A chitin nanocrystal multi-level oriented muscle repair scaffold and its preparation method

Abstract

This invention relates to the field of tissue engineering technology and discloses a multi-level oriented muscle repair scaffold made from chitin nanocrystals and its preparation method. The invention uses chitin derived from shrimp and crab shells as raw material to prepare chitin nanocrystals. Through steps such as preparing an aqueous suspension of chitin nanocrystals, ultrasonic dispersion, directional freezing to induce ice crystal orientation growth, freeze-drying to control ice crystal sublimation, and glutaraldehyde cross-linking fixation, a multi-level oriented scaffold with a macroscopic oriented pore structure and a microscopic oriented micro / nanofiber structure is prepared, with the chitin nanocrystals arranged along the orientation pore direction. This scaffold has a high specific surface area, can promote the orientation alignment and myogenic differentiation of C2C12 cells, and can be effectively used for the repair of volumetric muscle tissue loss. Furthermore, the preparation method is simple, requires low-end equipment, is inexpensive, and can be mass-produced.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a chitin nanocrystal multi-level oriented muscle repair scaffold and its preparation method. Background Technology

[0002] Volumetric muscle injuries exceeding the body's regenerative capacity are difficult to heal spontaneously, and their repair remains a significant clinical challenge. Currently, the primary clinical approach to repairing volumetric muscle injuries is autologous transplantation. However, this method suffers from limited donor availability and is prone to postoperative complications, severely restricting its practical effectiveness and applicability. Constructing tissue-engineered scaffolds with interconnected pores and sufficient porosity can ensure cell survival within the scaffold and promote angiogenesis, which is crucial for the repair of volumetric muscle injuries. Furthermore, constructing oriented scaffolds, by regulating cell orientation morphology and promoting the formation of highly aligned muscle fibers, can accelerate the generation of new muscle tissue.

[0003] Currently, methods for constructing oriented scaffolds for muscle injury repair mainly include electrospinning, directional freeze-drying, and 3D printing. These methods produce oriented structures primarily encompassing oriented pore structures, groove structures, oriented nanofibers, and oriented one-dimensional nanoparticles. These structures promote cell orientation through contact guidance mechanisms, or a combination of mechanical and biochemical signal guidance mechanisms. Constructing three-dimensional scaffolds with both oriented pores and oriented nanoparticles can create a "multi-level, multi-signal synergistic" scaffold system, possessing both cellular-scale spatial constraints from oriented pores and protein-scale contact guidance from one-dimensional nanoparticles. However, the limitations of precision and scalability in ultra-microfabrication techniques have become a bottleneck in realizing the construction of such multi-scale oriented scaffold structures. Therefore, finding a simple, low-requirement, and cost-effective strategy to construct tissue engineering scaffolds with both macroscopic and microscopic orientation is of significant practical importance and application value. Summary of the Invention

[0004] The present invention aims to provide a chitin nanocrystal multi-level oriented muscle repair scaffold and its preparation method, addressing the limitations of autologous transplantation in existing volumetric muscle injury repair methods and the difficulty in constructing multi-scale oriented scaffolds.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a chitin nanocrystal multi-level oriented muscle repair scaffold, which uses chitin nanocrystals as the main material and is in the form of a three-dimensional block. The scaffold has a polygonal oriented pore structure on a macroscopic scale, and the scaffold also has a micro-oriented micro-nano fiber structure.

[0006] Preferably, as an improvement, the chitin nanocrystals are prepared from chitin, and the scaffold is fixed with glutaraldehyde.

[0007] Preferably, as an improvement, the micro-oriented micro / nanofiber structure of the scaffold is distributed on the cavity wall of the cavity, and the chitin nanocrystals are arranged along the extension direction of the cavity.

[0008] Preferably, as an improvement, the chitin is derived from shrimp and crab shells.

[0009] The preparation method of the chitin nanocrystal multi-level oriented muscle repair scaffold according to any of the above-described methods includes the following steps: (1) Chitin was mixed with concentrated hydrochloric acid and heated in an oil bath to carry out acid hydrolysis. After the reaction, the mixture was centrifuged, washed with deionized water, dialyzed, and freeze-dried to obtain chitin nanocrystals. (2) Prepare a chitin nanocrystal aqueous suspension, disperse it evenly by ultrasonication, and inject it into a self-made mold. Introduce a copper block to control the growth rate of ice crystals, so that the ice crystals grow in orientation along the temperature gradient between liquid nitrogen and air. (3) After ice crystals form, place the support in the freeze dryer and adjust the pressure to control the sublimation rate of the ice crystals; (4) Crosslink the freeze-dried scaffold with glutaraldehyde aqueous solution, wash and freeze-dry again to obtain a scaffold with a multi-level orientation structure.

[0010] Preferably, as an improvement, in step (1), the amount of chitin nanocrystals used is 5-15 grams, the oil bath heating temperature is 104°C, the acid hydrolysis reaction time is 4 hours, after centrifugation, the chitin nanocrystals are washed three times with deionized water, dialysis is performed for three days using a dialysis bag, and after freeze-drying, chitin nanocrystals are obtained.

[0011] Preferably, as an improvement, in step (2), the concentration of the chitin nanocrystal aqueous suspension is 0.2-5% (w / v), the ultrasonic method is an ultrasonic cell disruptor, and the ultrasonic time is 40 minutes.

[0012] Preferably, as an improvement, in step (3), the pressure of the freeze dryer is 1 Pa to 80 Pa; in step (4), the concentration of the glutaraldehyde aqueous solution is 6.5%.

[0013] Preferably, as an improvement, the chitin nanocrystal multi-level oriented muscle repair scaffold prepared according to the above preparation method is used in the preparation of muscle tissue repair products with volumetric loss.

[0014] The advantages of this approach are: the macroscopic polygonal orientation structure and the microscopic orientation micro / nanofiber structure of the scaffold work synergistically to provide a "multi-level, multi-signal synergistic" microenvironment for cell growth. It guides cell orientation through the spatial constraint of the pores and promotes cell alignment through mechanisms such as contact guidance and mechanical signal guidance by one-dimensional chitin nanocrystals. The high specific surface area is conducive to cell adhesion and proliferation, promoting the orientation alignment and myogenic differentiation of C2C12 cells, thereby effectively repairing volumetric muscle defects. The preparation process does not require complex and precise equipment, the steps are simple, the raw materials are readily available, the cost is low, and batch production can be achieved. It solves the problems of high difficulty and high cost in constructing multi-scale orientation scaffolds in existing technologies, while avoiding the donor limitations and postoperative complications of autologous transplantation. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the fabrication process of the directional freeze-drying support structure used in this invention; Figure 2 SEM image of the cross-sectional structure of the orientation pores of the chitin nanocrystal multi-level oriented muscle repair scaffold prepared in this invention; Figure 3 SEM image of the longitudinal section fiber structure of the chitin nanocrystal multi-level oriented muscle repair scaffold prepared in this invention; Figure 4 Two-dimensional wide-angle X-ray diffraction pattern of chitin nanocrystals with multi-level orientation muscle repair scaffold prepared in this invention. Figure 5 Confocal microscope image of C2C12 cells stained after 3 days of culture on a scaffold; Figure 6 This is a schematic diagram illustrating the repair effect of the scaffold on muscle defects in rats. Detailed Implementation

[0016] The following detailed description illustrates the specific implementation method: I. For example Figure 1 As shown, the fabrication process of the chitin nanocrystal multi-level oriented muscle repair scaffold is as follows: Preparation of chitin nanocrystals: 15g of chitin raw material derived from shrimp shells (crushed to 100 mesh size) was mixed with concentrated hydrochloric acid (3mol / L) at a solid-liquid ratio of 1:30 (g / mL) until homogeneous. The mixture was placed in an oil bath at 104℃ for constant temperature acid hydrolysis for 4 hours, with magnetic stirring (300r / min) to ensure uniform reaction. After acid hydrolysis, the reaction solution was transferred to a centrifuge tube and centrifuged at 8000r / min for 5 minutes. The precipitate was collected and repeatedly washed with deionized water until the pH of the supernatant was 7.0. The precipitate was then transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed in deionized water for 3 days. Finally, after pre-freezing at -80℃ for 2 hours, the mixture was freeze-dried in a freeze dryer (1Pa pressure) for 24 hours to obtain white, fluffy chitin nanocrystals.

[0017] Preparation of chitin nanocrystal aqueous suspension: Weigh the above chitin nanocrystals and add deionized water to prepare an aqueous suspension with a concentration of 2% (w / v). Transfer the suspension into an ultrasonic cell disruptor, set the power to 300W, the working mode to "ultrasound 5 seconds - intermittent 5 seconds", and the total ultrasonic time to 40 minutes to ensure that the chitin nanocrystals are uniformly dispersed and without obvious agglomerates.

[0018] Directional cryoforming: Inject a uniformly dispersed aqueous suspension of chitin nanocrystals into a self-made Teflon mold (10mm in diameter and 20mm in height). Place the bottom of the mold in contact with a pre-cooled copper block and control the ice crystals to grow directionally along the temperature gradient formed by liquid nitrogen and air. Maintain this state for 30 minutes to align the chitin nanocrystals along the ice crystal growth direction.

[0019] Freeze-drying and cross-linking fixation: The freeze-formed scaffold, along with the mold, was placed in a freeze dryer, and the pressure was adjusted to 80 Pa for continuous freeze-drying for 48 hours to allow the ice crystals to sublimate and form a porous structure. Subsequently, the freeze-dried scaffold was immersed in a 6.5% glutaraldehyde aqueous solution and cross-linked and fixed at room temperature for 12 hours. After removal, it was washed three times with deionized water (15 minutes each time) to remove residual glutaraldehyde, and then placed in the freeze dryer again for 24 hours to finally obtain a chitin nanocrystal multi-level oriented muscle repair scaffold.

[0020] II. Structural Characteristics of Chitosan Nanocrystal Multilevel Oriented Muscle Repair Scaffold like Figure 1 As shown in the schematic diagram of the three-dimensional structure of scaffold C, the scaffold prepared by this invention has a regular three-dimensional block shape and an oriented cavity structure at the macroscopic scale, exhibiting good structural stability. At the microscopic scale, the surface of the pore walls is composed of interwoven micro- and nanofibers, and the chitin nanocrystals are highly oriented along the extension direction of the macroscopic pores, forming a multi-level orientation structure of "macroscopic oriented pores - microscopic oriented fibers". This structure can simultaneously provide spatial constraint and contact guidance signals for cells.

[0021] After the cross-section of the support was sprayed with gold, it was observed using a scanning electron microscope, and the results are as follows: Figure 2 As shown, the cross-section of the scaffold exhibits a uniformly distributed polygonal cavity structure with regular pore arrangement and a pore size distribution concentrated in the range of 10-50 μm. This pore size range can provide sufficient space for cell infiltration and nutrient transport, while the pore walls are continuous and intact without collapse.

[0022] SEM observation results of the longitudinal section of the stent are as follows Figure 3 As shown in the longitudinal section, the pores extend in a single direction, and the micro- and nanofibers on the pore walls are arranged in parallel, with uniform fiber diameter and no obvious aggregation. Chitosan nanocrystals are oriented along the pore extension direction, forming a continuous oriented structure. This microscopic orientation feature can induce cell growth along the fiber alignment direction through contact guidance, meeting the directional growth requirements of muscle tissue.

[0023] The crystal structure of the scaffold was analyzed using synchrotron wide-angle X-ray diffraction (2DWAXD), and the results are as follows: Figure 4 As shown in the figure, characteristic diffraction rings (corresponding to crystal plane (110)) appear, and the diffraction rings exhibit obvious intensity inhomogeneity. The diffraction intensity is significantly enhanced along a specific direction, indicating that the chitin nanocrystals form a highly preferred orientation in the scaffold, which is consistent with the micro-orientation results observed by SEM, confirming the successful construction of the multi-level orientation structure.

[0024] C2C12 cells (mouse myoblasts) were seeded onto a scaffold and cultured at 37°C in a 5% CO2 incubator for 3 days. Double staining was then performed using a Rhodamine–Phalloidin-labeled actin (F-actin) specific fluorescent probe and a nuclear dye (DAPI). The results were observed using laser confocal microscopy. Figure 5 As shown, the cell nuclei (blue fluorescence) are uniformly distributed within the scaffold channels and on the channel walls, while actin (red fluorescence) extends in fibrous form along the orientation of the scaffold's microfibers. The cell morphology exhibits obvious directional spreading characteristics, demonstrating that the multi-level orientation structure of the scaffold can effectively induce the oriented arrangement of C2C12 cells, laying the foundation for myoblastic differentiation.

[0025] III. Rat Muscle Defect Repair Experiment 1. Experimental group design Normal group: Healthy SD rats (male, weighing 200-250g) were selected without any muscle damage treatment and were used as a control standard for normal muscle tissue.

[0026] Control group: A 10mm×8mm×2mm full-thickness defect model of the tibialis anterior muscle (TA muscle) was created in SD rats. The surgery only sutured the wound and did not implant any repair material. The natural healing of the defect site was observed.

[0027] Materials Group: A tibial anterior muscle volume defect model identical to the blank group was constructed in SD rats. The chitin nanocrystal multi-level oriented muscle repair scaffold prepared in this invention was trimmed into blocks matching the defect size, sterilized, and implanted into the defect site. The wound was then sutured layer by layer.

[0028] 2. Experimental Results All rats were routinely fed for 4 weeks post-surgery. The repair effect was assessed by histological staining, muscle function testing, and immunohistochemical analysis. Results are as follows: Figure 6 As shown: Normal group: The tibialis anterior muscle has an intact tissue structure, with muscle fibers arranged tightly and in the same direction, sarcomeres clearly visible, no inflammatory cell infiltration, and normal muscle function.

[0029] The blank group: the defect area was mainly filled with fibrous scar tissue, and only a small number of scattered new muscle fibers were observed. The muscle fibers were disordered and non-directional, and the repair effect was extremely poor.

[0030] Material group: The scaffold partially degraded in vivo, and a large number of directionally aligned new muscle fibers formed at the defect site. The diameter of the muscle fibers was close to that of the normal group, the sarcomere structure was mature, and the inflammatory response was mild. Muscle function recovered to the level of the normal group, which was significantly higher than that of the blank group. Immunohistochemical quantitative analysis showed that there was no significant difference in the diameter of muscle fibers (based on MHC staining) and the vascular area (based on CD31 staining) of the regenerated tissue in the material group compared with the normal group.

[0031] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A chitosan nanocrystal multi-level oriented muscle repair scaffold, characterized in that: Using chitin nanocrystals as the main material, the overall structure is a three-dimensional block. The scaffold has a polygonal oriented pore structure on a macroscopic scale, and it also has a micro-oriented micro-nano fiber structure.

2. The chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 1, characterized in that: The chitin nanocrystals are prepared from chitin, and the scaffold is fixed with glutaraldehyde.

3. The chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 2, characterized in that: The micro-oriented micro / nanofiber structure of the scaffold is distributed on the cavity wall, and the chitin nanocrystals are arranged along the extension direction of the cavity.

4. The chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 3, characterized in that: The chitin is derived from shrimp and crab shells.

5. The method for preparing a multi-level oriented muscle repair scaffold using chitosan nanocrystals according to any one of the above-described methods, characterized in that, Includes the following steps: (1) Chitin was mixed with concentrated hydrochloric acid and heated in an oil bath to carry out acid hydrolysis. After the reaction, the mixture was centrifuged, washed with deionized water, dialyzed, and freeze-dried to obtain chitin nanocrystals. (2) Prepare a chitin nanocrystal aqueous suspension, disperse it evenly by ultrasonication, and inject it into a self-made mold. Introduce a copper block to control the growth rate of ice crystals, so that the ice crystals grow in orientation along the temperature gradient between liquid nitrogen and air. (3) After ice crystals form, place the support in the freeze dryer and adjust the pressure to control the sublimation rate of the ice crystals; (4) Crosslink the freeze-dried scaffold with glutaraldehyde aqueous solution, wash and freeze-dry again to obtain a scaffold with a multi-level orientation structure.

6. The method for preparing a chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 5, characterized in that: In step (1), the amount of chitin used is 5-15 grams, the oil bath heating temperature is 104℃, the acid hydrolysis reaction time is 4 hours, after centrifugation, it is washed three times with deionized water, dialysis is performed for three days using a dialysis bag, and chitin nanocrystals are obtained after freeze drying.

7. The method for preparing a chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 6, characterized in that: In step (2), the concentration of the chitin nanocrystal aqueous suspension is 0.2-5% (w / v), and the ultrasonic method is an ultrasonic cell disruptor with an ultrasonic time of 40 minutes.

8. The method for preparing a chitin nanocrystal multi-level oriented muscle repair scaffold according to claim 7, characterized in that: In step (3), the pressure of the freeze dryer is 1 Pa to 80 Pa; in step (4), the concentration of the glutaraldehyde aqueous solution is 6.5%.

9. The use of the chitin nanocrystal multi-level oriented muscle repair scaffold according to any one of claims 1-4 in the preparation of products for repairing volumetric muscle tissue loss.

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