord injury according to claim 1, it is characterized in that: the cell of adsorbed plantation is mesenchymal stem cells MSCs or NSC.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104258460A (en) * | 2014-10-14 | 2015-01-07 | 中山大学 | Construction of controlled-release TrkC receptor ligand gelatin sponge cylinder bracket for repairing spinal cord injury |
| CN104800885A (en) * | 2015-05-13 | 2015-07-29 | 中山大学 | Preparation method and application of bioactive bracket with chemotactic function |
| CN108452379A (en) * | 2018-03-29 | 2018-08-28 | 天津市天津医院 | The gelfoam material and its application method containing bone marrow stem cell for repairing intervertebral discs damage |
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| CN104258460A (en) * | 2014-10-14 | 2015-01-07 | 中山大学 | Construction of controlled-release TrkC receptor ligand gelatin sponge cylinder bracket for repairing spinal cord injury |
| CN104800885A (en) * | 2015-05-13 | 2015-07-29 | 中山大学 | Preparation method and application of bioactive bracket with chemotactic function |
| CN108452379A (en) * | 2018-03-29 | 2018-08-28 | 天津市天津医院 | The gelfoam material and its application method containing bone marrow stem cell for repairing intervertebral discs damage |
| CN109938875A (en) * | 2019-03-07 | 2019-06-28 | 宁波光远致信生物科技有限公司 | A kind of nerve prosthesis and its preparation method and application |
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