Periodontal complex stent with bionic structure and preparation method of periodontal complex stent

Abstract

The invention discloses a periodontal complex stent with a bionic structure and a preparation method. The preparation method comprises the following steps: step 1, preparing a polycaprolactone-polylactic acid nanofiber membrane by an electrostatic spinning method; 2, the polycaprolactone-polylactic acid nanofiber membrane obtained in the step 1 is coated with chitosan layer by layer, the fiber membrane is stacked, and after freeze drying, a polycaprolactone-polylactic acid nanofiber scaffold is obtained; 3, immersing the polycaprolactone-polylactic acid nanofiber scaffold obtained in the step 2 into a collagen solution, and carrying out self-assembly to form a composite scaffold; then immersing into a hydroxyapatite solution, and mineralizing to obtain the required composite scaffold; according to the invention, a directionally arranged PCL-PLA nanofiber scaffold is taken as a basis, the surface layer is coated with porous collagen, and after mineralization, a mineralized structure with gradient decreasing from a collagen layer in an external phase to a middle fiber layer is obtained; the obtained composite scaffold simulates a heterogeneous structure of periodontal membrane-bone interface tissue, and provides possibility for collaborative regeneration of soft and hard tissues of a periodontal complex.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a periodontal complex scaffold with a bionic structure and a preparation method thereof. Background Art

[0002] The destruction of tooth supporting tissues (including periodontal ligament, alveolar bone and cementum) is the main feature of periodontitis. Currently, guided tissue regeneration (GTR) surgery is commonly used clinically to repair the epithelial attachment and alveolar bone regeneration mediated by bone powder of periodontal supporting tissues. However, compared with normal periodontal tissues, the differentiation ability of progenitor cells in the infected area of periodontitis patients is reduced, and a weak long epithelial connection is mostly established by the ingrowth of gingival epithelial cells, making it difficult to induce a large amount of new periodontal connective tissue to attach to the alveolar bone / cementum. The physiological connection between the periodontal ligament and the bone tissues at both ends is the key for the normal function of periodontal tissues. Therefore, a treatment method that can effectively achieve the regeneration of a functional periodontal ligament-to-bone interface is urgently needed. In science and technology, although there are many reports on periodontal tissue engineering, there is no very ideal bionic material that can meet the requirements of periodontal tissue function regeneration.

[0003] Currently commonly used periodontal tissue scaffold materials generally draw on bone tissue engineering, including chitosan (CHI), collagen (Col), polycaprolactone (PCL), polyethylene glycol (PEG), polylactic acid (PLA), hydroxyapatite (HA), and β-tricalcium phosphate (β-TCP), etc., which can synthesize scaffolds with good mechanical properties, biocompatibility and biodegradability. However, it is difficult to obtain a heterogeneous structure similar to that of the periodontal ligament-bone interface tissue. Summary of the Invention

[0004] The present invention provides a periodontal complex scaffold with a bionic structure and a preparation method thereof for the problems existing in the prior art.

[0005] The technical solution adopted by the present invention is: a preparation method of a periodontal complex scaffold with a bionic structure, comprising the following steps:

[0006] Step 1: Prepare a polycaprolactone-polylactic acid nanofiber membrane by electrospinning;

[0007] Step 2: Coat the polycaprolactone-polylactic acid nanofiber membrane obtained in Step 1 with chitosan layer by layer. After stacking and freeze-drying the fiber membranes, a polycaprolactone-polylactic acid nanofiber scaffold is obtained.

[0008] Step 3: Immerse the polycaprolactone-polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to form a composite scaffold by self-assembly; then immerse it into a hydroxyapatite solution and mineralize it to obtain the required composite scaffold.

[0009] Further, in Step 1, the mass ratio of polycaprolactone to polylactic acid is 5:1.

[0010] Further, in Step 1, the voltage of electrospinning is 21 kV and the rotation speed is 2000 rpm.

[0011] Further, in Step 1, the concentration of the electrospinning solution is 20 wt.%.

[0012] Further, in Step 2, the mass concentration of the chitosan solution is 1 wt.%.

[0013] Further, in the composite scaffold of Step 3, the mass ratio of collagen to hydroxyapatite is 16:3.2.

[0014] Further, in Step 3, the concentration of the hydroxyapatite solution is 40 mg / mL to 100 mg / mL.

[0015] A periodontal complex scaffold with a bionic structure, the internal of the composite scaffold is a layered polycaprolactone-polylactic acid nanofiber structure, and the surface is covered with a collagen-hydroxyapatite gradient mineralized structure extending from the outside to the inside.

[0016] Advantages of the present invention:

[0017] (1) Based on the PCL-PLA nanofiber scaffold with oriented arrangement, the present invention has a porous collagen layer on the surface, and after mineralization, a mineralized structure with a gradient decrease from the outer collagen layer to the middle fiber layer is obtained;

[0018] (2) The composite scaffold obtained by the present invention simulates the heterogeneous structure of the periodontal ligament-bone interface tissue, providing the possibility for the coordinated regeneration of the hard and soft tissues of the periodontal complex. Description of the Drawings

[0019] Figure 1 It is a schematic process diagram of the preparation method of the present invention.

[0020] Figure 2 It is a laser confocal image and scanning electron microscope image of the random and oriented fibers in Example 1 of the present invention.

[0021] Figure 3This is the SEM image of the composite scaffold obtained in Example 1 of the present invention.

[0022] Figure 4 This is the EDS image of the composite scaffolds obtained in Example 1 and Comparative Example 1 of the present invention. Detailed implementation manners

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation manners.

[0024] As Figure 1 shown, a preparation method of a periodontal composite scaffold with a bionic structure includes the following steps:

[0025] Step 1: Prepare a polycaprolactone - polylactic acid nanofiber membrane by electrospinning;

[0026] Among them, the mass ratio of polycaprolactone PCL to polylactic acid PLA is 5:1, and the two are mixed to form an electrospinning solution with a mass concentration of 20 wt.%.

[0027] Electrospinning is carried out at a voltage of 21 ± 0.5 kV, and by adjusting the rotation speed of the collection device, it is set to 2000 rpm to achieve the oriented arrangement of nanofibers. During the fiber collection process, the rotation speed of the drum has an important influence on the arrangement and morphology of the fibers.

[0028] When the drum rotates at a certain speed, the fibers will be subjected to a tangential tensile force exerted by the drum during the collection process. This tensile force can cause the fibers to be oriented on the surface of the drum. From a mechanical perspective, the fibers will deform during the stretching process, and their molecular chains tend to be parallel under the action of the tensile stress. This oriented arrangement of molecular chains makes the fibers macroscopically show the characteristics of oriented arrangement, thus endowing the fiber aggregate with better mechanical properties and anisotropy.

[0029] During the fiber collection process, the fibers are not completely dry and the solvent is still volatilizing. The volatilization of the solvent will cause changes in the stress distribution inside the fibers. On the one hand, the volatilization of the solvent will cause the fibers to gradually shrink, increasing the stress inside the fibers; on the other hand, there is an interaction between the volatilization rate of the solvent and the stretching rate of the fibers. If the rotation speed of the drum is too low, the fibers cannot generate enough tensile stress to overcome the shrinkage stress caused by solvent volatilization, and thus the oriented arrangement cannot be achieved. When the rotation speed of the drum is too high, the tensile stress on the fibers is too large, exceeding the tensile strength of the fibers, and the fibers are easily broken.

[0030] The tensile strength of the fiber is closely related to its diameter, material properties, and internal structure. During the drum collection process, the tensile stress on the fiber is proportional to the rotational speed of the drum. When the rotational speed of the drum exceeds a certain critical value, the tensile stress on the fiber exceeds its tensile strength, and the fiber will be broken. In addition, during the stretching process, the fiber is also affected by the shrinkage stress caused by the volatilization of the solvent. This shrinkage stress interacts with the tensile stress, further increasing the risk of fiber breakage.

[0031] After comprehensive calculation, combined with the actual situation of the fibers of the present invention, considering the requirements of fiber orientation and the risk of breakage, the rotational speed of the drum is fixed at 2000 rpm. At this rotational speed, the tensile stress exerted by the drum on the fiber can cause sufficient fiber orientation without exceeding the tensile strength of the fiber. In addition, this rotational speed can also ensure that the fiber maintains a relatively stable shape during the solvent volatilization process, avoiding fiber breakage caused by the shrinkage stress due to solvent volatilization.

[0032] In summary, the rotational speed of the drum has an important impact on the orientation and shape of the fiber. The rotational speed set in the present invention can achieve fiber orientation while the fiber is not completely dry and the solvent is still volatilizing, and at the same time avoid the risk of being broken.

[0033] Step 2: Coat the polycaprolactone-polylactic acid nanofiber membrane obtained in Step 1 layer by layer with chitosan, stack the fiber membranes, and obtain a polycaprolactone-polylactic acid nanofiber scaffold after freeze-drying; wherein the mass concentration of chitosan is 1 wt.%.

[0034] Step 3: Immerse the polycaprolactone-polylactic acid nanofiber scaffold obtained in Step 2 into the collagen Col solution. After a gel is formed on the side first immersed in the collagen solution, turn over the scaffold and immerse the other side in the collagen solution to form a composite structure with porous collagen on the outside and nanofibers in the middle.

[0035] Then immerse it in the hydroxyapatite HA solution, and obtain the required composite scaffold after mineralization. The concentration of the hydroxyapatite solution is 40 mg / mL to 100 mg / mL. The gradient change of the mineralization degree can be achieved through diffusion, and the mineralization time can be set as needed.

[0036] The calcium ions and phosphate ions in the HA mineralization solution will diffuse from the higher concentration Col layer to the lower concentration PCL-PLA nanofiber layer. Since the chemical properties of the PCL-PLA nanofiber layer are different from those of the Col layer, there are differences in the diffusion rate and binding ability of ions in it, resulting in a decrease in the degree of mineralization from the Col layer to the PCL-PLA layer. The Col layer has high hydrophilicity and ion adsorption capacity, which can quickly adsorb ions in the mineralization solution and form HA crystals. The PCL-PLA nanofiber layer is more hydrophobic, and the rate of ion diffusion and binding is slower, thus forming a gradient mineralization. The extension of the immersion time will cause the ions in the mineralization solution to gradually diffuse to the PCL-PLA nanofiber layer, but due to the longer diffusion path and greater resistance, the degree of mineralization gradually decreases in the PCL-PLA layer.

[0037] The outer HA-rich Col layer has high biological activity and can promote cell adhesion, proliferation and osteogenic differentiation. The middle PCL-PLA nanofiber layer provides good mechanical support and structural stability. This gradient structure can better simulate the structural characteristics of natural bone tissue and promote bone tissue regeneration. The gradient mineralization structure can improve the mechanical properties of the composite material. The HA mineralized layer has high hardness and stiffness, which can enhance the compressive strength and wear resistance of the material. At the same time, the flexibility and elasticity of the PCL-PLA nanofiber layer can buffer stress and avoid brittle fracture of the material when it is stressed. The PCL-PLA nanofiber layer has good biodegradability and can gradually degrade in the body and release nutrients to promote tissue regeneration. The degradation rate of the HA mineralized layer is slow, which can provide mechanical support for a long time. This gradient structure can balance the degradation rate and mechanical properties of the material.

[0038] The mineralization degree of the composite material of the present invention presents a gradient change from the outer Col layer to the middle PCL-PLA nanofiber layer. The outer Col layer has a higher mineralization degree, densely distributed HA crystals, and has higher hardness and biological activity. The middle PCL-PLA nanolayer has a lower mineralization degree, sparsely distributed HA crystals, but has good mechanical properties and flexibility. This gradient structure can better simulate the structural characteristics of natural bone tissue, promote bone tissue regeneration, and achieve a balance between mechanical properties and biological activity.

[0039] Example 1

[0040] A method for preparing a periodontal complex scaffold with a bionic structure comprises the following steps:

[0041] Step 1: Prepare a polycaprolactone - polylactic acid nanofiber membrane by electrospinning; polycaprolactone and polylactic acid are configured into an electrospinning solution with a mass concentration of 20 wt.% according to a mass ratio of 5:1. Adjust the voltage of the spinning equipment to 21 ± 0.5 kV, and set the rotation speed of the collection device to 2000 rpm.

[0042] Step 2: Layer - by - layer coat the polycaprolactone - polylactic acid nanofiber membrane obtained in Step 1 with chitosan, stack the fiber membranes, and obtain a polycaprolactone - polylactic acid nanofiber scaffold after freeze - drying; as Figure 2 shown. The concentration of the chitosan solution is 1 wt.%.

[0043] Step 3: Immerse the polycaprolactone - polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to form a composite scaffold by self - assembly; then immerse it into a hydroxyapatite solution with a concentration of 60 mg / mL, and obtain the required composite scaffold after mineralization (mineralization group); the mass ratio of collagen to hydroxyapatite in the composite scaffold is 16:3.2.

[0044] The SEM image of the composite scaffold obtained in this example is as Figure 3 shown. It can be clearly seen from the figure that the middle is PCL - PLA fiber and the outer surface is a mineralized Col layer.

[0045] Example 2

[0046] A method for preparing a periodontal complex scaffold with a bionic structure, comprising the following steps:

[0047] Step 1: Prepare a polycaprolactone - polylactic acid nanofiber membrane by electrospinning; polycaprolactone and polylactic acid are configured into an electrospinning solution with a mass concentration of 20 wt.% according to a mass ratio of 5:1. Adjust the voltage of the spinning equipment to 21 ± 0.5 kV, and set the rotation speed of the collection device to 2000 rpm.

[0048] Step 2: Layer - by - layer coat the polycaprolactone - polylactic acid nanofiber membrane obtained in Step 1 with chitosan, stack the fiber membranes, and obtain a polycaprolactone - polylactic acid nanofiber scaffold after freeze - drying. The concentration of the chitosan solution is 1 wt.%.

[0049] Step 3: Immerse the polycaprolactone - polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to form a composite scaffold by self - assembly; then immerse it into a hydroxyapatite solution with a concentration of 100 mg / mL, and obtain the required composite scaffold after mineralization; the mass ratio of collagen to hydroxyapatite in the composite scaffold is 16:3.2.

[0050] Example 3

[0051] A method for preparing a periodontal complex scaffold with a bionic structure, comprising the following steps:

[0052] Step 1: Prepare a polycaprolactone - polylactic acid nanofiber membrane by electrospinning; polycaprolactone and polylactic acid are configured into an electrospinning solution with a mass concentration of 20 wt.% according to a mass ratio of 5:1. Adjust the voltage of the spinning equipment to 21 ± 0.5 kV, and set the rotation speed of the collection device to 2000 rpm.

[0053] Step 2: Layer - by - layer coat the polycaprolactone - polylactic acid nanofiber membrane obtained in Step 1 with chitosan, stack the fiber membranes, and obtain a polycaprolactone - polylactic acid nanofiber scaffold after freeze - drying. The concentration of the chitosan solution is 1 wt.%.

[0054] Step 3: Immerse the polycaprolactone - polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to self - assemble into a composite scaffold; then immerse it into a hydroxyapatite solution with a concentration of 40 mg / mL, and obtain the required composite scaffold after mineralization; the mass ratio of collagen to hydroxyapatite in the composite scaffold is 16:3.2.

[0055] Comparative Example 1

[0056] A preparation method of a periodontal complex scaffold with a bionic structure, comprising the following steps:

[0057] Step 1: Prepare a polycaprolactone - polylactic acid nanofiber membrane by electrospinning; polycaprolactone and polylactic acid are configured into an electrospinning solution with a mass concentration of 20 wt.% according to a mass ratio of 5:1. Adjust the voltage of the spinning equipment to 21 ± 0.5 kV, and set the rotation speed of the collection device to 2000 rpm.

[0058] Step 2: Layer - by - layer coat the polycaprolactone - polylactic acid nanofiber membrane obtained in Step 1 with chitosan, stack the fiber membranes, and obtain a polycaprolactone - polylactic acid nanofiber scaffold after freeze - drying; as Figure 2 shown. The concentration of the chitosan solution is 1 wt.%.

[0059] Step 3: Immerse the polycaprolactone - polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to self - assemble into a composite scaffold (non - mineralized group).

[0060] Figure 4 For the EDS diagrams of the composite scaffolds obtained in Example 1 and Comparative Example 1, the upper two are the analysis diagrams of P, Ca, O, and C, and the lower one is the corresponding Ca element analysis diagram. It can be seen from the figure that the position of the calcium element is the position where hydroxyapatite appears. The analysis of the calcium element in the composite scaffold obtained in Example 1 is Figure 3 consistent with the position of the porous material in

[0061] The present invention combines PCL-PLA nanofibers with collagen and hydroxyapatite to prepare a composite scaffold material with gradient mineralization characteristics. It simulates the heterogeneous characteristics of the natural periodontal ligament-bone interface. The degree of mineralization decreases from the surface collagen layer to the middle nanofiber layer, providing a gradually changing microenvironment for cells. This gradient mineralized structure can effectively promote the directional migration and differentiation of cells, and significantly improve the effect of periodontal tissue regeneration compared with traditional single materials or uniformly mineralized materials. By adjusting the parameters of electrospinning, aligned PCL-PLA nanofibers are obtained; this alignment enables periodontal ligament cells to grow along the fiber alignment direction on the aligned fiber scaffold, showing obvious directionality. It effectively guides the growth direction of cells, simulates the arrangement of natural periodontal ligament fibers, and promotes the functional regeneration of periodontal tissue.

Claims

1. A preparation method of a periodontal complex scaffold with a bionic structure, characterized in that, It includes the following steps: Step 1: Prepare a polycaprolactone-polylactic acid nanofiber membrane by electrospinning; Step 2: Layer-by-layer coat the polycaprolactone-polylactic acid nanofiber membrane obtained in Step 1 with chitosan. After stacking the fiber membranes and freeze-drying, a polycaprolactone-polylactic acid nanofiber scaffold is obtained; Step 3: Immerse the polycaprolactone-polylactic acid nanofiber scaffold obtained in Step 2 into a collagen solution to self-assemble a composite scaffold; Then immerse it into a hydroxyapatite solution and mineralize to obtain the required composite scaffold.

2. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, wherein, In Step 1, the mass ratio of polycaprolactone to polylactic acid is 5:

1.

3. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, characterized in that, In Step 1, the voltage of electrospinning is 21 kV and the rotation speed is 2000 rpm.

4. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, characterized in that, In Step 1, the concentration of the electrospinning solution is 20 wt.%.

5. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, characterized in that, In Step 2, the mass concentration of the chitosan solution is 1 wt.%.

6. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, characterized in that, In the composite scaffold of Step 3, the mass ratio of collagen to hydroxyapatite is 16:3.

2.

7. The preparation method of a periodontal complex scaffold with a bionic structure according to claim 1, characterized in that, In Step 3, the concentration of the hydroxyapatite solution is 40 mg / mL to 100 mg / mL.

8. The periodontal complex scaffold with a bionic structure obtained by any of the preparation methods according to claims 1 to 7, characterized in that, The interior of the composite scaffold is a layered polycaprolactone-polylactic acid nanofiber structure, and the surface is covered with a collagen-hydroxyapatite gradient mineralized structure extending from the outside to the inside.

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