CN112755249A

CN112755249A - Mechanical property enhanced acellular spinal cord biomaterial stent and preparation method and application thereof

Info

Publication number: CN112755249A
Application number: CN202110086054.4A
Authority: CN
Inventors: 曾湘; 马瑗锾; 曾园山
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2021-05-07
Anticipated expiration: 2041-01-22
Also published as: CN112755249B

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a mechanical property enhanced acellular spinal cord biomaterial scaffold as well as a preparation method and application thereof. Compared with a pure DSC bracket, the biomaterial bracket has enhanced mechanical property and better pressure resistance, and can prevent the material from collapsing and being pressed by surrounding tissues after being transplanted; researches show that the spinal cord injury can be effectively repaired by transplanting the biomaterial scaffold to a rat full-transection spinal cord injury area, so that the problem of poor mechanical property of the decellularized spinal cord is solved, and the in-situ repair of the spinal cord injury is further promoted.

Description

Mechanical property enhanced acellular spinal cord biomaterial stent and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a mechanical property enhanced acellular spinal cord biomaterial scaffold as well as a preparation method and application thereof.

Background

Spinal Cord Injury (SCI) is a disease of central nervous system injury, and the incidence rate is further increased with the progress of economy and the aging of society. Statistically, the incidence of spinal cord injuries worldwide is 23/million per year, and the incidence in Asian countries, particularly China and Korea, is higher, 12.06-61.6/million. At present, about 200 million paraplegic patients caused by spinal cord injury exist in China, the number of paraplegic patients increases at the speed of about 5 million people every year, the tendency of high morbidity, high disability rate and low aging is presented, and great economic pressure is brought to families and society. In china alone, government direct economic expenses due to spinal cord injury are as high as billions, and thus research and treatment of spinal cord injury become hot spots for research in various countries around the world.

In recent years, regenerative medicine has a wide prospect in treating spinal cord injury, and has two major factors: one is seed cells and the other is biological materials. The selection of ideal biological materials is crucial for tissue engineering repair of spinal cord injury, and the materials currently used in the field of spinal cord injury research include natural biological materials (represented by collagen, gelatin, alginate, etc.), degradable polymer synthetic materials (represented by poly-D, L-lactic acid (PLLA)), and acellular materials (represented by acellular sciatic nerve, acellular optic nerve, and acellular spinal cord). It is believed that the in situ decellularization of organs is used to repair the damaged organs, which most conform to the original structure and extracellular matrix components of the organs, and a great deal of decellularized materials are currently used in the research of organs, such as blood vessels, bones, lungs, heart, skin and peripheral nerves. Similarly, the acellular spinal cord (DSC) can be used as a scaffold for spinal cord tissue engineering to treat spinal cord injury. Although studies have shown that DSC implanted into the spinal cord after spinal cord injury shows some structural or functional improvement, a significant obstacle to such scaffolds is their poor mechanical properties. In fact, the elastic modulus of fresh DSC is only 0.564MPa, and the shape of the stent cannot be well maintained due to softness and poor mechanical properties. The elastic modulus is improved to 1.541MPa after being modified by a crosslinking method (such as genipin crosslinking), but is only half of the elastic modulus of normal grey and white spinal cord matter, and the mechanical strength is difficult to overcome the tissue shrinkage caused by invasive fibrous scar after traumatic SCI. Thus, implantation of a DSC only within the spinal cord results in stent collapse and significant fibrosis at the implantation site. Therefore, the development of the acellular spinal cord stent with excellent mechanical properties is very important.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a mechanical property enhanced acellular spinal cord biomaterial scaffold, which comprises an inner acellular spinal cord (DSC) and a poly (D, L-lactic-co-glycolic acid, PLGA) shell surrounding the periphery of the acellular spinal cord, and the scaffold is prepared by spraying PLGA (polylactic-co-glycolic acid, polylactic-glycolic acid) to the periphery of the DSC for electrostatic spinning to form a thin shell.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a mechanical property enhanced acellular spinal cord biomaterial scaffold, which comprises an inner acellular spinal cord and a PLGA shell surrounding the periphery of the acellular spinal cord.

Preferably, the PLGA outer shell is sprayed on the periphery of the decellularized spinal cord by electrospinning, and forms a compact integrated structure.

According to the invention, the PLGA shell is added, so that the mechanical property of the whole biological scaffold is enhanced, the shape of the acellular spinal cord scaffold is maintained, the shell and the internal material can be better and firmer connected by the electrospinning method, the internal scaffold is not easy to slip, the enhancement of the mechanical property can prevent the scaffold from collapsing after being transplanted to the spinal cord injury, and the scaffold can be used as a novel biological material for effectively treating the spinal cord injury.

The invention also provides application of the mechanical property enhanced acellular spinal cord biomaterial scaffold in preparation of a product for repairing spinal cord injury.

The invention also provides a functional product for repairing spinal cord injury, which comprises the mechanical property enhanced acellular spinal cord biomaterial scaffold.

The invention also provides a preparation method of the mechanical property enhanced acellular spinal cord biomaterial scaffold, which comprises the following steps:

s1, preparing a decellularized spinal cord material by adopting an extraction method, wherein the decellularized spinal cord material removes cells and main inhibitory components and reserves other extracellular matrixes of the central nervous system;

s2, preparing a PLGA solution, and then electro-spinning a PLGA shell outside the decellularized spinal cord material to obtain the mechanical property enhanced decellularized spinal cord biomaterial scaffold.

PLGA has good biocompatibility, belongs to degradable high molecular biological materials, can be electrospun into a required shape, and has good mechanical properties. Therefore, the invention adopts the electrospinning technology to electrically spin a PLGA shell outside the Decellularized Spinal Cord (DSC) to prepare the mechanical property enhanced decellularized spinal cord biomaterial scaffold (PLGA-DSC), the PLGA shell of the PLGA-DSC enhances the overall mechanical property of the biomaterial and improves the pressure resistance of the material, compared with the DSC material, the stress of the PLGA-DSC is increased, the compression modulus is greatly improved, and the compression modulus is improved by about 10 times; the mechanical reinforcement of the PLGA shell obviously enhances the capability of PLGA-DSC in resisting the invasion of alpha-SMA positive myofibroblasts (the cells are main tension generating cells in a fibrous scar), prevents the collapse of the transplanted material and the compression of surrounding tissues, thereby maintaining the neurogenic ecological environment and being beneficial to the targeted migration and residence of endogenous neural stem cells after SCI. Because the DSC in the interior removes cells and main inhibitory components and retains other extracellular matrixes of the central nervous system, the damage of the central nervous system can be better repaired, the problem of poor mechanical properties of the decellularized spinal cord is solved, and the in-situ repair of the spinal cord damage is further promoted.

Preferably, the rotational speed of electrospinning is 300rpm, the X-axis moving speed is 5.3mm/s, the flow rate of a syringe pump is 1mL/h, the voltage is 21.86kV, and the operation is not less than 40 minutes.

Preferably, the mass concentration percentage of the PLGA solution is 8-15%. The molecular weight of the PLGA was 10,0000.

Preferably, the manufacturing of the acellular spinal cord material comprises the following steps:

s11, obtaining a fresh spinal cord, cutting the spinal cord into small sections, soaking the small sections in an antibiotic solution, and standing the small sections at minus 80 ℃ overnight;

s12, taking out the tissue from-80 ℃, still soaking the tissue in an antibiotic solution, and shaking until a large number of histiocytes are separated out;

s13, transferring the mixture to an aqueous solution containing 1% double antibody, and shaking for 5-7 h;

s14, transferring the mixture into an aqueous solution containing 3% Triton-X-100 and 1% double antibody, and shaking for 10-14 h;

s15, taking out, washing, soaking in a 4% sodium deoxycholate aqueous solution, and shaking for 12-36 h;

s16, repeating the step S15;

s17, taking out, washing, soaking in an enzyme aqueous solution, and shaking at a constant temperature of 37 ℃ for 1-3 h;

s18, taking out, washing, soaking in water, freezing overnight, and freeze-drying to obtain the acellular spinal cord material.

Further, the antibiotic solution of steps S11 and S12 contains 2% (mass concentration percentage) of double antibody, 10. mu.g/mL gentamicin and 2.5. mu.g/mL amphotericin B. Specifically, the double antibody is penicillin and streptomycin.

Further, the aqueous enzyme solution in step S17 contains 50U/mL DNase, 1KU/mL RNase, 50mM Tris and 10mM MgCl₂·6H₂O。

Further, step S11 is to cut the spinal cord into small segments of 1-2cm in length.

Further, the oscillation time of step S13 is 6h, the oscillation time of step S14 is 12h, the oscillation time of step S15 is 24h, and the oscillation time of step S17 is 2 h.

Further, the washing described in steps S15-S17 is 3 times of washing with water, each time for 10 min.

Further, the washing in step S18 is performed by washing with water 3 times for 20min each time.

Further, the temperature of freezing overnight in step S18 was-80 ℃.

Further, the water used in the present invention is sterile ultrapure water.

Further, when the acellular spinal cord material is manufactured, the rotating speed for shaking is 150 r/min.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a mechanical property enhanced acellular spinal cord biomaterial scaffold which is prepared by electrospinning a PLGA shell outside an acellular spinal cord (DSC) by an electrospinning technology, namely, spraying PLGA electrospinning on the periphery of the DSC to form a thin shell as the outer layer of a soft DSC. Compared with a pure DSC bracket, the biomaterial bracket has enhanced mechanical property and better pressure resistance, and can prevent the material from collapsing and being pressed by surrounding tissues after being transplanted; researches show that the spinal cord injury can be effectively repaired by transplanting the biomaterial scaffold to a rat full-transection spinal cord injury area, so that the problem of poor mechanical property of the decellularized spinal cord is solved, and the in-situ repair of the spinal cord injury is further promoted.

Drawings

FIG. 1 is a schematic structural diagram of a PLGA-DSC scaffold;

FIG. 2 is a flow chart and SEM images of PLGA-DSC stent;

in FIG. 2, A is a manufacturing flow chart (showing the structure and overall design of a PLGA-DSC scaffold, i.e. an adult rat spinal cord is decellularized and then lyophilized to obtain a DSC material, and then the obtained DSC is spun with a PLGA shell on the periphery by an electrospinning method to obtain a PLGA-DSC scaffold); b is a three-dimensional view of the PLGA-DSC stent (front view and side view; D in the figure is PLGA shell, electrostatic spinning interwoven into a network at high power, E is acellular spinal cord, reticular matrix at high power, F is a junction area of the two, and the interwoven wires are tightly connected together); c is an SEM image of the overall shape of the PLGA-DSC stent; d is an SEM image of the electrospun PLGA nano fiber; e is SEM picture of the extracellular matrix network of DSC decellularized; and F is an SEM image of the binding site of the electrospun PLGA shell and the DSC matrix.

FIG. 3 is a mechanical property test chart of PLGA-DSC stent;

in FIG. 3, A is the stress-strain curve of the PLGA-DSC stent (a) and DSC stent (b) in response to compression; b is a histogram of PLGA-DSC scaffold and DSC scaffold compressive modulus values (n-5, p < 0.05).

FIG. 4 is a graph showing the detection of infiltration of surrounding connective tissue into a PLGA-DSC scaffold 7 days after the implantation of the scaffold into a spinal cord injured rat;

in fig. 4, a, B represent H & E staining patterns of the transplanted area 7 days after PLGA-DSC scaffolds and DSC scaffolds were implanted into the injured spinal cord rat, respectively (boxes show the approximate location of the scaffold in the injured/implanted area, double arrows indicate the longitudinal axis of the scaffold, the longitudinal axis of the PLGA-DSC scaffold is "parallel" to the host spinal cord, while the implanted DSC scaffold is squeezed into the center of the injured/implanted area and its longitudinal axis forms an angle with the longitudinal axis of the host spinal cord); c is an H & E stained image of PLGA skin under high power microscopy (PLGA skin in dotted box with asterisk, showing that PLGA thin shell remains on the outer layer of the scaffold, while meningeal derived fibroblasts are outside the PLGA thin shell); d is an H & E stained image of the DSC stent implantation area under a high power microscope (showing that a large number of fibroblasts gather under the meninges and migrate to the injury area, the migration trace is visible [ arrow ]); e is an immunofluorescence staining pattern of PLGA outer shell and surrounding alpha-SMA expression (PLGA outer shell in the dotted line frame with asterisk, indicating that cells outside the PLGA outer shell strongly express alpha-SMA [ arrow ], while the inner side of the PLGA outer shell lacks alpha-SMA positive cells); f is an immunofluorescent staining pattern of alpha-SMA expression in the region of DSC scaffold implantation (showing that a large number of alpha-SMA positives were present around the cell scaffold and inside the damaged region [ indicated by arrows ]). The scale bar in (A and B) is 1mm, and the scale bar in (C-F) is 40 μm.

FIG. 5 is a graph showing the polarization and cavitation of immune cells in the injured area 8 weeks after PLGA-DSC stent implantation into spinal cord injured rats.

In FIG. 5, A, B are CCR7 and CD68 immunostaining patterns (labeled M1 type macrophages/microglia; wherein A1-A3 and B1-B3 are high magnification images of boxed areas in A and B, respectively, showing cells doubly labeled with CCR7 and CD68 [ indicated by arrows ]) for PLGA-DSC and control groups, respectively; c, D are CD206 and CD68 immunostaining profiles (labeled M2 type macrophages/microglia; where C1-C3 and D1-D3 are high magnification images of the boxed area in C and D, respectively, showing CD206 and CD68 double labeled cells [ indicated by arrows ]) for the PLGA-DSC group and the control group, respectively; e is a bar graph of CCR7/CD68 and CD206/CD68 (× P <0.05[ n ═ 5 ]); f is an H & E staining chart showing the cavity condition of the spinal cord injury area of the PLGA-DSC group and the control group; g is a statistical plot of the void area (. P <0.05[ n-5 ]). (a, B and F) medium-scale 500 μm, (a1-A3, B1-B3, C1-C3 and D1-D3) medium-scale 20 μm.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 preparation of mechanical property enhanced acellular spinal cord biomaterial scaffold and application thereof in rats with complete spinal cord transection

According to the operation flow chart shown in fig. 2, the preparation of the acellular spinal cord biomaterial scaffold and the application thereof in rats with complete spinal cord transection are carried out, and the specific operation method is as follows:

1. animal information

Rat: SD rats, provided by the Experimental animals center of university of Zhongshan.

2. Main instruments and reagents

High-voltage electrostatic spinning instrument (Shenzhen Tonglian micronano technology, TL-01), electronic universal tester (UTM6104), frozen microtome (Thormo), fluorescence microscope (Leica), scanning electron microscope (FIE), PLGA powder (Sigma, molecular weight is 10,0000), 0.01M PBS (China fir bridge), Hoechst33342 dye (Sigma), goat serum (GIBCO), primary antibody (alpha-SMA, Abcam), secondary antibody (Alex-555, Invitrogen).

3. Preparation of acellular spinal cord material

(1) After the SD rat is anesthetized to die, the whole spinal cord is quickly taken out on ice, cut into small sections of 1-2cm, and soaked in an antibiotic solution (containing 2% double antibiotics [ penicillin and streptomycin in ratio of 1:1] +10 mug/mL gentamicin +2.5 mug/mL amphotericin B, solvent is sterile ultrapure water) for overnight in a refrigerator at-80 ℃;

(2) taking out from the refrigerator, still soaking in antibiotic solution, shaking the shaking table (250r/min) until a large amount of tissue cells are separated out.

(3) The cells were removed and transferred to sterile ultrapure water containing 1% diabesin (penicillin and streptomycin) and shaken on a shaker (250r/min) for 6h, changing every 2 h.

(4) Taking out, transferring to an aqueous solution containing 3% Triton-X-100 (polyethylene glycol octyl phenyl ether) + 1% double antibody (penicillin and streptomycin), and shaking for 12h by a shaking table (250 r/min);

(5) taking out, washing with sterile ultrapure water for 3 times, each for 10 min;

(6) taking out and soaking in 4% sodium deoxycholate water solution, shaking in a shaking table (250r/min) for 24 h;

(7) taking out, washing with sterile ultrapure water for 3 times, each for 10 min;

(8) repeating the step (6) to the step (7) for 1 time;

(9) the cells were immersed in an aqueous enzyme solution (DNase 50U/mL + RNase 1KU/mL + Tris50mM + MgCl)₂·6H₂O10mM), shaking for 2h in a constant temperature shaking table (150r/min) at 37 ℃;

(10) taking out, washing with sterile ultrapure water for 3 times, each for 20 min;

(11) and (3) freeze drying: the decellularized material is placed in water (sterile ultrapure water) which is just enough to be spread, and is frozen overnight in a refrigerator with the temperature of minus 80 ℃, and then is frozen and dried overnight in a freeze dryer, so that the decellularized spinal cord material (DSC) is prepared.

4. Electrospinning of PLGA shells onto decellularized spinal cord material

(1) Preparation of PLGA solution: weighing 1mg of PLGA powder, dissolving the PLGA powder in 10mL of HFIP (hexafluoroisopropanol), preparing a PLGA solution with the mass fraction of 10%, placing the PLGA solution in a small bottle, placing the small bottle on a magnetic stirrer, and stirring overnight to fully dissolve the PLGA solution;

(2) sucking the dissolved PLGA solution into a 2.5mL needle tube, standing for a while until bubbles disappear completely;

(3) and (3) penetrating the decellularized spinal cord material prepared in the step (3) through a thin iron wire, installing the iron wire at the position of the original roller of the high-voltage electrostatic spinning instrument, fixing the needle tube, and enabling the distance from the needle head to the iron wire to be 7 cm.

(4) The operation process of the high-voltage electrostatic spinning instrument comprises the following steps:

opening main switch, light in sequence; set rotation speed Drum (rotation speed): 300 rpm; set X-axiort sliding (X-axis moving speed): 5.3 mm/s; setting the flow rate of the injection pump: 1 mL/h; voltage: 21.86kV and then the run was started for 40 minutes.

(5) When the operation is finished, the injection pump, the voltage and the sliding table are closed, then the wire collecting device is closed, and the material is carefully taken out;

(6) and (3) placing the prepared material in a vacuum drying oven, vacuumizing, and vacuumizing twice (each time overnight) to obtain a mechanical property enhanced acellular spinal cord biomaterial scaffold (PLGA-DSC), wherein the structure of the scaffold is shown in figure 1.

5. Scanning Electron Microscope (SEM) inspection

The morphological properties of PLGA-DSC scaffolds were examined by SEM (FEI Quanta 200). To prepare the imaged samples, the scaffolds were adhered to a conductive tape, freeze-dried, and gold-coated thereon for 120s, and observed under a scanning electron microscope.

As shown in fig. 2, adult rat spinal cord is decellularized and then lyophilized to obtain a decellularized spinal cord material (DSC), and then the obtained DSC is spun with a PLGA shell on the periphery by an electrospinning method to obtain a PLGA-DSC scaffold, wherein the PLGA shell and the Decellularized Spinal Cord (DSC) form a compact integrated structure.

6. Detection of mechanical properties of materials

The DSC scaffold material with or without a PLGA thin shell (i.e. the PLGA-DSC scaffold and the DSC material prepared in step 3) was subjected to a compression mechanical property test on an electronic universal tester (UTM6104, Sunstest corp., china)), with a preload of 0.1N, a maximum compression strain of 10%, a compression rate of 1mm/min, and shown as a stress-strain curve on a computer screen, and the mechanical properties of the material (N-5) were analyzed. The compressive modulus can be obtained by calculating the slope of the linear region of the stress strain.

As shown in FIG. 3, as the strain increases, the stress ratio of PLGA-DSC increases, and the compressive modulus also increases greatly, by about 10 times. As can be seen, the mechanical property of the PLGA-DSC stent material is improved.

7. Transplanting the mechanical property enhanced acellular spinal cord biomaterial scaffold into a rat with full spinal cord transection

Rats were anesthetized by pre-operative intraperitoneal injection of sodium pentobarbital (0.064mg/10 g). After fixing body position and skin preparation and disinfection, incising skin and superficial fascia under aseptic condition, using instruments to perform blunt muscle and ligament separation along the processes of spinal muscles of waist and spine on two sides of spinous processes on T8-T10(T represents a segment of thoracic vertebra), fixing an operation area by using a self-made draw hook, clearly exposing T9 spinous process and vertebral arch, then using dental forceps to slightly lift T9 spinous process, using ophthalmic needle holding forceps to slightly bite the root of vertebral arch along the gap of the vertebral arch of T9-T10, and gradually biting the vertebral arch of T9 to expose T10 spinal cord. And (3) cutting off a dura mater by using a straight-pointed trabecular scissors, inserting a side cutter foot to the bottom, quickly and completely transecting the whole spinal cord, transecting the spinal cord again at a position 2mm away from the transection part of the head end, carefully taking out the middle spinal cord tissue by using micro-forceps to ensure complete transection, and after sufficient hemostasis, filling the mechanical-property-enhanced acellular spinal cord biomaterial scaffold (PLGA-DSC scaffold) constructed in the step (4) into a spinal cord tissue defect area (taking the DSC scaffold as a contrast), and suturing layer by layer according to the sequence of a muscle layer, a subcutaneous tissue and skin. After the operation, marking is carried out, each animal is injected with penicillin (16 ten thousand units) 1mL/d intramuscularly for 3 consecutive days, and manual urination is carried out by pressing the animal in the bladder area moderately and 1-2 times a day. In order to prevent the undeveloped wound from being bitten, the patient is fed in a single cage after operation. Thereafter, the number of urination is gradually reduced according to the recovery of bladder function, and the animals are kept for 7 days and tested for material collection after 2 months (8 weeks), during which time the animals are kept warm, naturally illuminated and fully fed.

7.1 detection of infiltration of connective tissue into the scaffolds around the acute phase (7 days)

After implantation of PLGA-DSC and DSC scaffolds at the site of spinal cord full-lateral injury (defect area 2 mm), infiltration of surrounding connective tissue into the scaffolds was assessed by histological (H & E staining and immunofluorescence staining) examination during the acute phase (7 days).

(1) H & E (hematoxylin and eosin) staining

The H & E staining comprises the following steps:

1) a3 cm rat spinal cord containing the lesion/graft area was horizontally sectioned with a cryomicrotome, 30 microns thick. The cut frozen sections were stored at-30 ℃. Before staining, the spinal cord tissue cryosections were rewarmed for 15 minutes at room temperature and then rinsed 3 times for 10 minutes each in 0.01 MPBS; 2) the sections were washed in distilled water for 1 minute and then stained in hematoxylin for 10 minutes; 3) the sample was washed with distilled water for 1 minute to remove the excess dye solution; 4) soaking the slices in 1% acidic alcohol (1% HCl with 70% alcohol) for 20 seconds; 5) the sample was washed clean with tap water for about 30 minutes until the color turned blue; 6) soaking the slices in 95% alcohol for 5 min; 7) soaking in 1% eosin solution for 1 min; 8) soaking the sample in 80% alcohol twice, each for 20 seconds; (9) samples were dehydrated with increasing concentrations of alcohol (90% alcohol for 20 seconds; 95% alcohol for 2 minutes; 100% alcohol twice for 5 minutes each); 10) the sections were twice cleared in xylene for 15 minutes; 11) finally, the sample was covered with a neutral resin, the sections were sealed with a cover slip, and the sample was observed under a microscope after drying.

(2) Immunofluorescence staining (fluorescent nuclear dye staining)

The immunofluorescence staining comprises the following steps:

1) a3 cm rat spinal cord containing the lesion/graft area was horizontally sectioned with a cryomicrotome, 30 microns thick. The cut frozen sections were stored at-30 ℃. Before staining, the spinal cord tissue frozen section is rewarmed for 15 minutes at room temperature and then washed with 0.01MPBS for 10 minutes; 2) blocking with 10% goat serum at 37 deg.C for 30 min; 3) adding 0.3% Triton X-100 diluted primary antibody (alpha-SMA, CD206, CD68, CCR7, etc.) to incubate at 4 ℃ overnight; 4) the next day, sections were washed three times with PBS for 10 minutes each; 5) incubation with secondary antibody at 37 ℃ for 1 hour; 6) counterstaining the nucleus with the nuclear dye Hochst 33342; 7) finally, the sections were mounted with mounting medium and coverslips and observed under a confocal microscope (Zeiss LSM800, Germany).

H & E staining as shown in fig. 4 shows that the PLGA thin shell remains on the outer layer of the scaffold and has not degraded, and the longitudinal axis of the PLGA-DSC scaffold is on the same parallel line as the longitudinal axis of the host spinal cord. No significant compression by surrounding tissue was observed in the lesion/implant area (as indicated by the arrows in fig. 4A). However, without the protective PLGA shell, the DSC scaffolds were squeezed into the central injured/implanted region, surrounded by a dense layer of basophil and eosinophil collagen rich regions, and the implanted DSCs collapsed deforming and lost their original orientation (fig. 4B). This indicates that the PLGA thin outer shell provides sufficient mechanical strength to withstand the pressure from the surrounding fibrotic scar. High power images (FIG. 4C/D) show thin layers of meningeal fibroblasts outside the PLGA coat, with a few cells migrating to the PLGA coat and penetrating this barrier. In contrast, the DSC group of the control material had a large amount of fibroblasts accumulated under the meninges and had a distinct migration trace in the center of the injured/implanted area. Immunostaining for α -SMA (a myofibroblast marker, the primary mediator of scars, detected by immunostaining against α -smooth muscle actin) showed strong α -SMA expression by cells outside the PLGA shell, in contrast to which only negligible α -SMA positive cells were observed inside the PLGA shell (i.e. inside the DSC matrix) (fig. 4E), demonstrating that the PLGA shell provides a resilient barrier for DSC to prevent fibrotic tissue invasion. However, in the control group, myofibroblasts directly infiltrated the DSC scaffolds if there was no PLGA barrier (as shown in figure 4F, positive immunostaining for α -SMA appeared within the scaffold). These results indicate that a thin PLGA shell can act as an effective barrier to isolation of meningeal fibroblast infiltration, and that fibrotic invasion of nerve regeneration in the microenvironment will be relatively minor.

7.2 detection of polarization and cavitation of immune cells in the Chronic (2 month) lesion

PLGA-DSC and DSC scaffolds were implanted into rat spinal cord total transection injury sites, and the polarization of immune cells in the injury area was assessed by CCR7 and CD68, as well as CD206 and CD68 immunofluorescence double labeling during the chronic phase (2 months), and the cavitation in the injury area was assessed by H & E staining. The H & E staining and immunofluorescence staining methods are the same as 7.1.

As the results of fig. 5 show, implantation of PLGA-DSC scaffolds induced polarization of macrophages/microglia from M1 type to M2 type (M1 type was considered as pro-inflammatory and M2 type was considered as anti-inflammatory in both states M1 and M2 macrophages/microglia), and less CCR7/CD68 double positive cells (M1 type) were observed in the PLGA-DSC group (fig. 5A, E) relative to the SCI group (fig. 5B, D and E); meanwhile, more CD206/CD68 double positive cells (M2 type, as shown in fig. 5C, E) were found. Whereas in the SCI group, M1 remains the major macrophage/microglia subtype, the M1 subtype (pro-inflammatory) exhibits polarization to the M2 subtype (anti-inflammatory). It was shown that PLGA-DSC scaffolds may help macrophages/microglia infiltrated into macrophages to become inflammasome, which favors the microenvironment of tissue repair after SCI compared to the inflammatory environment typical of chronic diseases. HE staining showed that implantation of PLGA-DSC scaffolds was beneficial to reduce the luminal area of the spinal cord injury zone, and that the spinal cord luminal area was significantly smaller in the PLGA-DSC group than in the SCI group (P <0.05, n ═ 5; fig. 5F, G). The above results demonstrate that PLGA-DSC stent implantation can reduce inflammation and reduce cavity formation after spinal cord injury.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims

1. A biomaterial scaffold for acellular spinal cord with enhanced mechanical properties is characterized by comprising an inner acellular spinal cord and a PLGA shell surrounding the outer periphery of the acellular spinal cord.

2. The mechanical-property-enhanced acellular spinal cord biomaterial scaffold according to claim 1, wherein the PLGA outer shell is sprayed on the periphery of the acellular spinal cord by electrospinning and forms a compact and integrated structure.

3. Use of the mechanical property enhanced acellular spinal cord biomaterial scaffold of claim 1 or 2 in the preparation of a product for repairing spinal cord injury.

4. A functional product for repairing spinal cord injury, comprising the mechanical property-enhanced acellular spinal cord biomaterial scaffold of claim 1 or 2.

5. The method for preparing the mechanical property enhanced acellular spinal cord biomaterial scaffold as claimed in claim 1 or 2, is characterized by comprising the following steps:

6. The preparation method according to claim 5, wherein the rotational speed of electrospinning is 300rpm, the X-axis moving speed is 5.3mm/s, the flow rate of an injection pump is 1mL/h, the voltage is 21.86kV, and the operation is not less than 40 minutes.

7. The method according to claim 5, wherein the PLGA solution is used in an amount of 8 to 15% by mass.

8. The method for preparing the cell-free spinal cord material according to claim 5, wherein the manufacturing of the cell-free spinal cord material comprises the steps of:

s16, repeating the step S15;

9. The method of claim 8, wherein the antibiotic solutions of steps S11 and S12 comprise 2% double antibody, 10 μ g/mL gentamicin, and 2.5 μ g/mL amphotericin B.

10. The method according to claim 8, wherein the aqueous solution of the enzyme in step S17 contains 50U/mL DNase, 1KU/mL RNase, 50mM Tris and 10mM MgCl₂·6H₂O。