Patent Application
20220331496
The present application claims priority to Chinese Patent Application No. 201911312837.9, entitled “Tissue engineering material for nerve injury repair, preparation method therefor and application thereof”, and filed on Dec. 18, 2019, the entire disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to the technical field of tissue engineering, specifically, to a N-cadherin crosslinked biodegradable material for nerve injury repair and a preparation method therefor and application thereof.
Spinal cord, as an important part of the central nervous system, is the conduction pathway between the brain and peripheral nervous system. Spinal cord injury can lead to complete or partial loss of motor and sensory functions below the injured section of patients, which greatly reduces the quality of life of the patients and brings a huge burden to the society. At present, the clinical treatments for spinal cord injury are mainly medication such as neurotrophins and surgical decompression, but these means cannot regenerate the injured spinal cord, and thus cannot improve their motor functions, either.
With the development of tissue engineering, scaffold material combined with stem cells has brought new hope for the treatment of spinal cord injury. Studies have shown that 5 days after spinal cord injury in rats, Nestin+ neural stem cells will be activated in the intact segments adjacent to the injury region and spontaneously migrate to the injury region, but such migration is spatially limited, and it is difficult for neural stem cells to migrate to the central part of the injury region, so it is difficult to provide sufficient seed cells for tissue regeneration. At the same time, due to the existence of inhibitory factors such as myelin protein and chondroitin sulfate proteoglycan, the neural stem cells in the injury region tend to differentiate into glial cells rather than neurons.
Tissue engineering scaffolds are generally considered to have the characteristics of good biocompatibility. The spinal cord is composed of bundles of nerve fibers, so the scaffold material should also have the function of guiding the ordered growth of new neurons in the longitudinal direction. Collagen, as a kind of animal-derived extracellular matrix protein, has the advantages of biocompatibility and degradability, but the mechanical strength of which is poor, greatly limiting the application thereof. In addition, the scaffold materials used for the treatment of spinal cord injury should also maintain a stable morphology without bending when being immersed in cerebrospinal fluid, so as to provide a stable mechanical environment for the migration, differentiation and maturation of seed cells. At present, the facilitation role of tissue-engineered scaffolds for spinal cord injury repair in the migration of neural stem cells and neuronal differentiation still needs to be improved. Therefore, the development of tissue-engineered scaffolds that can efficiently promote the migration of neural stem cells and neuronal differentiation is of great significance for the repair of spinal cord injury and the recovery of motor functions.
In order to solve the technical problems in the prior art, the object of the present invention is to provide a tissue engineering material capable of effectively promoting the migration of neural stem cells and their differentiation into neurons, the tissue engineering material can be used for nerve injury repair; another object of the present invention is to provide a method for preparing a tissue engineering material capable of effectively promoting migration of neural stem cells and their differentiation into neurons.
In order to achieve the above objects, the present invention has carried out the screening of substances capable of effectively inducing the migration of neural stem cells when being used as tissue engineering materials, as well as the matching screening of suitable scaffold materials thereof. Through a large number of researches, the present invention has discovered that, the neural cadherin (N-cadherin) can not only form homotypic binding and mediate cell-to-cell adhesion through an extracellular domain, but can also efficiently induce the migration of the neural stem cells, enrich the neural stem cells and effectively promote the differentiation of the neural stem cells into neurons through the binding to the N-cadherin expressed by the neural stem cells when N-cadherin is crosslinked to the surface of the tissue engineering scaffolds, which is different from many factors that can only play efficient roles between cells; at the same time, the crosslinking treatment can also significantly improve the mechanical properties of the material.
Specifically, the technical solutions of the present invention are as follows: In a first aspect, the present invention provides a tissue engineering material for nerve injury repair, which is a N-cadherin crosslinked biodegradable material; the biodegradable material comprises one selected from collagen and gelatin.
Among many biodegradable materials, the present invention finds that the collagen material can better crosslinked with the N-cadherin, the structure and the mechanical properties of the collagen material after crosslinking treatment are remarkably improved while ensuring the amount of N-cadherin crosslinked, and a more stable mechanical environment is provided for the migration of neural stem cells.
Therefore, the biodegradable material of the present invention is preferably collagen, more preferably, is a linear ordered collagen scaffold (LOCS).
Considering comprehensively the crosslinking efficiency of the linear ordered collagen scaffold and the action effect of the N-cadherin, the linear ordered collagen scaffold and N-cadherin are preferably crosslinked in the following ratio: one linear ordered collagen scaffold having a diameter of 0.25 mm and a length of 4 cm: (1-6) μg of N-cadherin. More preferably, they are crosslinked in the following ratio: 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.
Preferably, N-cadherin is covalently crosslinked to the linear ordered collagen scaffold.
More preferably, the crosslinking agents used for covalent crosslinking are Traut's reagent and Sulfo-SMCC.
Using the above crosslinking agent, N-cadherin can be covalently crosslinked to the linear ordered collagen scaffold by crosslinking agents Sulfo-SMCC and Traut's reagent in sequence.
In a second aspect, the present invention provides a preparation method for a tissue engineering material for nerve injury repair, comprising: crosslink 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, that biodegradable material is collagen; more preferably is a linear ordered collagen scaffold.
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: (1-6) μg of N-cadherin.
More preferably, the 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 the N-cadherin.
Specifically, the preparation method of the tissue engineering material for nerve injury repair comprises the following steps:
(1) treating a 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.
The present invention has found that the linear ordered collagen scaffold modified by Traut's reagent can covalently bind to N-cadherin modified by Sulfo-SMCC more efficiently to improve the crosslinking efficiency, and the obtained linear ordered collagen scaffold crosslinked with N-cadherin has a stable and ordered topological structure and mechanical properties.
Preferably, the 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. The molar ratio of the N-cadherin to the Sulfo-SMCC is 1:(60-100).
In the above step (1), the concentration of the Traut's reagent is preferably 0.2 mg/mL to 5 mg/ml.
In the above step (2), the concentration of the N-cadherin is preferably 200 μg/mL to 400 μg/mL. The concentration of the Sulfo-SMCC is preferably 0.3 mg/mL to 3.5 mg/mL.
In the above step (1), the treatment time is preferably 1 h to 3 h.
In the above step (2), the treatment time is preferably 1 h to 3 h.
Both step (1) and the step (2) are preferably carried out at a temperature of 18° C. to 25° C.
In a third aspect, the present invention provides the use of the tissue engineering material for nerve injury repair or the preparation method of the tissue engineering material for nerve injury repair in preparing a material for nerve injury repair.
Preferably, that nerve injury is a spinal cord nerve injury.
In a fourth aspect, the present invention provides the use of the tissue engineering material for nerve injury repair or the preparation method of the tissue engineering material for nerve injury repair in promoting directed migration of neural stem cells or promoting differentiation of neural stem cells.
The advantageous effect of the present invention lies in that the tissue engineering material (LOCS-Ncad) prepared by crosslinking N-cadherin with the linear ordered collagen scaffold can efficiently induce migration of neural stem cells towards an injury region (especially a central region of the injury region), enrich the neural stem cells in the injury region, and effectively inhibit the deposition of inhibitory factors such as chondroitin sulfate proteoglycan and the like, promote differentiation of the neural stem cells into neurons, and further promote the recovery of motor functions; at the same time, the obtained N-cadherin crosslinked linear ordered collagen scaffold also has a stable and ordered topology structure and excellent mechanical properties (the tensile modulus is remarkably improved, and the linear state is stable), so it can be used to repair nerve injuries such as spinal cord injury and the like.
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.
Unless otherwise specified, the experimental methods used in the following Examples are conventional methods.
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.
In the following Examples, the linear ordered collagen scaffold was prepared as follows:
(1) the isolated bovine fascia tissue was taken, and muscle and fat were removed;
(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);
(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);
(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);
(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);
(6) the resultant was subjected to freeze-drying (at a vacuum degree of 0.1 Mpa for 48 hours) to obtain a collagen scaffold.
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.
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
(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
(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.
(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
(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.
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.
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:
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
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
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
The structure of LOCS-Ncad prepared in Example 1 was detected by a scanning electron microscope, the result is shown in E of
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
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
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.
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 5×105/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
Cell migration experiment was performed according to the schematic diagram shown in C of
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
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.
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+ neural stem cells migrated to the center of the injury region (A1 to A13 of
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.
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
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.
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911312837.9 | Dec 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/085855 | 4/21/2020 | WO |
