CN1589913A - Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method - Google Patents
Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method Download PDFInfo
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Abstract
A tissue-engineered peripheral nerve for reparing the demaged peripheral nerve is composed of neural canal and neuroglia cells or stem cells, and is prepared through preparing neural canal, preparing neurotrophic factor release controllable microspheres, and configuring the tissue-engineered peripheral nerve.
Description
Technical field
The invention belongs to the biomaterial for medical purpose technical field in the human implantable, relate to a kind of tissue engineered peripheral nerve of repairing peripheral nerve defection and preparation method thereof that is used to.
Background technology
The peripheral nerve defection of the long distance that usually runs in the clinical treatment, because nerve lacks flexibility, most applications can't be directly identical to drawing, nerve autograft remains most popular clinically method.Yet there is the shortcoming that is difficult to overcome in this method, after the donor nerve cuts, must cause sensory disturbance for the district, and traumatic neuroma might take place, cause local pain, additional nerve cuts art prolongs operating time, increase cicatrix for the district, and the variation of neural thickness makes the nerve that cuts may not be fit to coupling, also might cause the regeneration of nervus motorius and sensorineural misorientation, the neural limited application that also limits the nerve autograft art greatly of originating of donor in addition.
The eighties in 20th century, there is neural chemotaxis in Lundborg etc. by Y type silica gel tube experiment confirm peripheral nerve regeneration, have established theoretical basis for using nerve trachea to repair neurologic defect.Along with the development of tissue engineering technique, use biomaterial and seed cell to become developing direction to replace nerve autograft at the external structure tissue engineered peripheral nerve.The application organizes engineering makes up tissue engineered peripheral nerve, the core of research is a simulation peripheral nerve natural structure, seed cell and biologic bracket material are organically combined the structure that becomes similar B ü ngner band,, thereby promote neural regeneration for regenerating nerve provides good growing environment.The Method of Tissue Engineering reparation of peripheral nerve defection can overcome the shortcoming of nerve autograft, reaches the ideal goal that neural growth, function are fast recovered fully.
Tissue engineered peripheral nerve still is in animal experiment stage at present, the principal element that restricts its development is very huge as the schwann cell requirement of seed cell, yet schwann cell propagation slowly, and the In vitro culture difficulty often takes place by fibroblastic being infected with.
In addition, studies show that neurotrophic factor plays an important role to survival, the promotion neuranagenesis of neuroprotective unit.Yet, directly use neurotrophic factor to be difficult to keep the effectiveness of its effect in the later stage the working in early days of tissue repair; Be owing to, make local concentration be difficult to reach valid density on the one hand easily by body fluid dilution, absorption; On the other hand if the excessive danger that then can cause general action of dosage that awards, and because intravital environment and inactivation does not reach desired biological effect, perhaps at short notice with regard to approach exhaustion.
Chinese patent application with the more approaching prior art patent documentation disclosed Japanese Shimizu Yasuhiko in 2002 of the present invention, this invention relates generally to a kind of preparation of artificial neural canal, constitute by the absorbable material of biology, surface-coated collagen, inner chamber has the microfibre collastromin, fills laminine in the space.The Chinese patent (application number is 00123465.X) that Wang Shenguo is arranged again, major defect is: (1) has ignored the central role that seed cell and neurotrophic factor rise in neuranagenesis, the undue facilitation that relies on material to neuranagenesis, clinical efficacy is uncertain; (2) do not relate to the complex method of cell and neurotrophic factor and biomaterial.
Summary of the invention
At the prior art situation, the object of the invention is to make up a kind of tissue engineered peripheral nerve, but the stem cell of using the differentiation of neurogliocyte or neurad glial cell is as seed cell, Biodegradable material is as support, use the Controlled Release System of compound multiple neurotrophic factor simultaneously, set up a microenvironment that helps neuranagenesis, nerve is regenerated, for clinical treatment provides feasible program.
The present invention is used to repair the tissue engineered peripheral nerve of peripheral nerve defection, it is characterized in that: tissue engineered peripheral nerve is formed by nerve trachea and neurogliocyte or stem cell constructing; Described nerve trachea is made of Biodegradable material; But described neurogliocyte or stem cell are from the neurogliocyte in body or allogeneic source or the stem cell of neurad system cells differentiation; Include the Controlled Release System of cell or neurotrophic factor and collagen, laminin (LN), fiber adhesion albumen extracellular matrixs such as (FN) in it.Described Biodegradable material comprises natural macromolecular material such as collagen, chitosan; With high molecular synthetic material such as polylactic acid, polyglycolic acid, and their copolymer; Described stem cell comprises schwann cell, embryonic stem cell and adult stem cell, as mesenchymal stem cells MSCs, and hematopoietic stem cell, skin progenitor cell, mescenchymal stem cell, muscle stem cell, liver stem cells, neural stem cell.Described nerve trachea has 100 μ m-300 μ m micropores, and porosity is 50%-90%.The cell concentration that is comprised is 10
5-10
7/ ml; The neurotrophic factor that is comprised refers to one or more among neurotrophic factor NGF, neurenergen 3 NT-3, Brain Derived Neurotrophic Factor BDNF, ciliary neurotrophic factor CNTF, glial cell line-derived neurotrophic factor GDNF, transforming growth factor TGF 'beta ' family, leukaemia inhibitory factor LIF, basic fibroblast growth factor bFGF and the insulin-like growth factor I GF.Described Controlled Release System adopts Biodegradable material compound to neurotrophic factor, and utilize the selection permeability of macromolecule to medicine, realization is to the sustained release of neurotrophic factor, the sustained release microsphere diameter is 100nm-100 μ m, degradation time was about 30 days-150 days, can adjust according to the speed and the regenerated distance of neuranagenesis; Comprise in the tissue engineered peripheral nerve and be full of the gel that collagen, laminin (LN) and fiber adhesion albumen extracellular matrix components such as (FN) constitute.
Nerve trachea of the present invention has certain permeability, can allow a large amount of refuses diffusion dischargings that produce owing to metabolism in cell and the axon regeneration process, stop a large amount of invasions of fibroblast and the passage of obstruction nerve growth, prevent the loss of local nerve trophic factors, near the tiny blood capillary of the blood circulation conduit of growing into being beneficial to.
Neurotrophic factor among the present invention is discharged by Controlled Release System.The adding trophic factors mainly acts on and is in nerve trachea: the differentiation of induced dry-cell neurad glial cell; Keep neuronic survival, promote the regeneration of neural axon.Yet regular meeting when directly using trophic factors is because intravital environment and inactivation does not reach desired biological effect, and can not continue to discharge.We use the Controlled Release System of the cytotrophy factor, can continue to discharge, and keep the concentration of the cytotrophy factor, make it continue to play a role.Controlled Release System adopts Biodegradable material compound to neurotrophic factor, and the cytotrophy factor is discharged gradually along with the degraded of material.In addition; utilize the selection permeability of macromolecule to medicine; can make neurotrophic factor in the polymer protection layer, dissolve, spread with certain speed; enter body then to bring into play its biological agent; therefore can make the biological agent of neurotrophic factor keep considerable time, thereby realize sustained release neurotrophic factor.The sustained release microsphere diameter of employed neurotrophic factor is between the 100nm-100 μ m among the present invention, and degradation time is 30 days-150 days, can adjust according to the speed and the regenerated distance of neuranagenesis.
Fill the main component that the extracellular matrix that adds is a basement membrane in the nerve trachea among the present invention, can play important guiding function the regenerated directivity of aixs cylinder.
The advantage that tissue engineered peripheral nerve of the present invention is compared with prior art is, neurogliocyte and neurotrophic factor necessary in the neuranagenesis process have been added, and to neurotrophic factor realization sustained release, being beneficial to neurotrophic factor plays a role for a long time, by the combined effect of neurogliocyte and neurotrophic factor, can play neuroprotective unit, promote the regenerated effect of neural axon; Also comprise extracellular matrixs such as collagen, LN, FN in the tissue engineered peripheral nerve simultaneously, the regenerated directivity of aixs cylinder is played important guiding function.
The preparation method that the present invention is used to repair the tissue engineered peripheral nerve of peripheral nerve defection comprises the preparation method of nerve trachea, the preparation method of neurotrophic factor sustained release microsphere, the construction method of tissue engineered peripheral nerve, the preparation method of described nerve trachea is by control solvent evaporation method or solution technique preparation, it is characterized in that:
1) the preparation method step of described neurotrophic factor sustained release microsphere is as follows:
One or more neurotrophic factors, Biodegradable material are dissolved in (as dichloromethane, chloroform, acetic acid, hydrochloric acid etc.) in the solvent, be added drop-wise in the glycerol, dispersed with stirring is even, in impouring 0.5% aqueous gelatin solution, disperse emulsion droplet, the eluting organic solvent, biomaterial formation of deposits microsphere.Centrifugal collection, distilled water wash filters, and constant pressure and dry is promptly.
2) construction method of described tissue engineered peripheral nerve comprises the steps:
The solution of extracellular matrix adds the minimum essential culture fluid DMEM of the hyclone of 1/10 volume, 10 times of concentration under ice bath, regulate pH value to 7.2-7.4, microsphere with seed cell and compound various kinds of cell trophic factors, fill with in the nerve trachea behind the mixing, after treating its formation gel, add the DMEM culture medium culturing.
Description of drawings
Fig. 1: tissue engineered peripheral nerve structural representation of the present invention.
Wherein: the 1 neural broken ends of fractured bone, 2 nerve tracheas, 3 neurotrophic factor NTF.Be filled with Controlled Release System and the extracellular matrixs such as collagen, laminin and fiber adhesion albumen of cell and neurotrophic factor NTF in the conduit.
Fig. 2: tissue engineering nerve of the present invention is repaired SD rat sciatic nerve 15mm after damaged 12 weeks, and regenerated nerve has passed through the laboratory animal photo of defective region.
The specific embodiment
Now the present invention is described further in conjunction with specific embodiments.
1 schwann cell is cultivated
10 of SD filial mices are put to death the back alcohol disinfecting, get the bilateral sciatic nerve, place the ice bath operating board, remove epineurium and adhesion organization, after adding pancreatin/collagenase mixture slaking, collect top cell suspension, the centrifugal supernatant of abandoning adds culture fluid and suspends again, is inoculated in the culture bottle and cultivates.Add 10 in the culture fluid after 24 hours
-5M arabinose born of the same parents-, act on and be replaced by the G-418 culture fluid that contains 100 μ g/ml, continuous culture 5 days after 48 hours.Add 2 μ M fluorine screw thread woodss subsequently and continue to cultivate, changed liquid once in per 3 days, converge to cell.
The preparation of 2 nerve tracheas
Prepare the nerve trachea that contains 100 μ m-300 μ m holes according to control solvent evaporation method or solution technique, according to defect length decision nerve trachea length, general nerve trachea length is than the long 5mm of neurologic defect length, so that the socket of neural two broken ends of fractured bone.
The preparation of 3 neurotrophic factor sustained release microspheres
The preparation of microsphere divided for two steps carried out, i.e. the eluting of the dispersion of organic facies and organic solvent.One or more neurotrophic factors, Biodegradable material are dissolved in the solvent as dichloromethane, chloroform, acetic acid, hydrochloric acid etc., are added drop-wise in the glycerol, dispersed with stirring is even, in impouring 0.5% aqueous gelatin solution, disperse emulsion droplet, eluting organic solvent, biomaterial formation of deposits microsphere.Centrifugal collection, distilled water wash filters, and constant pressure and dry is promptly.As disperse medium, can under the mixing speed of 500r/min, obtain the emulsion droplet of particle diameter with glycerol less than 30 μ m.Eluent uses 0.5% aqueous gelatin solution, has both helped the diffusion eluting of solvent, can prevent that again emulsion droplet from merging or the microsphere adhesion.When microsphere was degraded, neurotrophic factor discharged gradually, sustainable 30 days-150 days time.
The structure of 4 tissue engineered peripheral nerves
The solution of extracellular matrix adds the hyclone of 1/10 volume, the DMEM culture medium of 10 times of concentration under ice bath, regulate pH value to 7.2-7.4, microsphere with seed cell and compound various kinds of cell trophic factors, fill with in the nerve trachea behind the mixing, after treating its formation gel, add the DMEM culture medium culturing.
It is damaged that 5 usefulness tissue engineered peripheral nerves are repaired the SD rat sciatic nerve
Using-system through engineering approaches peripheral nerve bridge joint sciatic nerve is damaged, detects neuranagenesis and sciatic nerve functional rehabilitation situation by methods such as SABC, electric physiology, transmission electron microscope, horseradish peroxidase retrograde tracing.Find that nerve fiber marshalling, number are many, nerve fiber is thick, myelin is thick, the flaggy densification in the myelin of regenerating less of interfascicular connective tissue, myelin obviously thickens, be rich in neurofilament and microtubule in the axoplasm, electric physiology can detect sciatic nerve mixing action potential, and the sciatic nerve function index shows the most of (see figure 2) of recovering of sciatic nerve function.
Claims (8)
1, a kind of tissue engineered peripheral nerve that is used to repair peripheral nerve defection is characterized in that: tissue engineered peripheral nerve is formed by nerve trachea and neurogliocyte or stem cell constructing; Described nerve trachea is made of Biodegradable material; But described neurogliocyte or stem cell are from the neurogliocyte in body or allogeneic source or the stem cell of neurad system cells differentiation, include the Controlled Release System and the extracellular matrix of cell or neurotrophic factor in it.
2, tissue engineered peripheral nerve according to claim 1 is characterized in that: described Biodegradable material comprises natural macromolecular material such as collagen, chitosan; With high molecular synthetic material such as polylactic acid, polyglycolic acid, and their copolymer; Described neurogliocyte is meant schwann cell; Described stem cell comprises embryonic stem cell and adult stem cell.
3, tissue engineered peripheral nerve according to claim 1 is characterized in that: the tissue engineered peripheral nerve conduit has 100 μ m-300 μ m micropores, and porosity is 50%-90%.
4, tissue engineered peripheral nerve according to claim 1 is characterized in that: the cell concentration that is comprised in the tissue engineered peripheral nerve is 10
5-10
7Individual cell/ml.
5, tissue engineered peripheral nerve according to claim 1 is characterized in that: the neurotrophic factor that is comprised in the tissue engineered peripheral nerve refers to one or more among neurotrophic factor NGF, neurenergen NT, Brain Derived Neurotrophic Factor BDNF, ciliary neurotrophic factor CNTF, glial cell line-derived neurotrophic factor GDNF, transforming growth factor TGF 'beta ' family, leukaemia inhibitory factor LIF, basic fibroblast growth factor bFGF and the insulin-like growth factor I GF.
6, tissue engineered peripheral nerve according to claim 1, it is characterized in that: described Controlled Release System adopts Biodegradable material compound to neurotrophic factor, and utilize the selection permeability of macromolecule to medicine, realization is to the sustained release of neurotrophic factor, the sustained release microsphere diameter is 100nm-100 μ m, and degradation time is 30 days-150 days.
7, tissue engineered peripheral nerve according to claim 1 is characterized in that: comprise the gel that is full of collagen, laminin (LN) and fiber adhesion albumen (FN) extracellular matrix components formation in the tissue engineered peripheral nerve.
8, a kind of preparation method that is used to repair the tissue engineered peripheral nerve of peripheral nerve defection, comprise the preparation of nerve trachea, the preparation of neurotrophic factor sustained release microsphere, the structure of tissue engineered peripheral nerve, the preparation of described nerve trachea is to finish by control solvent evaporation method or solution technique, it is characterized in that:
1) the preparation method step of described neurotrophic factor sustained release microsphere is as follows:
One or more neurotrophic factors, Biodegradable material are dissolved in the solvent as dichloromethane, chloroform, acetic acid, hydrochloric acid etc., are added drop-wise in the glycerol, dispersed with stirring is even, in impouring 0.5% aqueous gelatin solution, disperse emulsion droplet, eluting organic solvent, biomaterial formation of deposits microsphere.Centrifugal collection, distilled water wash filters, and constant pressure and dry is promptly.
2) the construction method step of described tissue engineered peripheral nerve is as follows:
The solution of extracellular matrix adds the commercial minimum necessary culture fluid DMEM culture medium of the hyclone of 1/10 volume, 10 times of concentration under ice bath, regulate pH value to 7.2-7.4, microsphere with seed cell and compound various kinds of cell trophic factors, fill with in the nerve trachea behind the mixing, after treating its formation gel, add commercial minimum necessary culture fluid DMEM culture medium culturing.
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| CN100462105C (en) * | 2006-10-19 | 2009-02-18 | 中国人民解放军第四军医大学 | Method for preparing recombinant neuro surrogate |
| CN100560037C (en) * | 2007-12-06 | 2009-11-18 | 崔树森 | Artificial nerve catheter from the functional amplification of body |
| CN101579247B (en) * | 2009-06-23 | 2012-02-29 | 许和平 | I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof |
| CN102727936A (en) * | 2012-06-20 | 2012-10-17 | 中山大学 | Construction method of sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries |
| CN102836016A (en) * | 2011-06-20 | 2012-12-26 | 中山大学附属第一医院 | Implanting type degradable device for promoting nerve regeneration after ambient nerve implanting |
| WO2014114043A1 (en) | 2013-01-25 | 2014-07-31 | 南通大学 | Cell matrix modified tissue engineering nerve graft for repairing peripheral nerve injury and preparation method thereof |
| CN104055599A (en) * | 2013-10-17 | 2014-09-24 | 上海交通大学 | Degradable magnesium alloy nerve conduit for nerve defect repair and preparing method of degradable magnesium alloy nerve conduit |
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