TISSUE ENGINEERING MATERIAL FOR NERVE INJURY REPAIR, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

A tissue engineering material for nerve injury repair, a preparation method therefor and an application thereof. The tissue engineering material for nerve injury repair is an N-cadherin crosslinked linear ordered collagen scaffold. By crosslinking N-cadherin with a linear ordered collagen scaffold, the prepared tissue engineering material can efficiently induce migration of neural stem cells towards an injury region so that the neural stem cells are enriched in the injury region, and can effectively inhibit deposition of inhibitory factors such as chondroitin sulfate proteoglycan, promote differentiation of the neural stem cells into neurons, and then promote recovery of electrophysiological and motion functions. The N-cadherin crosslinked linear ordered collagen scaffold also has a stable ordered topological structure and excellent mechanical properties, and can be used to repair nerve injuries such as spinal cord injury.

Claims

1. A tissue engineering material for nerve injury repair, wherein, the tissue engineering material for nerve injury repair is a N-cadherin crosslinked biodegradable material; the biodegradable material comprises one selected from collagen and gelatin.

2. The tissue engineering material of claim 1, wherein the biodegradable material is collagen; preferably a linear ordered collagen scaffold; more preferably, a ratio of the linear ordered collagen scaffold to the N-cadherin is one linear ordered collagen scaffold having a diameter of 0.25 mm and a length of 4 cm: (1-6) μg of N-cadherin.

3. The tissue engineering material according to claim 1, wherein the N-cadherin is covalently crosslinked with the linear ordered collagen scaffold; preferably, the crosslinking agent for covalent crosslinking is Traut's reagent and Sulfo-SMCC.

4. A preparation method for a tissue engineering material for nerve injury repair, characterized by comprising: crosslinking N-cadherin with a biodegradable material covalently to obtain a N-cadherin crosslinked biodegradable material; the biodegradable material comprises one selected from collagen and gelatin; preferably, the biodegradable material is collagen; more preferably a linear ordered collagen scaffold.

5. The preparation method according to claim 4, wherein a ratio of the linear ordered collagen scaffold to N-cadherin is one linear ordered collagen scaffold having 0.25 mm in diameter and 4 cm in length: (1-6) μg of N-cadherin; preferably, a ratio of the linear ordered collagen scaffold to N-cadherin is one linear ordered collagen scaffold having a diameter of 0.25 mm and a length of 4 cm: (2.5-5) μg of N-cadherin.

6. The preparation method according to claim 4 or 5, characterized by comprising the following steps: (1) treating the linear ordered collagen scaffold with Traut's reagent to obtain a linear ordered collagen scaffold modified by Traut's reagent; and (2) treating the linear ordered collagen scaffold modified by Traut's reagent with Sulfo-SMCC and N-cadherin.

7. The preparation method according to claim 6, wherein a ratio of the linear ordered collagen scaffold to the Traut's reagent is one linear ordered collagen scaffold having a diameter of 0.25 mm and a length of 4 cm: (0.05-0.15) mg of Traut's reagent; a molar ratio of the N-cadherin to the Sulfo-SMCC is 1:(60-100); preferably, a concentration of the Traut's reagent is 0.2 mg/mL to 5 mg/mL.

8. The preparation method according to claim 6, wherein the treatment time in step (1) is 1 h to 3 h; and the treatment time in the step (2) is 1 h to 3 h; preferably, both step (1) and step (2) are carried out under the condition of 18° C. to 25° C.

9. A method for preparing a material for nerve injury repair, comprising using a tissue engineering material for nerve injury repair, wherein, the tissue engineering material for nerve injury repair is a N-cadherin crosslinked biodegradable material; the biodegradable material comprises one selected from collagen and gelatin; preferably, the nerve injury is a spinal cord nerve injury.

10. A method for promoting directed migration of neural stem cells or promoting differentiation of neural stem cells into neurons, comprising using a tissue engineering material for nerve injury repair, wherein, the tissue engineering material for nerve injury repair is a N-cadherin crosslinked biodegradable material; the biodegradable material comprises one selected from collagen and gelatin.

11. The tissue engineering material according to claim 2, wherein the N-cadherin is covalently crosslinked with the linear ordered collagen scaffold; preferably, the crosslinking agent for covalent crosslinking is Traut's reagent and Sulfo-SMCC.

12. The preparation method according to claim 5, characterized by comprising the following steps: (1) treating the linear ordered collagen scaffold with Traut's reagent to obtain a linear ordered collagen scaffold modified by Traut's reagent; and (2) treating the linear ordered collagen scaffold modified by Traut's reagent with Sulfo-SMCC and N-cadherin.

13. The preparation method according to claim 12, wherein a ratio of the linear ordered collagen scaffold to the Traut's reagent is one linear ordered collagen scaffold having a diameter of 0.25 mm and a length of 4 cm: (0.05-0.15) mg of Traut's reagent; a molar ratio of the N-cadherin to the Sulfo-SMCC is 1:(60-100); preferably, a concentration of the Traut's reagent is 0.2 mg/mL to 5 mg/mL.

14. The preparation method according to claim 7, wherein the treatment time in step (1) is 1 h to 3 h; and the treatment time in the step (2) is 1 h to 3 h; preferably, both step (1) and step (2) are carried out under the condition of 18° C. to 25° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows the preparation and performance characterization of LOCS-Ncad in Experimental Examples 1 and 2 of the present invention. (A) shows a schematic diagram of two-step crosslinking method for the preparation of LOCS-Ncad; (B) shows the concentration of sulfhydryl groups after treatment with different concentration of Traut's reagent detected by Ellman's reaction; (C) shows the concentration of sulfhydryl groups in untreated LOCS (LOCS), LOCS after treatment with 5 mg/mL of Traut's reagent (Thiolation LOCS), and after further reaction with Sulfo-SMCC/N-cadherin (LOCS-Ncad); (D) shows the binding amounts of N-cadherin of crosslinking treatment (LOCS-Ncad) and natural adsorption (LOCS/Ncad) detected by ELISA; (E) shows the linear ordered structure of LOCS-Ncad confirmed by scanning electron microscopy; (F) shows stress-strain curves of LOCS-Ncad and LOCS; (G) shows Young's modulus of LOCS-Ncad and LOCS; and (H) shows morphology of LOCS-Ncad and LOCS after immersion in normal saline for 30 min; * represents p<0.05, and ** represents p<0.01.

[0034] FIG. 2 shows that a two-dimensional simulation material of LOCS-Ncad promotes the adhesion and migration of neural stem cells in Experimental Example 3 of the present invention. (A1) to (A4) show that immunofluorescence staining confirms the expression of N-cadherin in neural stem cells, Hoechst represents Hoechst staining, Nestin represents Nestin protein, and N-cadherin represents neural cadherin; (B1) to (B4) show the adhesion of neural stem cells on different modified surfaces, Col-Ncad represents a two-dimensional simulation material of LOCS-Ncad, Col-BSA represents a two-dimensional simulation material of LOCS-BSA, and Col represents a collagen gel; (C) shows schematic diagram of migration experiment design; and (D1) to (D6) show migration experiment results; * represents p<0.05, and ** represents p<0.01.

[0035] FIG. 3 shows that a two-dimensional simulation material of LOCS-Ncad promotes differentiation of neural stem cells into neurons in Experimental Example 3 of the present invention. (A1) to (A4) show the proportion of Tuj-1 (a neuron marker) positive cells determined by immunofluorescence staining after neural stem cells differentiated on the surfaces of two-dimensional simulation material of LOCS-Ncad (Col-Ncad), two-dimensional simulation material of LOCS-BSA (Col-BSA) and two-dimensional simulation material of LOCS (Col) for 7 days, respectively; (B1) to (B4) show the proportion of GFAP (an astrocyte marker) positive cells determined by immunofluorescence staining after neural stem cells differentiate on the surface of two-dimensional simulation material of LOCS-Ncad (Col-Ncad), two-dimensional simulation material of LOCS-BSA (Col-BSA) and two-dimensional simulation material of LOCS (Col) for 7 days, respectively; and (C) shows changes in expression of Tuj-1 and GFAP in neural stem cells on the surfaces of two-dimensional simulation material of LOCS-Ncad (Col-Ncad), two-dimensional simulation material of LOCS-BSA (Col-BSA) and two-dimensional simulation material of LOCS (Col) detected by qPCR; * represents p<0.05, and ** represents p<0.01.

[0036] FIG. 4 shows that LOCS-Ncad promotes the migration of endogenous neural stem cells to the center of an injury region in a spinal cord injury model in Experimental Example 4 of the present invention. (A1) to (A13) show migration of endogenous neural stem cells on LOCS-Ncad and LOCS scaffolds after 10 days of complete spinal cord transection in rats; (B1) to (B13) show the proportion of endogenous neural stem cells differentiated into neuron cells on different scaffolds after 10 days of complete spinal cord transection in rats; (C1) to (C13) show the proportion of endogenous neural stem cells differentiated into neuron cells on LOCS-Ncad and LOCS scaffolds after 30 days of complete spinal cord transection in rats; Control represents the group without any transplantation treatment; Rostral represents the headend portion of the graft material, Center represents the central portion of the graft material, and Caudal represents the caudal portion of the graft material. * represents p<0.05, and ** represents p<0.01.

[0037] FIG. 5 shows that LOCS-Ncad inhibits the deposition of chondroitin sulfate proteoglycans (CSPGs) in the spinal cord injury model in Experimental Example 4 of the present invention; wherein A, B and C show the CSPGs deposition on LOCS-Ncad, LOCS and the control group after 30 days of complete spinal cord transection in rats, respectively; D shows an enlarged detail view of CSPGs deposition in the control group; and E shows the statistical result of the CSPGs deposition.

[0038] FIG. 6 shows that the transplantation of LOCS-Ncad promotes electrophysiological and motor functions recovery in rats with complete transection spinal cord injury in Experimental Example 5 of the present invention. (A) shows the electrophysiological representative curves of LOCS-Ncad and LOCS scaffold transplantation groups, control group (Control) without any transplantation treatment, and normal group (Normal) 30 days after complete spinal cord transection in rats; (B) shows the changes in the latency after the transplantation of LOCS-Ncad and LOCS scaffold 30 days after complete spinal cord transection in rats; (C) shows the BBB score results of rats after the transplantation of LOCS-Ncad and LOCS scaffolds 30 days after complete spinal cord transection; and (D) shows the results of the oblique plate test in rats after the transplantation of LOCS-Ncad and LOCS scaffolds 30 days after complete spinal cord transection; * represents p<0.05, and ** represents p<0.01.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0039] Preferred embodiments of the present invention will be described in detail below with reference to Examples. It is to be understood that the following Examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Various modifications and substitutions can be made to the present invention by a person skilled in the art without departing from the purpose and the spirit of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following Examples are conventional methods.

[0041] Unless otherwise specified, the materials, reagents and the like used in the following examples are commercially available, wherein Traut's reagent was purchased from Sigma corporation under Art. No. 16256; N-cadherin recombinant protein was purchased from R&D Co., Ltd., under Art. No. 6626-NC-050; and Sulfo-SMCC was purchased from Sigma corporation under Art. No. M6035.

[0042] In the following Examples, the linear ordered collagen scaffold was prepared as follows:

[0043] (1) the isolated bovine fascia tissue was taken, and muscle and fat were removed;

[0044] (2) the resultant was immersed in acetone, stood at 16° C. for 24 hours, and then placed in deionized water for immersion cleaning for three times (2 hours each time);

[0045] (3) the resultant was immersed in 0.2% by mass sodium lauryl sulfate aqueous solution, stood at 16° C. for 24 hours, and then placed in deionized water for immersion cleaning for 3 times (2 hours each time);

[0046] (4) the resultant was immersed in 1% by mass TritonX-100 aqueous solution, stood at 16° C. for 24 hours, and then placed in deionized water for immersion cleaning for 3 times (2 hours each time);

[0047] (5) the resultant was immersed in 0.25% by mass trypsin aqueous solution, stood at 37° C. for 30 minutes, and then placed in deionized water for immersion cleaning for 3 times (2 hours each time);

[0048] (6) the resultant was subjected to freeze-drying (at a vacuum degree of 0.1 Mpa for 48 hours) to obtain a collagen scaffold.

Example 1: Preparation of N-Cadherin Crosslinked Linear Ordered Collagen Scaffolds (1)

[0049] The present invention provides an N-cadherin crosslinked LOCS (LOCS-Ncad), which is obtained by crosslinking N-cadherin to LOCS by using crosslinking agents Traut's and Sulfo-SMCC.

[0050] The preparation method of the above-mentioned N-cadherin crosslinked LOCS was specifically as follows (a schematic diagram of the reaction principle of crosslinking N-cadherin on a linear ordered collagen scaffold is shown in FIG. 1A):

[0051] (1) a bunch of LOCS (the size of a bunch of LOCS is 4 cm in length and 4 mm in diameter, and the size of each LOCS is 4 cm in length and 0.25 mm in diameter) was immersed in 300 μL of Traut's reagent (PBS, pH=8, 3 mM EDTA) with a concentration of 5 mg/mL at room temperature for 2 h; after immersing, the resultant was subjected to immersion cleaning with PBS for 3 times, 5 minutes each time, to obtain a LOCS modified by Traut's reagent; and

[0052] (2) the LOCS modified by Traut's reagent obtained in step (1) was immersed in 200 μL of PBS solution (PBS, pH=7.2, 3 mM EDTA) containing N-cadherin recombinant protein and Sulfo-SMCC (in which the final concentration of the N-cadherin recombinant protein was 200 μg/mL, and the final concentration of Sulfo-SMCC was 1.6 mg/mL) for 2 h at room temperature to obtain an N-cadherin crosslinked LOCS.

Example 2: Preparation of N-Cadherin Crosslinked Linear Ordered Collagen Scaffolds (2)

[0053] (1) a bunch of LOCS (the size of a bunch of LOCS is 4 cm in length and 4 mm in diameter, and the size of each LOCS is 4 cm in length and 0.25 mm in diameter) was immersed in 300 μL of Traut's reagent (PBS, pH=8, 3 mM EDTA) with a concentration of 5 mg/mL at room temperature for 2 h; after immersing, the resultant was subjected to immersion cleaning with PBS for 3 times, 5 minutes each time, to obtain a LOCS modified by Traut's reagent; and

[0054] (2) the LOCS modified by Traut's reagent obtained in step (1) was immersed in 200 μL of PBS solution (PBS, pH=7.2, 3 mM EDTA) containing N-cadherin recombinant protein and Sulfo-SMCC (in which the final concentration of the N-cadherin recombinant protein was 400 μg/mL, and the final concentration of Sulfo-SMCC was 3.2 mg/mL) for 2 h at room temperature to obtain an N-cadherin crosslinked LOCS.

Comparative Example 1: Preparation of BSA Crosslinked Linear Ordered Collagen Scaffold

[0055] The present Comparative Example provides a BSA (bovine serum albumin) crosslinked LOCS, and the present Comparative Example is the same as Example 1 expected that the N-cadherin recombinant protein was replaced with BSA.

Experimental Example 1: Effect of Amount of Traut's Reagent and N-Cadherin Recombinant Protein on Crosslinking Efficiency

[0056] Based on the principle of crosslinking N-cadherin to LOCS by crosslinking agents Traut's reagent and Sulfo-SMCC, the crosslinking of N-cadherin was analyzed through the detection of sulfhydryl group concentration, which is specified as follows:

[0057] In the preparation process of the N-cadherin crosslinked linear ordered collagen scaffold, a bundle of LOCS (the size of which is 4 cm in length and 4 mm in diameter) was immersed in 300 μL of Traut's reagent (PBS, pH=8, 3 mM EDTA) with a concentration of 0, 1, 2.5, 5, 7.5 and 10 mg/mL at room temperature for 2 h respectively, to obtain a LOCS diluted with Traut's reagent. The concentration of sulfhydryl group after treatment with different concentration of Traut's reagent were detected, and the results are shown in FIG. 1B. The results show that the concentration of sulfhydryl group in LOCS (Example 1 and Example 2) treated with 5 mg/mL of Traut's reagent reaches the highest.

[0058] At the same time, the concentration of sulfhydryl group after treatment with Sulfo-SMCC and N-cadherin was detected, and the results are shown in FIG. 1C. The results show that compared with LOCS modified by Traut's reagent, the concentration of sulfhydryl group of N-cadherin crosslinked LOCS is significantly reduced, demonstrating the effectiveness of the crosslinking method.

[0059] The crosslinking amounts of N-cadherin when different amounts of N-cadherin were added (a volume of 200 μL, and concentrations of 12.5, 25, 50, 100, 200 and 400 m/mL, respectively) were detected by ELISA method, and the natural adsorption group with no cross linking agent added and only LOCS and N-cadherin was used as a control, the results are shown as D in FIG. 1. The results showed that the crosslinking amount of N-cadherin reached the highest when 200 m/mL of N-cadherin was added, and it was significantly higher than that in the natural adsorption group. It further demonstrates the effectiveness of the crosslinking methods provided in Examples 1 and 2.

Experimental Example 2: Performance Test of LOCS-Ncad

[0060] The structure of LOCS-Ncad prepared in Example 1 was detected by a scanning electron microscope, the result is shown in E of FIG. 1, and the ordered topology structure of LOCS-Ncad can be seen.

[0061] Further, the mechanical properties of LOCS-Ncad prepared in Example 1 were tested, and the stress-strain curves of which were shown in F of FIG. 1 and the Young's modulus was shown in G of FIG. 1. The results showed that compared with LOCS that no N-cadherin is crosslinked, the tensile modulus of LOCS-Ncad was significantly improved. The LOCS-Ncad was immersed in normal saline for 30 min to observe its morphological changes, and the results are shown as H in FIG. 1, after being immersed in normal saline for 30 min, LOCS-Ncad still maintained a linear state without bending.

Experimental Example 3: Adhesion, Migration and Differentiation into Neurons of Neural Stem Cells In Vitro Promoted by LOCS-Ncad

[0062] The rat neural stem cells were cultured in three-dimensional culture method in vitro, and the results of the immunofluorescence experiment showed that the neural stem cells highly expressed N-cadherin, which provided a basis for the adhesion of LOCS-Ncad to the neural stem cells (A1 to A4 in FIG. 2).

[0063] In order to facilitate clear imaging and prove that the collagen materials modified with N-cadherin could increase adhesion to neural stem cells and promote their migration and differentiation into neurons at the same time, a two-dimensional simulation material (Col-Ncad) of LOCS-Ncad was prepared, and the preparation method is as follows: the rat tail collagen tissues of adult rats were peeled off, and dissolved with 0.5 M acetic acid for 72 h, the resultant was centrifuged to obtain the supernatant, a 48-well plate was coated with the obtained supernatant at 200 μL per well, after overnight at 4° C., the supernatant was aspirated and air-drying was performed (a Col obtained after air-drying was used as a control of the two-dimensional simulation material without crosslinking any substance). After air-drying, two-step crosslinking treatment was continuously performed using the preparation method of the crosslinked N-cadherin linear ordered collagen scaffold of Example 1 to obtain Col-Ncad. The preparation method of Col-BSA is the same as that of Col-Ncad.

[0064] After culturing the neural stem cells into spheres for 7 days, the resultant was digested into single cells, the cells were inoculated on the surface of Col-Ncad at a density of 5×10.sup.5/mL, and subjected to FDA staining after standing at 37° C. for 2 hours, with BSA-crosslinked LOCS (Col-BSA) of Comparative Example 1 and Col without crosslinking any substance as controls. The statistical results of the FDA staining experiment showed that the number of neural stem cells adhered to the surface of LOCS-Ncad was significantly more than that of the controls (B1 to B4 in FIG. 2).

[0065] Cell migration experiment was performed according to the schematic diagram shown in C of FIG. 2, neural stem cells were obliquely inoculated on the side of crosslinked BSA, observation was performed at different time points after horizontal placement, and it is found that more neural stem cells migrate to the Col-Ncad side (D1 to D6 in FIG. 2).

[0066] After 7 days' differentiation of neural stem cells on the surface of Col-Ncad, immunofluorescence staining was used to determine the proportion of Tuj-1 (a marker of neuron) and GFAP (an astrocyte marker) positive cells. Further, the changes in the expression levels of Tuj-1 and GFAP in neural stem cells on the surface of Col-Ncad were detected using qPCR. The results are shown in FIG. 3, and the results show that the proportion of Tuj-1 positive cells on the surface of Col-Ncad is significantly higher than that of Col-BSA and uncrosslinked Col, while the proportion of GFAP positive cells is significantly lower than that of Col-BSA and uncrosslinked Col, proving that Col-Ncad can efficiently promote the differentiation of neural stem cells into neurons.

Experimental Example 4: Migration of Neural Stem Cells and their Differentiation into Neurons In Vivo Promoted by LOCS-Ncad

[0067] LOCS-Ncad prepared in Example 1 was transplanted into a rat model of complete transection spinal cord injury, and the effects of LOCS-Ncad on promoting the migration of neural stem cells and their differentiation into neurons were analyzed in vivo.

[0068] Sample slices were taken 10 days after injury for immunofluorescence staining, and the results showed that as compared with the LOCS transplantation group and control group without any treatment, more Nestin.sup.+ neural stem cells migrated to the center of the injury region (A1 to A13 of FIG. 4), and more newborn neurons appeared in the central region (B1 to B13 of FIG. 4) at the same time. The result of staining at 30 days post-injury showed that as compared with the LOCS transplantation group and the control group without any treatment, more newborn neurons appeared in the center of the injury region (C1 to C13 in FIG. 4), and LOCS-Ncad significantly inhibited the deposition of chondroitin sulfate proteoglycan (CSPG) (FIG. 5).

Experimental Example 5: Recovery of Electrophysiological and Motor Functions in Rats with Complete Transected Spinal Cord Injury Promoted by LOCS-Ncad

[0069] LOCS-Ncad prepared in Example 1 was transplanted into the rat model of complete transected spinal cord injury to analyze the effects of LOCS-Ncad transplantation on the recovery of electrophysiological and motor functions in rats with complete transected spinal cord injury.

[0070] The electrophysiological test of cortical evoked potential was performed 30 days after injury. The results showed that as compared with the LOCS transplantation group and control group without any treatment, the latency of the LOCS-Ncad transplantation group was significantly reduced (A and B in FIG. 6). BBB score and oblique plate test were conducted once a week, and the test data of the LOCS-Ncad transplantation group was also significantly higher than that of the control groups (C and D in FIG. 6).

[0071] The LOCS-Ncad prepared in Example 2 was also subjected to the experimental verification of Experimental Examples 2 to 5, and the results showed that the LOCS-Ncad prepared in Example 2 can achieve substantially the same performance and effect as the LOCS-Ncad prepared in Example 1.

[0072] Although the general description, specific embodiments and experiments have been used to describe the present invention in detail above, some modifications or improvements can be made on the basis of the present invention, which is obvious to a person skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

INDUSTRIAL APPLICABILITY

[0073] The present invention provides a tissue engineering material for nerve injury repair, a preparation method and an application thereof. The tissue engineering material for nerve injury repair provided by the present invention is an N-cadherin crosslinked linear ordered collagen scaffold. The tissue engineering material prepared by crosslinking N-cadherin with a linear ordered collagen scaffold can efficiently induce the migration of the neural stem cells towards an injury region, so that the neural stem cells are enriched in the injury region, and can effectively inhibit the deposition of inhibitory factors such as chondroitin sulfate proteoglycan, promote the differentiation of the neural stem cells into neurons, and further promote the recovery of electrophysiological and motor functions. At the same time, the N-cadherin crosslinked linear ordered collagen scaffold also has a stable and ordered topology structure and excellent mechanical properties, can be used to repair nerve injuries such as spinal cord injury, and has good economic value and application prospect.