CN105561390A - Functional biomaterial capable of guiding peripheral nerve regeneration and preparation method thereof - Google Patents

Abstract

The invention discloses a method for preparing a collagen material used for repairing peripheral nerve injury. The preparation method provided by the invention comprises the following steps: co-incubating vascular endothelial growth factors (VEGF) with collagen binding domains and an ordered collagen material, so as to obtain the collagen material used for repairing peripheral nerve injury. Experiment results show that the collagen material containing VEGF-CBD factors and used for repairing peripheral nerve injury can promote peripheral nerve regeneration, as well as exponential recovery of impaired peripheral nerve functions, electrophysiological recovery of peripheral nerve transaction parts, expression of S-100 proteins and/or neurofilament proteins of peripheral nerve injured parts, increase of diameter and/or wall thickness of myelin sheaths of the peripheral nerve injured parts and recovery of target organs, which proves that the functional material has more practical guiding significance on future clinical application to peripheral nerve injury repairing.

Description

A kind of functional biological material guiding peripheral nervous to regenerate and preparation method thereof

Technical field

The invention belongs to field of medical materials, relate to a kind of functional biological material guiding peripheral nervous to regenerate and preparation method thereof.

Background technology

Peripheral nerve injury (PNI) usually causes the forfeiture of patient's correlation function, affects the quality of life of patient.The peripheral nerve injury caused due to maternal infuries, traffic accident or violence wound etc. can cause feeling or the degeneration necrosis of motor neuron.Although peripheral nerve has the ability of regeneration, its functional rehabilitation is very undesirable.The development of artificial neuron research, effectively facilitates regeneration and the functional rehabilitation of neural axon.Collagen-based materials is one of good material in organizational project.Collagen-based materials is widely used in tissue regeneration because of its reduced immunogenicity and good degradability.

In nervous system, somatomedin plays an important role to promotion nerve growth around.Its VEGF VEGF is the glycoprotein that a class is rich in cysteine, wherein VEGF ₁₆₅be most widely used, it can promote vascular endothelial cell proliferation, differentiation, suppress endotheliocyte apoptosis, promote the functions such as the migration of vascular smooth muscle cell, research confirms that VEGF angiogenesispromoting effect also plays an important role in peripheroneural Regeneration and Repair.But in VEGF solution body after application because its half-life short, spread in a large number after body fluid washes away, thus can not obtain satisfied therapeutic effect.In order to reach effective drug level at therapentic part, periodic injections medication or heavy dose of administration are conventional methods.But such method can cause infecting or serious side effect.

Summary of the invention

An object of the present invention is to provide a kind of method of the collagen-based materials for the preparation of reparation peripheral nerve injury.

Method for the preparation of the collagen-based materials for repairing peripheral nerve injury provided by the present invention, specifically can comprising the steps: jointly to hatch with the VEGF of collagen land and orderly collagen-based materials, obtaining described for repairing the collagen-based materials of peripheral nerve injury.

In the process, the proportioning of the described VEGF with collagen land (VEGF-CBD) and described orderly collagen-based materials is: 0.2nmol:1-10mg.In one embodiment of the invention, the proportioning of the described VEGF with collagen land (VEGF-CBD) and described orderly collagen-based materials is specially 0.2nmol:5mg.

In the process, hatch described in and can be 10-37 DEG C (as 25 DEG C) and hatch 0.5-1 hour (as half an hour).

In the process, liquid environment when hatching described in is specially 1 × PBS.

The pH of described 1 × PBS is 7.4, and solvent is water, solute and concentration as follows: potassium dihydrogen phosphate 0.27g/L, sodium hydrogen phosphate 1.42g/L, sodium chloride 8g/L, potassium chloride 0.2g/L.

In the present invention, described hatching is specially: be dissolved in 1 × PBS described in 5 microlitres by the VEGF (VEGF-CBD) with collagen land described in 0.2nmol, then be added drop-wise on orderly collagen-based materials described in 5mg, hatch half an hour for 25 DEG C.

In the present invention, the aminoacid sequence of the described VEGF with collagen land (VEGF-CBD) specifically can be following (a) or (b):

The 1-184 position of sequence 1 in (a) sequence table;

Sequence 1 in (b) sequence table.

Wherein, sequence 1 is made up of 192 amino acid residues.Be the aminoacid sequence of VEGF from N-terminal 1-166 position; From the aminoacid sequence that N-terminal 178-184 position is collagen binding domain (CBD); Be histidine-tagged from N-terminal 187-192 position.

In the present invention, the preparation method of described orderly collagen-based materials (LOCS), specifically can comprise the steps:

1) by tendon Bovis seu Bubali film concentration expressed in percentage by volume be tributyl phosphate solution-treated 24-72 hour (as 48 hours) of 1-1.5% (as 1%);

2) 25-100mmol/L (as 50mmol/L), the pH of use containing 0.5-1.5mol/L (as 1mol/L) NaCl is Tris-HCl buffer process 24-72 hour (as 48 hours) of 7.6-8.5 (if pH is 8.0);

3) with 1.25 × 10 ⁵-3.75 × 10 ⁵u is (as (2.5 × 10 ⁵u)) trypsin/100mL buffer process 48-96 hour (as 72 hours), obtains described orderly collagen-based materials; Described buffer is 25-100mmol/L (as 50mmol/L) Tris-HCl, pH is 7-8 (if pH is 8);

The temperature of above-mentioned process is 2-8 DEG C (as 4 DEG C).

Further, in step 3) with after trypsin treatment, also can comprise by 0.5-1.5mol/L strong base solution (as 1MNaOH aqueous solution) (as the 5 minutes) step that processes 5-10 minute.

Step 1) described in the solvent of tributyl phosphate solution be PBS buffer or Tris-HCl buffer, if 25-100mmol/L (as 50mmol/L), pH are the Tris-HCl buffer of 7.6-8.5 (if pH is 8.0).

Step 1) in, described tendon Bovis seu Bubali film also will remove the muscular tissue of internal layer adhesion and the fat of outer layer adheres before treatment.

What utilize said method to prepare also belongs to protection scope of the present invention for the collagen-based materials repairing peripheral nerve injury.

Another object of the present invention is to provide a kind of for collagen scaffold repairing peripheral nerve injury and preparation method thereof.

The preparation method of the collagen scaffold for repairing peripheral nerve injury provided by the present invention, specifically can comprise the steps:

(1) according to the radius at peripheral nerve injury position to be repaired, collagem membrane is adopted to prepare the collagen tube of same radius;

(2) filling described for repairing the collagen-based materials of peripheral nerve injury in the collagen tube prepared to step (1), namely obtaining described for repairing the collagen scaffold of peripheral nerve injury.

In the process, step (1) specifically can be: according to the radius at peripheral nerve injury position to be repaired, find the mould of same radius (material can be glass or plastics), collagem membrane water (as tri-distilled water) is soaked (as soaked 1 ~ 2min), the collagem membrane soaked is rolled on described mould and forms tubulose (as being wound around 5/4 circle), the wrapped circle of the liquid collagen of right use, air-dry, 10 ~ 30 DEG C (as 25 DEG C) are placed and are made it crosslinked in 12 hours, remove described mould, obtain described collagen tube.

In step (1), after described removal mould, also can comprise and successively adopt NaH respectively ₂pO ₄(0.1M, solvent is water) solution and water (deionized water) carry out the step of washing.Specifically can be and first use NaH ₂pO ₄(0.1M, solvent is water) solution washing 5 times, then use water (deionized water) to wash 5 times.

In step (1), also comprise the step of described collagen tube lyophilizing, irradiation (radiation dose specifically can be 8kGy) after the described mould of removal.

In the process, be filled to described collagen tube to be filled to described in step (2) and account for a half space.

What utilize described method to prepare also belongs to protection scope of the present invention for the collagen scaffold repairing peripheral nerve injury.

In addition, for repairing the collagen-based materials of peripheral nerve injury, or also belong to protection scope of the present invention for the application in arbitrary as follows of the collagen scaffold of repairing peripheral nerve injury:

(a1) preparation promotes the material of peripheral nervous regeneration;

(a2) preparation promotes the material that impaired peripheral nervous function index recovers;

(a3) preparation promotes the material that peripheral nervous cross-section position electro physiology recovers;

(a4) preparation promotes the material that peripheral nervous damaged part S-100 albumen and/or neural thread protein NTP (NF) are expressed;

(a5) preparation promotes the material that peripheral nervous damaged part myelin diameter and/or myelin wall thickness increase;

(a6) preparation promotes the material that target organ recovers.

In (a6), described target organ is the organ that the described peripheral nervous of damaged is directly arranged.In the present invention, described peripheral nervous is specially sciatic nerve; Described peripheral nerve injury is specially sciatic nerve transection lesion.More concrete, in one embodiment of the invention, described sciatic nerve transection lesion refers to the damage formed afterwards apart from piriformis outlet 5mm place with the nerve deducting 3mm length by the sciatic nerve of SD rat (as adult male SD rats).Accordingly, described target organ is specially gastrocnemius.Described peripheral nervous function index is specially sciatic nerve function index (Sciaticnervefunctionindex, SFI); Its computing formula is:

SFI＝109.5(ETS-NTS)/NTS-38.3(EPL-NPL)/NPL+13.3(EIT-NIT)/NIT-8.8

In formula, ETS represents the toes width of right side foot, and EPL represents the footmark length of right side foot, and EIT represents the middle toes distance of right side foot; NTS represents the toes width of left side foot, and NPL represents the footmark length of left side foot, and NIT represents the middle toes distance of left side foot.SFI=0 is normal, and SFI=-100 is completely losing of function.

The VEGF-CBD factor is linked on orderly collagen as tissue engineering scaffold by the present invention first, by setting up animal peripheral nervous-sciatic nerve nerves transected damage model, the effect that the peripheral nerve injury reparation collagen-based materials that evaluation contains the VEGF-CBD factor plays in the repairing of neural injury of transverse section.Result confirms, peripheral nerve injury containing the VEGF-CBD factor provided by the present invention repairs collagen-based materials, can promote that peripheral nervous regenerates, promotes that impaired peripheral nervous function index recovers, promotes that peripheral nervous cross-section position electro physiology recovers, promotes peripheral nervous damaged part S-100 protein expression, promote that peripheral nervous damaged part myelin diameter and/or myelin wall thickness increase, promote the recovery of target organ.Visible, this functional material has even more important practical guided significance in the application of future clinical peripheral nerve injury reparation.

Accompanying drawing explanation

Fig. 1 is the form that peripheral nerve injury repairs collagen as tissue engineering scaffold.Wherein, A is the form that collagen catheter is cross-linked precollagen film; B is the form of collagen fiber, and C is the Electronic Speculum figure of collagen fiber.

Fig. 2 is collagen tube and collagen fiber.Wherein, A is collagen tube; B is collagen fiber Electronic Speculum figure.

Fig. 3 is the sciatic nerve transection lesion model of SD rat.Wherein, when A just completes for performing the operation; B is 12 weeks after operation.

Fig. 4 is sciatic nerve function index testing result.Wherein, A is rat footprint figure; B is sciatic nerve function index.In B, * represents compared with OCFs+VEGF-CBD group, significant difference in P<0.05 level; * represents compared with OCFs+VEGF-CBD group, significant difference in P<0.01 level; # to represent compared with blank pipe group significant difference in P<0.05 level.

Fig. 5 is electrophysiology nerve conduction velocity testing result.* represents significant difference in P<0.01 level.

Fig. 6 is fluorogold retrograde tracing testing result.

Fig. 7 is that NF, RECA-1 and S-100 expression detects.Wherein, A is fluoroscopic examination result figure (NF is neurofilament coloration result, and RECA-1 is blood vessel coloration result, the nuclear staining of Hoechst showed cell, and Merge is the superposition synthesis of first three figure); B is the SABC collection of illustrative plates of S-100; C is unit are epineural fiber number statistical result in SABC collection of illustrative plates (positive area of NF dyeing and the visual field under the ratio of the picture gross area); D is blood vessel number statistical result in unit are in SABC collection of illustrative plates (positive area of RECA-1 dyeing and the visual field under the ratio of the picture gross area).E is the ratio of the picture gross area under the positive area of S-100 dyeing and the visual field.* significant difference in P<0.05 level is represented; * represents significant difference in P<0.01 level.

Fig. 8 is the myelin analysis result of morphology and regenerating nerve.Wherein, A is Hematoxylin-eosin coloration result; B is myelin fast blue coloration result; C is transmission electron microscope observing result; D is the myelin diameter statistical result of regenerating nerve; E is the Myelin thickness statistical result of regenerating nerve.* significant difference in P<0.05 level is represented; * represents significant difference in P<0.01 level.

Fig. 9 is gastrocnemius recovery rate and Ma Song trichrome stain result.Wherein, A is horse pine trichrome stain result; B is the statistical result of gastrocnemius recovery rate; C is muscle positive area ratio.In B and C, * represents significant difference in P<0.05 level; * represents significant difference in P<0.01 level.

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Embodiment 1, for repairing the preparation of the collagen scaffold of peripheral nerve injury

One, the preparation of orderly collagen-based materials

Prepared by this laboratory, method is as referenced patent ZL200510098731.5, and concrete steps are as follows:

(1) draw materials and process

1, pretreatment fascia

Get the Adult Bovine muscle of fresh band adularescent fascia, with cold deionized water rinsing 3 times; Isolate fascia with tweezers and scalpel, remove the muscular tissue that internal layer adheres to as far as possible, and the tissue such as outer field fat.

2, material preparation

Pretreatment fascia step 1 obtained processes as follows:

1) be placed in 1% (percent by volume) TnBP (tributyl phosphate) solution (50mMTris-HCl buffer, pH8.0) and process 48 hours;

2) 48 hours are processed in 50mMTris-HCl buffer (pH8.0, containing 1MNaCl);

3) 1g (2.5 × 10 ⁵u) 72 hours are processed in trypsin/100ml (50mMTris-HCl buffer, pH8.0); Wherein, trypsin is Amresco Products, and its catalog number is 9002-07-7.

4) process 5 minutes in 1MNaOH, fully cleaning is to neutral.

Lyophilizing, obtains orderly collagen-based materials (Linearorderedcollagenscaffolds, LOCS) of the present invention.

(2) form of material

The orderly collagen-based materials LOCS of gained is white plates form, can also be prepared as orderly tubulose or collagen fiber.As shown in Figure 1, in Fig. 1, A, B represent that in lamellar and fibrous LOCS, Fig. 1, C is the stereoscan photograph of LOCS to the aspect graph of LOCS respectively.As can be seen from the figure, LOCS maintains the structure of natural collagen fibre, and its electromicroscopic photograph shows it and has orderly surface texture, and its coarse surface texture is also conducive to the adhesion of cell.

Two, with the preparation of the VEGF (VEGF-CBD) of collagen land

1, the expression vector with VEGF-CBD is built

(1) gene order (the 3-554 position of sequence 2) of synthetic coding VEGF-CBD albumen (the 1-184 position of sequence 1), and increase nucleotide CC at 5 ' end, form the recognition sequence of restriction enzyme site NcoI, the recognition sequence CTCGAG of restriction enzyme site XhoI is increased at 3 ' end, obtain the 1-560 position of sequence 2 in sequence table, DNA fragmentation shown in it is designated as DNA fragmentation 1.

(2) with restricted enzyme NcoI and XhoI double digestion DNA fragmentation 1, reclaim endonuclease bamhi and be connected with the skeleton large fragment of the pET28a carrier (purchased from Novagen company, article No.: 69864-3) through same double digestion, obtain recombiant plasmid.The recombiant plasmid called after pET-VEGF-CBD of DNA fragmentation shown in 7-554 position small fragment between showing restriction enzyme site NcoI and XhoI of pET28a carrier through order-checking being replaced with sequence 2 in sequence table.In recombinant expression carrier pET-VEGF-CBD, the downstream of VEGF-CBD gene is connected with 6 His labels.Aminoacid sequence with the VEGF-CBD-6His fusion rotein of 6 His labels is sequence 1 in sequence table, and the sequence of the encoding gene (ORF) of its correspondence is the 3-581 position of sequence 2 in sequence table.

Build with reference to said method and obtain recombinant expression carrier pET-VEGF.The structure of recombinant expression carrier pET-VEGF is described as: the recombiant plasmid obtained after DNA fragmentation shown in the 7-500 position small fragment between restriction enzyme site NcoI and XhoI of pET28a carrier being replaced with sequence 2 in sequence table.

2, the Expression and purification of VEGF-CBD

Recombinant expression carrier pET-VEGF-CBD step 1 built proceeds to e. coli bl21 (DE3) competent cell (Quan Shi King Company product, its catalog number is CD601-01).Go in LB culture medium, at 37 DEG C, 200rpm cultivates 12 hours, then in the ratio of 2ml/100ml, the recombinant bacterium of cultivation is inoculated into 150ml fresh LB, and at 37 DEG C, 200rpm cultivates 3 hours, works as OD ₆₀₀when reaching 0.8-1, add inducer IPTG (IPTG final concentration 1mM) and induce 5 hours.The centrifugal 10min of 8000g collects thalline.100W ultrasonication thalline, centrifugalize precipitates, and is precipitated and dissolved in the Tris buffer containing carbamide (final concentration 8M), carries out protein renaturation with redox system.Albumen after renaturation is with affinity chromatography, and salt is removed with desalting column after being separated by imidazole gradient eluting, obtains VEGF-CBD.

With reference to said method, recombinant expression carrier pET-VEGF is proceeded to e. coli bl21 (DE3) competent cell, obtain vegf protein.

3, the biological activity determination of VEGF-CBD

In order to measure the biological activity of VEGF-CBD, be separated human endothelial cell, isolated culture method: after people's umbilical cord is separated, with PBS wash buffer inwall, hemocyte is removed, then the pancreatin of 0.25% is poured in umbilical cord, both sides mosquito forceps is closed, place 15 minutes for 37 DEG C, then by the liquid collecting after trypsinization, and with PBS rinse inwall tens of under, liquid is collected in the lump, centrifugal rear gained cell is cultivated in the M199 culture medium of 20% hyclone and 10ng/mlVEGF and 10ng/mlbFGF, it is namely people's umbilical-cord endothelial cells after amplification, be incubated in 48 porocyte culture plates, to add etc. mole (0, 1, 2, 3, 4 and 5nM) VEGF-CBD or VEGF, detected the increment situation of cell by MTT after three days.

Can see that the concentration with VEGF and VEGF-CBD increases, endotheliocyte is dependency propagation in gradient, and compared with VEGF, the activity of VEGF-CBD does not have significant difference, proves that VEGF-CBD has biological activity.

Three, containing the preparation of the peripheral nerve injury function reparation collagen scaffold of the VEGF-CBD factor

1, the preparation of collagen tube

The glass tubing that cut-off footpath is 1mm is (according to the radius at wanted repairing nerve damage position, find the mould of same radius), collagem membrane is cut into suitable size, 2min is soaked with tri-distilled water, to make collagem membrane soft, collagem membrane winding mold tool is wound around 5/4 circle, winds the wrapped circle of the liquid collagen of rear use, spend the night air-dry.Room temperature (25 DEG C) spend the night (12 hours) be cross-linked, after crosslinked, collagen tube is taken off from glass tubing, has been cross-linked and has first used NaH afterwards ₂pO ₄solution (0.1M, solvent is water) washs 5 times, then uses deionized water wash 5 times, and lyophilizing, radiation (radiation dose specifically can be 8kGy) are for subsequent use afterwards.

The preparation of the collagen-based materials of the reparation peripheral nerve injury 2, containing the VEGF-CBD factor

(pH of 1 × PBS is 7.4 VEGF-CBD after 0.2nmol purification to be dissolved in 5 microlitre 1 × PBS, solvent is water, solute and concentration as follows: potassium dihydrogen phosphate 0.27g/L, sodium hydrogen phosphate 1.42g/L, sodium chloride 8g/L, potassium chloride 0.2g/L) in, then the orderly collagen-based materials of 5mg step one tendon Bovis seu Bubali film preparation is added drop-wise to, hatch half an hour for 25 DEG C, solution is all absorbed by LOCS, obtain the collagen-based materials of the reparation peripheral nerve injury containing the VEGF-CBD factor.

3, assemble

The collagen-based materials of the reparation peripheral nerve injury containing VEGF-CBD factor step 2 obtained, is cut into 5 millimeters long; Collagen tube step 1 obtained is cut into 7 millimeters long, and is radiated degerming (Fig. 2) by 8kGy.Be filled in by the collagen-based materials cut in the collagen tube cut, fill and account for a half space (because neural regeneration also needs space), the peripheral nerve injury function namely obtained containing the VEGF-CBD factor repairs collagen scaffold.

Four, not containing the preparation of the peripheral nerve injury function reparation collagen scaffold of the factor

1, the preparation of collagen tube

With step 31.

2, not containing the preparation of the collagen-based materials of the reparation peripheral nerve injury of the factor

5 microlitre 1 × PBS (formula is the same) are added drop-wise to the orderly collagen-based materials of 10mg step one tendon Bovis seu Bubali film preparation, hatch half an hour for 25 DEG C, solution is all absorbed by LOCS, obtain not containing the collagen-based materials of the reparation peripheral nerve injury of the factor.

3, assemble

The collagen-based materials not containing the reparation peripheral nerve injury of factor step 2 obtained, is cut into 5 millimeters long.Collagen tube step 1 obtained is cut into 7 millimeters long, and is radiated degerming by 8kGy.The collagen-based materials cut is filled in the collagen tube cut, fills and account for a half space (because neural regeneration also needs space), namely obtain and do not repair collagen scaffold containing the peripheral nerve injury function of the factor.

Embodiment 2, application peripheral nerve injury function repair the cross-section injury of sciatic nerve of collagen scaffold treatment SD rat

One, prepare the sciatic nerve transection lesion model of SD rat, peripheral nerve injury function is repaired collagen as tissue engineering scaffold bridge joint at nerve injury two ends

1, Preparatory work of experiment

Buy adult male SD rats 60, body weight is between 200 to 220g, and SD rat is purchased from company of dimension tonneau China.Consumptive material before surgery prepares as follows: surgical needles line, aseptic filter paper, Mus plate, gel ink pen, hands table, elbow scissors, probe-pointed scissors, straight forceps, staight scissors, eye scissors, ophthalmic tweezers, knife blade, handle of a knife, mosquito forceps, clock and watch tweezer, aseptic hole-towel, syringe needle filter, disposable syringe, disposable antibacterial ware, pentobarbital sodium, normal saline, PBS, 75% ethanol, small beaker, distilled water, refuse bag, gentamycin, 50ml centrifuge tube, vial, rifle head, 500 μ LEP manage, sealed membrane, liquid-transfering gun, micropipettor, masking foil, ruler, rubber band, camera, rope, timer, sthptic sponge, gauze, label paper, pen, sterile gloves, mask, cotton ball soaked in alcohol, ice chest, charger.

2, the cross-section wound operation of sciatic nerve

The concentration of pentobarbital sodium by 10 mg/ml is dissolved in the normal saline of 0.9%.SD rat is undertaken anaesthetizing (40mg/kg body weight) by intraperitoneal injection pentobarbital sodium.SD rat rubber band is fixed on Mus plate.Adopt routine disinfection paving single, get right lateral thigh rear portion stringer otch, skin is cut with scalpel, by muscle blunt separation, right sciatic nerves is appeared by musculus lateralis externi gap after stock, deduct the nerve of 3mm length at sciatic nerve apart from piriformis outlet 5mm place microscissors, the retraction due to nerve can form the defect of 5 millimeters.

According to the difference of Therapeutic Method, the present invention comprises 4 experimental grouies altogether.Often organize rat at least 5:

(1) VEGF-CBD group: the peripheral nerve injury function containing VEGF-CBD factor embodiment 1 prepared repairs two broken ends of fractured bone closely far away of the band pin stitching thread Bridging nerve of collagen scaffold 9-0, two broken ends of fractured bone are respectively moved into 1mm in conduit.

(2) PBS group: two broken ends of fractured bone closely far away not repairing the band pin stitching thread Bridging nerve of collagen scaffold 9-0 containing the peripheral nerve injury function of factor embodiment 1 prepared, two broken ends of fractured bone are respectively moved into 1mm in conduit.

(3) blank pipe group (Emptytube): only transplant the 7mm collagen tube that empty embodiment 1 prepares, not containing LOCS.

(4) nerve autograft group (Autograft): autologous sciatic nerve is cut off rear stitching.

Finally the muscle of rat and skin are sewed up respectively.Post operation is by the Animal House of rat feeding in standard.Between raising, temperature maintains about 25 DEG C, quiet, aseptic.Between feeding period, rat gives feedstuff and water regularly.Observe the upgrowth situation of animal every day.

Two, the detection of postoperative peripheral nervous regeneration situation

1, the observation of peripheral nervous regeneration situation

When operation just completes as shown in A in Fig. 3.The 12nd week after surgery, VEGF-CBD group and PBS group, can see that used collagen tube is degraded, replace by the tissue of similar nerve.Normally connect between the neural broken ends of fractured bone near far away.Without serious adhesion between neural and surrounding tissue, neural two broken ends of fractured bone are not formed and significantly expand (in Fig. 3 B).

2, sciatic nerve function index (Sciaticnervefunctionindex, SFI) detects

(1) experimental technique

According to (Bain, J.R. such as Bain to the detection of rat sciatic nerve functional rehabilitation; Mackinnon, S.E.; Hunter, D.A., Functionalevaluationofcompletesciatic, peroneal, andposteriortibialnervelesionsintheratPlasticandreconstr uctivesurgery198983 (1) 129-38) early-stage Study carry out, detect rat sciatic nerve function index weekly after surgery.Process is summarized as follows.Do a darkroom with carton, an opening in darkroom, opening part connects a pasteboard closed aisle.Length, the width in aisle, be highly respectively 60 centimetres, 12 centimetres, 12 centimetres.Before functional test, prepare the large blank sheet of paper that a length is 70 centimetres, be laid at the bottom of aisle.First allow rat free traveling on the ground, by black for the bilateral flippers black dyes of rat dye after a period of time, one end rat being placed on aisle allows it freely along aisle walking until creep in darkroom.The footprint of bilateral is stayed on blank sheet of paper.After being regained by blank sheet of paper, measure the correlation values on left side foot (normal parapodum) (N) and right side foot (experiment parapodum) (E) respectively.Wherein footmark length (printlength, PL) is the distance from heel to toe; Toes width (widthbetweenthefirstandfifthtoes, TS) is that the 1st toe is to the 5th toe line distance; Middle toes distance (inter-toesdistance, IT) are that the 2nd toe is to the 4th toe line distance.The computing formula of sciatic nerve function index (Sciaticnervefunctionindex, SFI) is:

SFI＝109.5(ETS-NTS)/NTS-38.3(EPL-NPL)/NPL+13.3(EIT-NIT)/NIT-8.8

ETS represents the toes width of right side foot, and EPL represents the footmark length of right side foot, and EIT represents the middle toes distance of right side foot; NTS represents the toes width of left side foot, and NPL represents the footmark length of left side foot, and NIT represents the middle toes distance of left side foot.SFI=0 is normal ,-100 completely losing for function.

(2) result

Sciatic nerve function index can reflect the situation of rat functional rehabilitation, and when the 12nd week, the footprint of its Ipsilateral is shown in A in Fig. 4.The sciatic nerve function index respectively organized made curve is calculated to obtain by formula.When the recovery completely that sciatic nerve function index is 0 interval scale function of nervous system, and represent the disappearance completely of function of nervous system when sciatic nerve function index is-100.The first Wednesday after the cross-section wound of rat sciatic nerve a group sciatic nerve function index all close-100, this represents that operation modeling is successful.Three groups, As time goes on sciatic nerve function index all has the trend of increase, but it increases obviously faster than other two groups in OCFs+VEGF-CBD group, similar with nerve autograft group (in Fig. 4 B).

3, electrophysiology detection

(1) experimental technique

Post operation has carried out the test of electrophysiology nerve conduction velocity (nerveconductionvelocity, NCV) for 12 weeks to rat.The instrument adopted during experiment is that the RM6240USB2.0Z type Muscle Electricity Level Inducing Apparatus that Chengdu Instruement Factory produces traces NCV.By rat by method anesthesia before, cut skin and again expose sciatic nerve, with plastic glove pad under sciatic nerve, make tissue that is neural and surrounding separate to reduce interference.Adjust the various parameters of instrument.Two stimulating electrode is placed on the sciatic near-end of regeneration, two recording electrodes are placed on the nearly far-end that distance sews up line segment respectively, and wherein the recording electrode of near-end should keep suitable distance with two stimulating electrode.Reference electrode is clipped in subcutaneous rat.Distance between record two recording electrodes.Two stimulating electrode stimulates.Recording electrode will present different amplitude change respectively, according to the distance between near-end and far-end 2, calculates NCV.

(2) result

At the 12nd week of sciatic nerve injury and repair, to the statistics of electro physiology test, each group all has recovery in various degree after surgery.Its nerve conduction velocity presents the trend increased progressively from blank pipe group to normal nervous tissue.Reparation group is lower than the conduction velocity of normal neuronal, and its growth of OCFs+VEGF-CBD group is obvious faster than other two groups, similar with nerve autograft group (Fig. 5).

4, fluorogold retrograde tracing detects

(1) experimental technique

After surgery 10 weeks.By rat anesthesia, again expose sciatic nerve.The fluorogold (5 milligrams/100 microlitres, Fluorochrome, Inc., Denver, CO) of 3 microlitres 5% microsyringe is slowly expelled to neural far-end, stops 30 seconds after injection, slowly extract microsyringe.After muscle and skin closure, rat is still raised by situation before.After two weeks, rat is put to death by excessive anesthesia.Be separated and take out the dorsal root ganglia dorsalrootganglia (DRGs) of L4, L5, L6.The Dorsal ganglion of taking-up is saved the embedding of freezing embedding medium, used liquid nitrogen freezing as frozen section subsequently.Each dorsal root ganglion is cut into 10 micron thickness, and is affixed on microscope slide, observe under fluorescence inverted microscope (Axiovert200, Germany).Counting has the neuronic quantity of the concurrent golden yellow fluorescence of cellular morphology.

(2) result

Postoperative, retrograde tracing is carried out to rat, and observation of drawing materials in the 12nd week.Find, under burst of ultraviolel, presented golden yellow by the neuron of fluorogold labelling.Known by the counting of the sensory neuron to institute's labelling.In each group, the neuronal quantity that blank pipe group is labeled is minimum, and the neuronal quantity be labeled in the dorsal root ganglion of normal rat is the highest.In OCFs+VEGF-CBD group, it increases obviously faster than other two groups the sensory neuron be labeled, similar with nerve autograft group (Fig. 6).

5, histology is observed

(1) experimental technique

A. fluoroscopic examination

Transplantation treatment 12 weeks, after rat is put to death by excessive anesthesia, rapidly new life's nerve is taken out, the paraformaldehyde (purchased from Beijing chemical reagents corporation) 4 DEG C of 4% fixedly spends the night (more than 12 hours), transfer to the sucrose (4 DEG C of 20%, 3 days, reagent was purchased from Beijing chemical reagents corporation) and 30% sucrose (4 DEG C, 3 days).After liquid nitrogen freezing, be cut into 10 μm of slabs, every sheet tissue drips Normal Goat Serum (Xin Bosheng Products, its catalog number is ENS004.120) confining liquid and closes, and hatches 15 minutes at putting into 37 DEG C, wet box.Suck closed serum, do not wash.(NF, 1:500 dilute, Abcam for specimen and neural thread protein NTP-200 one antibody staining, article No. ab7795) or vascular endothelial cells Cell Plasma Antigen (RECA-1,1:100 dilution, Serotec company, article No. MCA970R) 4 DEG C spend the night, PBS rinsing 3 times, each 5 minutes.Add donkey anti-mouse antibody (the 1:100 dilution of Alexa495-donkey anti-rabbit secondary antibodies (1:100 dilutes, Invitrogen company, article No. A-11055) or Alexa594 labelling, Invitrogen company, article No. A-11020) hatch 1h, PBS rinsing 3 times, each 5 minutes.Adding 0.5-10 μ g/mlHoechst33342 (purchased from green skies company, article No. C1022) is for nuclear staining.Stained is at laser scanning co-focusing fluorescence microscope (come card).

B. immunohistochemical staining

Transplantation treatment 12 weeks, after rat is put to death by excessive anesthesia, takes out new life's nerve rapidly, in 10% formalin fixative, fixes 48 hours.Cut into slices after paraffin embedding, carry out the immunohistochemical staining of S-100 albumen.Use the slide bonding die of poly-D-lysine process, will cut into slices and toast 3 hours with 56 DEG C on roasting sheet machine.Need when carrying out immunohistochemical staining to experience following step: be followed successively by dimethylbenzene I, 15 minutes; Dimethylbenzene II, 15 minutes; Dimethylbenzene/ethanol (volume ratio 1:1), 5 minutes; 100% ethanol, 5 minutes; 95% ethanol, 5 minutes; 80% ethanol, 5 minutes; 70% ethanol, 5 minutes; 50% ethanol, 5 minutes; Tap water, 5 minutes; Distilled water, 5 minutes.PBS soaks 3 times, each 5 minutes.By antigen retrieval buffers (compound method: trisodium citrate 3 grams, citric acid 0.4 gram, adding distil water is to 1L, adjust pH=6.0, all reagent is all purchased from Beijing chemical reagents corporation) boil, tissue is put into the antigen retrieval buffers that boils and is maintained 10 minutes, allows tissue slowly cool to room temperature.3 times are washed, each 3 minutes with PBS.Draw a circle around tissue with SABC pen, blot with absorbent paper around sample.Drip 3%H ₂o ₂deionized water, hatches 10 minutes at putting into 37 DEG C, wet box, to block Endogenous peroxidase.PBS rinsing 3 times, each 5 minutes.Every sheet tissue drips Normal Goat Serum (Xin Bosheng Products, its catalog number is ENS004.120) confining liquid and closes, and hatches 15 minutes at putting into 37 DEG C, wet box.Suck closed serum, do not wash.Add the anti-S-100 (Sigma Products, its catalog number is S2644, dilution volume ratio 1:100) in mice source, after 4 DEG C of overnight incubation, PBS rinsing 3 times, each 5 minutes.Add biotinylation two anti-(Xin Bosheng Products, its catalog number is ENS004.120), put into after 37 DEG C, wet box hatches 15 minutes, PBS rinsing 3 times, each 5 minutes.Add three anti-(Xin Bosheng Products, its catalog number is ENS004.120), put into after 37 DEG C, wet box hatches 15 minutes, each sample adds DAB solution (the Xin Bosheng Products of proper amount of fresh configuration, its catalog number is ENS004.120), room temperature reaction 10 minutes, discards reactant liquor, and tap water stops.Place 5 minutes in 50% ethanol; Place 5 minutes in 70% ethanol; Place 5 minutes in 80% ethanol; Place 5 minutes in 95% ethanol; Place 5 minutes in 95% ethanol; Place 5 minutes in 100% ethanol; Place 5 minutes in 100% ethanol; Place 5 minutes in ethanol/dimethylbenzene (volume ratio 1:1); Place 10 minutes in dimethylbenzene I; Place 5 minutes in dimethylbenzene II; Neutral gum mounting, microscopic examination, takes a picture.

NF is the specific stain to aixs cylinder, and RECA-1 dyeing can show revascularization situation, and S-100 specific stain can demonstrate the regeneration situation of Scs, analyzes in statistics by the ratio of the picture gross area under the positive area of dyeing and the visual field.

(2) result

As can be seen from the figure, in blank pipe group, the ratio of NF, RECA-1 and S-100 positive area in each group is all minimum.And aobvious the highest at LOCS+VEGF-CBD group positive area, the positive area ratio the most close to normal neuronal in three reparation groups.Detected by this, can know, OCFs+VEGF-CBD more effectively can promote blood vessel and neuranagenesis (Fig. 7).

6, the myelin analysis of morphology and regenerating nerve

(1) experimental technique

After surgery the 12nd week, regenerating nerve is drawn materials and it is carried out after cutting into slices to the observation of tectology.First the situation that routine hematoxylin eosin stains (HE) grows in conduit to observe regenerating nerve is done.Then, detected by fast blue dyeing and Electronic Speculum, to the diameter of newborn nerve, quantity number and the thickness of myelin analyze, three indexs are added up respectively.

A. Hematoxylin-eosin dyeing (HE)

After injury of sciatic nerve 12 weeks, after respectively rat being put to death with excessive anesthesia, take out neural for new life and be fixed in 10% formalin fixative rapidly.After carrying out paraffin embedding, piece of tissue cut into slices, thickness is 5 microns.Thin slice is attached on the slide of use poly-D-lysine process.Microscope slide is placed on roasting sheet machine in 56 DEG C of bakings 3 hours.Dewax with dimethylbenzene.Be followed successively by dimethylbenzene I, 15 minutes; Dimethylbenzene II, 15 minutes; Dimethylbenzene/ethanol (volume ratio 1:1), 5 minutes; Anhydrous alcohol I, 5 minutes; Anhydrous alcohol II, 5 minutes; 95% ethanol, 5 minutes; 80% ethanol, 5 minutes; 75% ethanol, 5 minutes; 50% ethanol, 5 minutes; ddH ₂o washs 3 times, each 5 minutes; Haematoxylin dyeing, 5 minutes; Tap water 5 minutes; ddH ₂o soaks 2 times, each 5 minutes; Acid water breaks up, and about 3 seconds, reddens; Tap water number minute to a few hours, until biopsy tissues presents blueness; 50% ethanol, 5 minutes; 70% ethanol, 5 minutes; 80% ethanol, 5 minutes; Eosin stains, 3 minutes; 95% ethanol, 5 minutes; 95% ethanol, 5 minutes; Anhydrous alcohol I, 5 minutes; Anhydrous alcohol II, 5 minutes; Ethanol/dimethylbenzene (volume ratio 1:1), 5 minutes; Dimethylbenzene I, 10 minutes; Dimethylbenzene II, 5 minutes; Neutral gum mounting, microscopic examination, takes a picture.

B. sciatic myelin fast blue dyeing (LFB)

After rat being put to death by excessive anesthesia in 12nd week after surgery, rapidly sciatic nerve damaged part is taken out, in 10% formalin fixative, fix two days.By sample being cut into after paraffin embedding the semithin section of 5 microns, use the slide bonding die of poly-D-lysine process, roasting sheet a few hours on roasting sheet machine.By tissue successively by following process: dimethylbenzene I, 15 minutes; Dimethylbenzene II, 15 minutes; Dimethylbenzene/ethanol (volume ratio 1:1), 5 minutes; 100% ethanol, 5 minutes; 95% ethanol, 5 minutes; Tissue is put into prison indigo plant to spend the night with 37 DEG C; 95% washing with alcohol twice, each 3 minutes.Distilled water wash clean; Draw a circle around tissue with SABC pen; By the LiCO of 0.05% ₃dripped in tissue upper 5 second; Distilled water wash; 70% ethanol breaks up 10 seconds; Distilled water wash; Basis of microscopic observation, repeats 5-7 time until myelin and surrounding tissue form a sharp contrast; Tissue is soaked 1 second in 0.5% Yihong; Distilled water wash; Soak 1 second in the crystal violet of 0.25%; Wash in distilled water; 70% ethanol breaks up 10 seconds; Distilled water wash; Place 5 minutes in 50% ethanol; Place 5 minutes in 70% ethanol; Place 5 minutes in 80% ethanol; Place 5 minutes in 95% ethanol; Place 5 minutes in 95% ethanol; Place 5 minutes in 100% ethanol; Place 5 minutes in 100% ethanol; Ethanol/dimethylbenzene 1:1 (volume ratio 1:1), 5 minutes; Dimethylbenzene I, 10 minutes; Dimethylbenzene II, 5 minutes; Neutral gum mounting, microscopic examination, takes a picture.Image-ProPlus software (MediaCybernetics) is utilized to add up myelinogenetic number and diameter again.

C. the transmission electron microscope observing (TEM) of regenerating nerve

The 12nd week of transplantation treatment, by rat by after excessive anesthesia execution, newborn sciatic nerve is removed for doing transmission electron microscope observing.Concrete operations are as follows: be placed in by nerve in 2.5% glutaraldehyde solution (volume fraction, solvent is water) and fix 2 hours; By 0.1M phosphoric acid rinsing liquid (compound method: NaCl80g, Na ₂hPO ₄12H ₂o32.3g, NaH ₂pO ₄2H ₂o4.5g, is dissolved in 1L water, pH7.0.All chemical reagent are all purchased from Beijing chemical reagents corporation) rinsing three times, each 15 minutes; In 1% osmic acid fixative (compound method: 1g osmic acid is dissolved in 100ml water.Osmic acid purchased from sigma company, catalog number 419494) in fix 2 hours; With 0.1M phosphoric acid rinsing liquid (formula is the same) rinsing three times, each 15 minutes; Serial dehydration is carried out by the ethanol being organized in variable concentrations: place 15-20 minute in 50% ethanol in 4 DEG C of refrigerators; 15-20 minute is placed in 70% ethanol; 15-20 minute is placed in 90% ethanol; 15-20 minute is placed in 90% ethanol+90% acetone (volume ratio 1:1) ethanol; 15-20 minute is placed in 90% acetone ethanol; 15-20 minute is placed in room temperature, altogether placement three times in 100% acetone; Organization embedding: pure acetone+embedding liquid (volume ratio 2:1) room temperature 4 hours; Pure acetone+embedding liquid (volume ratio 1:2) ambient temperature overnight; Pure embedding liquid, 37 DEG C 3 hours; Solidification: spend the night in 37 DEG C of baking ovens; 45 DEG C of baking ovens 12 hours, in 60 DEG C of baking ovens 24 hours; With ultramicrotome, tissue is cut into the section of 50-60nm.(1% uranium acetate dye liquor preparation method: claim uranium acetate 0.2g, add distilled water to 10mL, sealed membrane seals, and 4 DEG C keep in Dark Place to use 1% acetic acid uranium-citrate.1% lead citrate dye liquor preparation method: claim plumbi nitras 0.265g, sodium citrate (containing 2 molecular crystalline water) 0.352g, adds distilled water to 10mL, sealed membrane seals, and 4 DEG C keep in Dark Place.Name of product: lead citrate, article No.: GA10701; Name of product: uranium acetate, article No.: GS02625.) two dyeing.Wherein, embedding liquid (middle mirror tech Products, name of product EMbed812 epoxy resin embedding suit article No.: GE14120).Use transmission electron microscope observing after completing above-mentioned steps and take pictures.The therapeutic effect of each group is evaluated by the myelin diameter of more each treatment group and myelin wall thickness.Eachly organize random selecting 3 different visuals field, carry out quantitatively by Image-ProPlus software to myelin diameter and myelin wall thickness, quantitative block diagram is by GraphPadPrism5 Software on Drawing.

(2) result

Haematoxylin eosin stains (HE) result shows, and neural presents orderly growth, the similar normal neuronal of the cell arrangement in tissue employing orderly collagen-based materials at each group.This illustrates, orderly collagen-based materials can guide neural along material ordering growth effectively.Can be drawn by fast blue dyeing and Electronic Speculum, the aixs cylinder of regenerating nerve presents high density in each group, the morphological characteristic of clear-cut.Visible by the thickness measuring myelin, the thickness of regenerating nerve is greater than other two groups (Fig. 8) in OCFs+VEGF-CBD group.

7, gastrocnemius recovery rate and Ma Song trichrome stain

(1) experimental technique

After surgery the 12nd week, rat is put to death by excessive anesthesia.Be separated immediately and cut the gastrocnemius of rats with bilateral, electronic balance claims to obtain the weight of weight in wet base record bilateral gastrocnemius, the weight of right side gastrocnemius being obtained a ratio divided by the weight of left side gastrocnemius, is referred to as the recovery rate of gastrocnemius.After weighing, the stage casing of gastrocnemius taken off and be fixed in 10% formalin fixative.The slice of 5 microns is cut into through paraffin embedding.Section carries out horse pine trichrome stain according to the prompting step of producer.Tissue to be put in dimethylbenzene I 15 minutes; 15 minutes are placed afterwards at dimethylbenzene II; Place 5 minutes in dimethylbenzene/ethanol (volume ratio 1:1); In 100% ethanol 5 minutes; In 95% ethanol 5 minutes; In 80% ethanol 5 minutes; In 70% ethanol 5 minutes; In 50% ethanol 5 minutes; Wash 5 minutes in tap water; Soak 5 minutes in distilled water.PBS washs 3 times, each 5 minutes; To dye 5-10 minute with haematoxylin dyeing liquid/ferric chloride aqueous solutions (volume ratio 1:1); Incline rear distilled water wash 5 minutes; Acidic alcohol differentiation liquid breaks up 3-5 second; Incline the rear distilled water wash several seconds; Add ammonia and return indigo plant for macroscopic blueness; Incline rear distilled water wash 5 minutes; Ponceaux acid fuchsin dyeing liquor dyeing 5-10 minute; Incline rear distilled water wash 5 minutes; Add acetic acid aqueous solution 1 minute; Incline rear distilled water wash 5 minutes; Add phosphomolybdic acid aqueous solution 1-2 minute; Incline rear distilled water wash 5 minutes; Add acetic acid aqueous solution 1 minute; Directly put into aniline blue dyeing liquor dyeing 1-2 minute; Incline rear distilled water wash 5 minutes; Acetic acid aqueous solution washs 1 minute; Incline rear distilled water wash 5 minutes; In 50% ethanol 5 minutes; In 70% ethanol 5 minutes; In 80% ethanol 5 minutes; In 95% ethanol 5 minutes; In 95% ethanol 5 minutes; In 100% ethanol 5 minutes; In 100% ethanol 5 minutes; In ethanol/dimethylbenzene (volume ratio 1:1) 5 minutes; In dimethylbenzene I 10 minutes; In dimethylbenzene II 5 minutes; Neutral gum mounting, microscopic examination, takes a picture.

(2) result

The 12nd week after function of nervous system's material is transplanted.Can there is atrophy in gastrocnemius, along with the regeneration of nerve, the muscle of atrophy also can along with recovery after denervation.Weigh the weight in wet base of gastrocnemius and the ratio of the rate that is restored (damage side/strong side) normal rat bilateral gastrocnemius muscle weight is 1.Learn from figure, recovery rate is high compared with other two groups in OCFs+VEGF-CBD group.Carried out horse pine trichrome stain to gastrocnemius afterwards, in figure, redness is meat fiber, and green is collagen fiber, analyzes the positive area ratio (red area area/gross area) of muscle.In OCFs+VEGF-CBD group, the positive area ratio of muscle is close to normal muscle.These results are pointed out, and the new function of nervous system's material built more effectively can promote the recovery (Fig. 9) of target organ.

Claims (10)

1., for the preparation of the method for collagen-based materials of repairing peripheral nerve injury, comprise the steps: the VEGF with collagen land jointly to hatch with orderly collagen-based materials, for repairing the collagen-based materials of peripheral nerve injury described in obtaining.

2. method according to claim 1, is characterized in that: the proportioning of the described VEGF with collagen land and described orderly collagen-based materials is: 0.2nmol:1-10mg.

3. method according to claim 1 and 2, is characterized in that: described in hatch as 10-37 DEG C hatches 0.5-1 hour.

4., according to described method arbitrary in claim 1-3, it is characterized in that: described in liquid environment when hatching be 1 × PBS.

5. according to described method arbitrary in claim 1-4, it is characterized in that: the aminoacid sequence of the described VEGF with collagen land is following (a) or (b):

The 1-184 position of sequence 1 in (a) sequence table;

Sequence 1 in (b) sequence table.

6. utilize the collagen-based materials for repairing peripheral nerve injury that in claim 1-5, arbitrary described method prepares.

7., for repairing the preparation method of the collagen scaffold of peripheral nerve injury, comprise the steps:

(1) according to the radius at peripheral nerve injury position to be repaired, collagem membrane is adopted to prepare the collagen tube of same radius;

(2) filling the collagen-based materials for repairing peripheral nerve injury according to claim 6 in the collagen tube prepared to step (1), namely obtaining described for repairing the collagen scaffold of peripheral nerve injury.

8. method according to claim 7, it is characterized in that: step (1) is: according to the radius at peripheral nerve injury position to be repaired, find the mould of same radius, collagem membrane is soaked in water, the collagem membrane soaked is rolled on described mould and forms tubulose, so with the wrapped circle of liquid collagen, air-dry, 10 ~ 30 DEG C of placements make it crosslinked in 12 hours, remove described mould, obtain described collagen tube.

9. the collagen scaffold for repairing peripheral nerve injury utilizing method described in claim 7 or 8 to prepare.

10. the collagen-based materials for repairing peripheral nerve injury according to claim 6, or the application of the collagen scaffold for repairing peripheral nerve injury according to claim 9 in arbitrary as follows:

(a1) preparation promotes the material of peripheral nervous regeneration;

(a2) preparation promotes the material that impaired peripheral nervous function index recovers;

(a3) preparation promotes the material that peripheral nervous cross-section position electro physiology recovers;

(a4) preparation promotes the material that peripheral nervous damaged part S-100 albumen and/or neural thread protein NTP are expressed;

(a5) preparation promotes the material that peripheral nervous damaged part myelin diameter and/or myelin wall thickness increase;

(a6) preparation promotes the material that target organ recovers.