CN103751845B - Tissue engineering biological material for transplanting and restoring peripheral nerve defect - Google Patents
Tissue engineering biological material for transplanting and restoring peripheral nerve defect Download PDFInfo
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Abstract
The invention discloses a tissue engineering biological material for transplanting and restoring a peripheral nerve defect. The tissue engineering biological material is prepared through the following steps: firstly, preparing an acellular nerve allograft scaffold by using a chemical extraction method; then culturing an adipose derived stem cell by using fatty tissues collected by a target receptor in an early debridement; finally carrying out induction in vitro by using an exogenous schwann cell endogenus neurotrophic factor to construct a tissue engineered peripheral nerve for later use. The acellular nerve allograft scaffold subject to chemical extraction has no immunogenicity and has a network structure most similar to an organism nerve and excellent biocompatibility, and meanwhile, a stem cell is controllably induced in vitro by using the schwann cell endogenus neurotrophic factor and is differentiated into schwann-liked cells in combination with an ideal scaffold and then transplanted in vivo to become effectiveness.
Description
Technical field
The present invention relates to biological technical field, specifically with biomaterial prepared by organizational engineering method.This Tissue Engineering Biomaterials is used for implant into body, repairs human peripheral nerve defection.
Background technology
Peripheral nerve is for nervus centralis, refers to all nerves beyond brain and spinal cord.The peripheral nerve injury run in clinical treatment or disappearance generally occur in body position, cause patient to feel and dyskinesia.The patient of peripheral nerve injury often needs Repeated Operation to treat.The process such as one-stage operation carries out debridement, bone reduction is fixed, delay to carry out CO2 laser weld.At present conventional clinically and effective Peripheral nerve repair method is nerve autograft, its way " is robbed Peter to pay Paul " after weighing the advantages and disadvantages, and is therefore severely limited.The reparation peripheral nerve that appears as that application organizational engineering method prepares human implantation's material technology brings new hope.Application organizational engineering method builds the peripheral nerve biomaterial can transplanted in human body, need possess three primary conditions: seed cell, timbering material and cytokine.At present all there are some technological difficulties not yet broken through at this in three:
1. about seed cell.Build organizational project peripheral nerve can seed cell have schwann cell (Schwann cells, SCs) and stem cell.Autologous schwann cell effect is good, but need win autologous normal peripheral nerve, and cause patient to be cut the new artificial damage in neural place, gains and losses balance each other, unsuitable multiplex.Available stem cell comprises mesenchymal stem cells MSCs (bone marrow stem cells, BMSCs) and neural stem cell (neural stem cells, NSCs).Wherein, prepare mesenchymal stem cells MSCs and need bone marrow extraction, patient is painful large, and the marrow quantity that can extract usually dislikes few, and the amplification in vitro time is long.The subject matter that neural stem cell exists is, the Schwann-liked cells after induction is long-term expression schwann cell specificity marker and stability thereof in vivo.Further, heterology neural stem cell has immunogenicity and ethnics Problem, the operation wound that the neural stem cell increase of autologous is larger.
2. about Nerve Scaffold.Nerve Scaffold is as the carrier guiding peripheral nerve regeneration, and its inner ultrastructure and immunogenicity are the keys that peripheral nerve injury is repaired.Desirable natural scaffold materials is autologous nerve, but donor lacks and the side effect that disables is long-standing problem.Fresh allogeneic nerve is because immunological rejection cannot be applied, and multiple physics, chemical process process heterology are neural, all can not thoroughly eliminate its immunogenicity.That relative efficacy is good is method [the Sondell M that Mariann Sondell etc. utilizes Triton X-100 sanitising agent and Sodium desoxycholate, Lundborg G, Kanje M. Regeneration of the rat sciatic nerve into allografts acellular through chemical extraction. Brain Res. 1998,795 (1-2): 44-54.] adopt the Allogeneic nerve graft reparation Sciatic processed in this way, immunogenicity is extremely low.But because schwann cell is the key that the rear aixs cylinder of transplanting is distally moved, this method eliminates schwann cell, cannot breed traction and form Bungner ' s band, therefore have the shortcoming of regenerating nerve conduct impulses insufficiency of function simultaneously.
3. about cytokine.The cytokine of differentiation of stem cells is various neurotrophic factor, and they are the most important biologically active factorss of neural system.But the transformation period of neurotrophic factor is short, be difficult to long-term role in vivo, what cannot need with neurotization mates for a long time.
Summary of the invention
Technical problem to be solved by this invention is, needs in putting into practice for current medical in reparation peripheral nerve defection and shortcoming, the deficiency of prior art, design a kind of Method of Tissue Engineering of biological technical field, the bioengineered tissue material prepared by the method can implant into body, repairs peripheral nerve defection.The present invention's alleged " Tissue Engineering Biomaterials " because itself being the nerve for transplanting prepared by Method of Tissue Engineering, therefore also known as " tissue engineering nerve ".
The Tissue Engineering Biomaterials that the present invention is used for repair of peripheral nerve defects with transplantation makes by following processing step:
Step one---chemical extraction prepares acellular nerve allograft support
Acellular nerve allograft derives from donator of cadaver, all through the agreement of patient's mandate before death and the lineal family members of patient, and by the license of Hospital Ethical Committee, meets the requirement of medical ethics.Select donator of cadaver before death without communicable diseases such as hepatitis B, hepatitis C, acquired immune deficiency syndrome (AIDS), syphilis.Corpse peripheral nerve is cut in one hour after patient death.The peripheral nerve of intercepting is inserted PBS liquid 4 DEG C and preserve 8-12 hour, then take out, aseptic finishing peripheral nerve, comprise structures such as carefully wiping out fat subsidiary on peripheral nerve, manadesma and blood vessel, retain complete epineurium, under 26-28 DEG C of envrionment temperature, by following operation, chemical extraction carried out to peripheral nerve material go antigen process:
(1) soak 10-14 hour in sterile distilled water, cell and myelin are expanded in hypotonic fluid;
(2) vibrate 10-14 hour in 5%Triton X-100 solution, cell and myelin broken completely, degrades and deviate from part cell disruption fragment;
(3) again soak 3 hours in sterile distilled water, deviate from cell debris further;
(4) vibrate 10-14 hour in 5% sodium deoxycholate solution, further destroys cell and myelin, and digest, degrade and deviate from cell debris.
After above process, around neural material inserts PBS liquid damping fluid (PH=7.2) 4 DEG C temporarily preservation
.
In this step, draw materials preferred femoral nerve and the sciatic nerve of corpse nerve.The nerve trunk material of particularly suitable should at diameter 7mm-8mm, length 80-100mm.
As optimization, " soaking → 5% sodium deoxycholate solution vibration in sterile distilled water in immersion → 5%Triton X-100 solution vibration → sterile distilled water " four step programs that this step prepares acellular nerve allograft support repeat, often repeat once, the antigenic substance in peripheral nerve material can be removed further, reduce its immunogenicity.Preferred repetition round is 2 times, comprises once initial, repeats this four steps procedure operation totally for 3 times.
Step 2---cultivate autologous fat stem cell (adipose derived stem cells, ADSCs) and make seed cell
The patient that peripheral nerve injury occurs is application organizational engineering biomaterial of the present invention, carries out the target body of peripheral nerve graft reparation.So generation peripheral nerve injury, normally wound causes.Contemporary medical science, to the process of wound, must carry out early stage debridement treatment, and fatty tissue must be had when carrying out early stage debridement surgical to be eliminated.Technical solution of the present invention is the material that Regular retains residual broken tissue work that patient is eliminated in debridement surgical and prepares fat stem cell.Specific practice is, chooses wherein without the shallow-layer fatty tissue of pathology, and remove the reticular tissue such as blood vessel, coating, PBS liquid rinses repeatedly, shreds into 1mm
3size tissue block.After PBS liquid rinses, digest 1 hour in 0.1% collagenase 37 DEG C incubator, centrifugal after filtering, be resuspended in DMEM/F12(1:1) cultivate, filter.After cell counting 1 × 10
5/ ml is inoculated in 25cm
2cultivate culture in glassware.After cytogamy, go down to posterity with 0.25% tryptic digestion attached cell.Utilize direct immunofluorescence labelling method and Flow cytometry fat stem cell relative special molecular E-cadherin, CD105, CD44, CD29, fat stem cell is defined as to sorting and continues to cultivate, go down to posterity.
The Tissue Engineering Biomaterials external structure of step 3---cytokine induction
In common 6 orifice plates, autologous fat stem cell step 2 being cultivated amplification is positioned on acellular nerve allograft support prepared by step one, and cell concn is 2 × 10
5/ ml, is positioned over 5%CO
2, in 37 DEG C of incubators, cultivate, merge.Overturn acellular nerve allograft support after 12 hours, change fat stem cell suspension, be again placed in 5%CO
2, 37 DEG C of incubators cultivate, merge.Then containing exogenous schwann cell derived neurotrophic factor (Schwann cell derived neurotrophic factor, SDNF) inducing culture in differentiation cocktail, differentiation cocktail contains H-DMEM substratum, 10%FBS, 100ng/ml SDNF, 10
-5m all-trans retinoic acid, 200ng/ml Heregulin-β.Induction Process is 26-30 days, within every 48-72 hour, changes once above-mentioned differentiation cocktail.Described schwann cell derived neurotrophic factor adopts commercially available product, and specification is 5ml/500ml or 10ug.Above-mentioned steps makes its formative tissue engineered material in vitro, for repairing peripheral nerve defection.After tissue engineering nerve external structure completes, if estimate to be implanted into object patient at 2-3 days, the tissue engineering nerve prepared is positioned over 5%CO
2, 37 DEG C of incubators are for subsequent use.The object accepting to transplant as intended implemented time of implant surgery uncertain or date of surgery more than 4 days, be then positioned in frozen storing liquid by the tissue engineering nerve prepared, successively through 4 DEG C 30 minutes ,-20 DEG C 2 hours ,-80 DEG C can be saved to 1 month.Application of thawing can be taken out at any time during this.
The present invention removes the allogeneic nerve support non-immunogenicity of cell through chemical extraction, has the spatial grid structure closest to organism nerve, and has good biocompatibility.Autologous fat stem cell makes seed cell non-immunogenicity, do not relate to the advantage that heteroplastic transplantation or non-treatment object cause the ethnics Problem of additional injuries.Gordian technique of the present invention innovation is to utilize schwann cell derived neurotrophic factor controlled induced dry-cell in vitro, after associated ideal support complex body is divided into Schwann-liked cells, then is transplanted in body and plays a role.Support, seed cell, cytokine three aspect are well combined the regeneration playing tissue engineered peripheral nerve by the present invention, to solve, the differentiation-inducing time is long, phenotype is uncertain, program is complicated, spend the problems such as large, while maintenance tissue engineering nerve morphological continuity recovers with promotion functions, meet clinical treatment needs.
Embodiment
Be that experimental subjects illustrates the present invention below with dog.
Concrete technological method comprises following processing step:
One. prepare acellular nerve allograft
With the sciatic nerve of dog in dead 1 hour for material, after drawing materials, nerve is inserted PBS liquid 4 DEG C and preserves 10 hours, then take out, aseptic finishing peripheral nerve, then under the environment of 26-28 DEG C, nerve is gone antigen process:
1., in sterile distilled water, soak 12 hours;
2. vibrate 12 hours in 5%Triton X-100 solution, degrade and deviate from part cell disruption fragment;
3. again soak 3 hours in sterile distilled water, deviate from cell debris further;
4. vibrate 12 hours in 5% sodium deoxycholate solution;
5. the peripheral nerve material processed through operation 4 is placed in sterile distilled water again to soak, repeats the process of operation 1-4 for twice.Four operation circulations carry out totally 3 times " to soak → 5% sodium deoxycholate solution vibration in sterile distilled water immersion → 5%Triton X-100 solution vibration → sterile distilled water ".Peripheral nerve material through above processing treatment is inserted PBS damping fluid (PH=7.2) 4 DEG C temporarily to preserve.
This experiment is through the license of laboratory animal Ethics Committee, and put to death in latter 1 hour at the experimental dog that other item scientific research is used, disposable peripheral nerve 2 of drawing materials, position is sciatic nerve, and the neural format diameter of the every bar cut is at 7mm, and length is 100mm.
Two. be separated, cultivate autologous fat stem cell
1) when row debridement surgical first to the target dog that limbs are subject to wound, the second phase need repair neurologic defect, cut the shallow-layer fatty tissue being equivalent to debridement surgical excision near wound, remove the reticular tissue such as blood vessel, coating, PBS rinses repeatedly, shreds into 1mm
3size tissue block.After PBS rinses, digest 1 hour in 0.1% collagenase 37 DEG C incubator, centrifugal after filtering, be resuspended in DMEM/F12(1:1) and carry out cultivation fat stem cell, filter.
2) after cell counting 1 × 10
5/ ml is inoculated in 25cm
2cultivate culture in glassware.After cytogamy, go down to posterity with 0.25% tryptic digestion attached cell.Utilize direct immunofluorescence labelling method and Flow cytometry fat stem cell relative specificity molecule E-cadherin, CD105, CD44, CD29 etc., fat stem cell is defined as to sorting and continues to cultivate, go down to posterity.Get growth conditions good the 3rd generation cell keep in stand-by in 37 DEG C of incubators.
Three. the organizational project peripheral nerve of the exogenous schwann cell derived neurotrophic factor induction of external structure
1) acellular nerve allograft of the dog processed is put into common 6 orifice plates, add previously cultivate the 3rd generation target dog fat stem cell cell suspension (cell concn is 1 × 10
5/ ml), cultivate, merge.Overturn Acellular nerve after 12 hours, change fat stem cell cell suspension, re-start immersion 12 hours, then change hole.
2) cell suspension of fat stem cell is combined with acellular nerve allograft, then in common six orifice plates, adds inducing containing the differentiation cocktail of schwann cell derived neurotrophic factor of buying from biotechnological formulation company, within every 24-48 hour, change nutrient solution.Differentiation cocktail contains H-DMEM substratum, 10%FBS, 100ng/ml SDNF, 10
-5m all-trans retinoic acid, 200 ng/ml Heregulin-β.Induction Process is 26-30 days, and every 48h-72h changes once above-mentioned differentiation cocktail.After tissue engineering nerve external structure completes, skin grafing and mending in the body completing object dog as estimated in 2-3 days, be then positioned over 5%CO
2, 37 DEG C of incubators.Organizational project peripheral nerve more than 4 days, is then positioned in frozen storing liquid by uncertain or repair time object repair time accepting to transplant as intended, successively through 4 DEG C 30 minutes ,-20 DEG C 2 hours ,-80 DEG C can be saved to 1 month.Application of thawing can be taken out at any time during this.Described frozen storing liquid composition is, 10% dimethyl sulfoxide (DMSO), 10% foetal calf serum, 80%DMEM/F12.
Result for the treatment of checking report: the tissue engineering nerve constructed by the present invention is transplanted in the body of object object, repairs peripheral nerve defection.In 24-36 week after the transfer, the recovery of gross function is assessed: main Testing index comprises Electrophysiology and detects the nerve conduction velocity of Nerve Graft section complex action potential, action potential peak swing, produces the Minimum Threshold intensity etc. of action potential; Gait analysis draws the sciatic nerve function index after reparation, Function of Evaluation recovery situation; Comprehensive above detection validation means, it is outstanding that evaluation show that the tissue engineering nerve of structure repairs the ability of Canine sciatic nerve defect in vivo, is substantially equal to the standard of nerve autograft.
Claims (8)
1., for a Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation, it is prepared by the following method:
Step one---chemical extraction prepares acellular nerve allograft support: the corpse being no more than a hour in death cuts peripheral nerve, insert PBS liquid 4 DEG C and preserve 8-12 hour, then take out, wipe out peripheral nerve and do subsidiary fat, manadesma and blood vessel, retain complete epineurium, under 26-28 DEG C of envrionment temperature, carry out chemical extraction according to the following steps go antigen process:
(1) soak 10-14 hour in sterile distilled water;
(2) vibrate 10-14 hour in 5%Triton X-100 solution;
(3) again soak 3 hours in sterile distilled water;
(4) vibrate 10-14 hour in 5% sodium deoxycholate solution;
Step 2---cultivate autologous fat stem cell and make seed cell: collect the residual broken tissue that patient is eliminated in debridement surgical, choose wherein without the shallow-layer fatty tissue of serious change, remove blood vessel, coating, PBS liquid rinses repeatedly, shreds into 1mm
3size tissue block, after PBS liquid rinses, digests 1 hour in 0.1% collagenase 37 DEG C incubator, centrifugal after filtering, and is resuspended in DMEM/F12 and cultivates, filter; After cell counting 1 × 10
5/ ml is inoculated in 25cm
2cultivate culture in glassware; After cytogamy, go down to posterity with 0.25% tryptic digestion attached cell, utilize direct immunofluorescence labelling method and Flow cytometry fat stem cell relative special molecular E-cadherin, CD105, CD44, CD29, fat stem cell is defined as to sorting and continues to cultivate, go down to posterity;
The Tissue Engineering Biomaterials external structure of step 3---cytokine induction: in common 6 orifice plates, autologous fat stem cell step 2 being cultivated amplification is positioned on acellular nerve allograft support prepared by step one, and cell concn is 2 × 10
5/ ml, is positioned over 5%CO
2, cultivate in 37 DEG C of incubators, merge; Overturn acellular nerve allograft support after 12 hours, change fat stem cell suspension, be again placed in 5%CO
2, 37 DEG C of incubators cultivate, merge; Then inducing culture in the differentiation cocktail containing exogenous schwann cell derived neurotrophic factor, every 48-72 hour changes once-combined induced liquid, complete Induction Process through 26-30 days, be the Tissue Engineering Biomaterials that can be used for repair of peripheral nerve defects with transplantation.
2. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 1, is characterized in that, perineural preferred femoral nerve and the sciatic nerve of drawing materials of corpse.
3. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 2, is characterized in that, peripheral nerve specification of drawing materials is diameter 7mm-8mm, length 80-100mm.
4. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 1, it is characterized in that, " soaking → 5% sodium deoxycholate solution vibration in sterile distilled water in immersion → 5%Triton X-100 solution vibration → sterile distilled water " four step programs that step one prepares acellular nerve allograft support repeat totally 3 times.
5. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 1, is characterized in that, step 3 differentiation cocktail used contains H-DMEM substratum, 10%FBS, 100ng/ml SDNF, 10
-5m all-trans retinoic acid, 200ng/ml Heregulin-β.
6. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 1, is characterized in that, the schwann cell derived neurotrophic factor specification that step 3 is used as cytokine is 5ml/500ml or 10ug.
7. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 1, it is characterized in that, the Tissue Engineering Biomaterials cultivated through step 3 to place in frozen storing liquid successively through 4 DEG C 30 minutes ,-20 DEG C 2 hours,-80 DEG C, save backup.
8. the Tissue Engineering Biomaterials for repair of peripheral nerve defects with transplantation according to claim 7, is characterized in that, described frozen storing liquid composition is: 10% dimethyl sulfoxide (DMSO), 10% foetal calf serum, 80%DMEM/F12.
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