Absorbable double-layer membrane for guiding regeneration and repair of oral gingival tissues and preparation method of absorbable double-layer membrane

Abstract

The invention relates to an absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair and a preparation method thereof, the double-layer membrane comprises a compact layer and a loose layer, the compact layer is formed by PCL fibers, and the loose layer is formed by recombinant collagen / polyhydroxyalkanoate blend fibers. Wherein the PCL compact layer can be well stitched, and plays a protective role during open type healing. The recombinant collagen / polyhydroxyalkanoate blend fiber loose layer solves the problems of hydrophobicity of polyhydroxyalkanoate and low strength and too fast degradability of single recombinant collagen, and is more beneficial to fixation of blood clots and ingrowth of soft tissue cells, and inflammatory response is reduced. According to the double-layer membrane disclosed by the invention, the degradation time and the strength are highly matched with gingival tissues by optimizing the components, the proportion and the like, so that the regeneration and the repair of the gingival tissues can be better promoted, and the double-layer membrane has a remarkable clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair and a preparation method thereof. Background Art

[0002] Oral tissue defects need to be repaired. Oral tissue defects mainly include periodontal tissue loss, insufficient or missing attached gingiva, defects after maxillofacial tumor resection, gingival recession, exposed root surfaces, abnormal frenulum attachment position / shallow vestibule groove, lip and cheek mucosal tissue defects, and prevention of gustatory sweating syndrome.

[0003] Currently, in the repair surgery for oral gingival tissue recession defects, autologous tissue transplantation is limited in material acquisition, and the technical level of doctors is also required to be high. The operation time is long, and it will cause additional pain to patients, and patients are more afraid. The decellularized dermal cell matrix derived from fetal bovine is rich in type I and type III collagen and is widely used in soft tissue augmentation around oral implants. However, xenogeneic decellularized dermal matrix has certain immunogenicity, which is easy to cause immune rejection reaction, severe inflammatory reaction, and has the disadvantages of limited donors, high price and poor sutureability. PRF (platelet-rich fibrin) is not suitable for large-scale material acquisition, and the existing centrifugal means and equipment also determine that only small areas of PRF can be prepared, which is difficult to use in large-scale defects. Therefore, it is urgent to develop oral soft tissue repair products that promote periodontal tissue regeneration and keratinized gingival augmentation, do not cause immune rejection reaction, have sufficient sources, low prices and are easy for patients to accept.

[0004] Chinese patent (publication number CN 109513046 A) "An absorbable oral repair membrane and its preparation method" discloses a method for preparing a gingival regeneration repair membrane, using gelatin and polycaprolactone as raw materials, respectively using electrospinning technology to obtain a membrane composed of nano-scale fibers, and pressing in the order of gelatin layer-polycaprolactone layer-gelatin layer to obtain a three-layer oral repair membrane with a gradual fiber diameter. The gelatin layer is prepared by adding 0.25-0.3wt% glutaraldehyde to the gelatin solution for cross-linking, which improves the cross-linking degree and strength of the gelatin layer. However, due to the low degree of cross-linking and fast degradation rate, it cannot meet the time requirement for tissue defect repair to be maintained in the body. Chinese patent (publication number CN117045870 A) "A method for preparing a polyhydroxyalkanoate oral guide regeneration membrane" discloses a method for preparing a polyhydroxyalkanoate oral guide regeneration membrane. However, the repair membrane prepared by this method is mainly used in GBR surgery, its hydrophobicity is too strong, and the degradation time is too long, which is also not suitable for oral tissue defect repair.

[0005] Therefore, it is an urgent need of clinicians and the market to develop a new type of gingival tissue regeneration and repair product that can obtain large amounts of materials, has a stable degradation rate, has good tissue compatibility, promotes cell growth and tissue regeneration, and reduces repair time. Summary of the invention

[0006] In view of the above technical deficiencies, the present invention provides an absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair and a preparation method thereof. Specifically, it is a new type of gingival tissue regeneration and repair product with stable degradation rate, good tissue compatibility, high strength, appropriate hydrophilicity and hydrophobicity, promoting cell growth and tissue regeneration, and reducing repair time. The specific scheme is as follows.

[0007] The first aspect of the present invention provides an absorbable double-layer membrane for guiding the regeneration and repair of oral gingival tissue, comprising: a dense layer and a loose layer, wherein the dense layer is formed by PCL fibers, and the loose layer is formed by recombinant collagen / polyhydroxyalkanoate blended fibers. 1. The mass ratio of recombinant collagen to polyhydroxyalkanoate is 2-5:8-15.

[0008] The dense layer is formed by PCL, which can be well sutured and plays a protective role during open healing. The loose layer is a recombinant collagen / polyhydroxyalkanoate blended fiber membrane, which improves the hydrophobicity of polyhydroxyalkanoates and the low strength and rapid degradation of single recombinant collagen. It is more conducive to the fixation of blood clots and the growth of soft tissue cells, and reduces inflammatory reactions. At the same time, its degradation time and strength are highly compatible with gingival tissue. The two membrane layers cooperate with each other and have significant clinical application value.

[0009] Preferably, the intrinsic viscosity of the PCL is 0.8-1.0 dL / g, and the test conditions are: 0.1% g / mL, chloroform, 25°C.

[0010] Preferably, the recombinant collagen is type I recombinant collagen.

[0011] Preferably, the polyhydroxyalkanoate is selected from medical grade polyhydroxyalkanoate powder with a purity greater than 99% and a weight average molecular weight of 15-30 Daltons.

[0012] Preferably, the fiber diameter of the dense layer is 1-3 μm and the thickness is 0.2-0.8 mm.

[0013] Further preferably, the thickness of the dense layer is 0.3-0.4 mm.

[0014] Preferably, the fiber diameter of the loose layer is 200-800 μm and the thickness is 1-1.6 mm.

[0015] Further preferably, the fiber diameter of the loose layer is 400-600 μm and the thickness is 1.4-1.5 mm.

[0016] Another aspect of the present invention further provides a method for preparing the absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair, comprising the steps of:

[0017] Preparing a PCL spinning solution and spinning to obtain the dense layer;

[0018] Recombinant collagen spinning solution and polyhydroxyalkanoate spinning solution are prepared respectively, and on the basis of the dense layer, the loose layer is obtained by blending.

[0019] Preferably, in the PCL spinning solution, the PCL content is 8-15wt%; in the recombinant collagen spinning solution, the recombinant collagen content is 2-5wt%; in the polyhydroxyalkanoate spinning solution, the polyhydroxyalkanoate content is 8-15wt%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The shortcomings of poor suturing properties of traditional commercially available acellular dermal matrix have been improved. The dense layer of PCL can be well sutured and plays a protective role during open healing.

[0022] 2) The recombinant collagen / polyhydroxyalkanoate blended fiber loose layer improves the hydrophobicity of polyhydroxyalkanoate and the low strength and rapid degradation of single recombinant collagen, which is more conducive to the fixation of blood clots and the growth of soft tissue cells, and reduces inflammatory response.

[0023] 3) The double-layer membrane of the present invention has a degradation time of 3-5 weeks and a tensile strength of 9-25 MPa by optimizing the components and ratios. The degradation time and strength are highly compatible with gingival tissue, which can better promote the regeneration and repair of gingival tissue and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For the fibroblast proliferation effect. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] Example 1

[0027] A double-layer membrane for guiding oral gingival tissue regeneration and repair comprises a PCL dense layer and a recombinant collagen / polyhydroxyalkanoate blended loose layer. The preparation method comprises the following steps:

[0028] S1 PCL dense layer preparation,

[0029] 1) Preparation of PCL spinning solution: A certain amount of polycaprolactone was added into an organic solvent and completely dissolved to obtain an 8 wt % polycaprolactone solution as the PCL spinning solution; the intrinsic viscosity of polycaprolactone was 0.8 dL / g (0.1% g / mL, chloroform, 25° C.).

[0030] 2) Spinning: Add the obtained PCL spinning solution into the syringe pump of the electrospinning equipment, install the needle, set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, and control the spinning temperature to 20-45°C and the humidity to 20-45% during the spinning process. Repeat the operation to obtain a dense layer with a fiber diameter of 1-2μm and a thickness of 0.3-0.4mm.

[0031] Preparation of S2 recombinant collagen / polyhydroxyalkanoate blended loose layer,

[0032] 1) Preparing a recombinant collagen spinning solution: adding a certain amount of recombinant collagen into an organic solvent and completely dissolving it to obtain a 2 wt % recombinant collagen solution as the recombinant collagen spinning solution; the recombinant collagen is preferably type I recombinant collagen.

[0033] 2) preparing a polyhydroxyalkanoate spinning solution: adding a certain amount of polyhydroxyalkanoate into an organic solvent and dissolving it completely to obtain an 8 wt % polyhydroxyalkanoate solution as the polyhydroxyalkanoate spinning solution, wherein the weight average molecular weight of the polyhydroxyalkanoate is 15w Daltons;

[0034] 3) Spinning: Inject equal volumes of collagen spinning solution and polyhydroxyalkanoate spinning solution into the syringe pump of the double-nozzle electrospinning machine respectively. On the basis of the dense layer, carry out the second spinning process: blending. Set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, control the spinning temperature to 20-45°C, and the humidity to 20-45% during the spinning process to obtain a loose layer with a fiber diameter of 400-600μm and a thickness of 1.4-1.5mm.

[0035] Example 2

[0036] A double-layer membrane for guiding oral gingival tissue regeneration and repair comprises a PCL dense layer and a recombinant collagen / polyhydroxyalkanoate blended loose layer. The preparation method comprises the following steps:

[0037] S1 PCL dense layer preparation,

[0038] 1) Preparation of PCL spinning solution: A certain amount of polycaprolactone was added to an organic solvent and completely dissolved to obtain a 15 wt % polycaprolactone solution as the PCL spinning solution; the intrinsic viscosity of polycaprolactone was 0.8 dL / g (0.1% g / mL, chloroform, 25° C.).

[0039] 2) Spinning: Add the obtained PCL spinning solution into the syringe pump of the electrospinning equipment, install the needle, set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, and control the spinning temperature to 20-45°C and the humidity to 20-45% during the spinning process. Repeat the operation to obtain a dense layer with a fiber diameter of 2-3μm and a thickness of 0.6-0.8mm.

[0040] Preparation of S2 recombinant collagen / polyhydroxyalkanoate blended loose layer,

[0041] 1) Preparing a recombinant collagen spinning solution: adding a certain amount of recombinant collagen into an organic solvent and completely dissolving it to obtain a 5 wt % recombinant collagen solution as the recombinant collagen spinning solution; the recombinant collagen is preferably type I recombinant collagen.

[0042] 2) preparing a polyhydroxyalkanoate spinning solution: adding a certain amount of polyhydroxyalkanoate into an organic solvent and dissolving it completely to obtain a 15 wt% polyhydroxyalkanoate solution as the polyhydroxyalkanoate spinning solution, wherein the weight average molecular weight of the polyhydroxyalkanoate is 20w Daltons;

[0043] 3) Spinning: Inject equal volumes of collagen spinning solution and polyhydroxyalkanoate spinning solution into the syringe pump of the double-nozzle electrospinning machine respectively. On the basis of the dense layer, carry out the second spinning process: blending. Set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, control the spinning temperature to 20-45°C, and the humidity to 20-45% during the spinning process to obtain a loose layer with a fiber diameter of 600-800μm and a thickness of 1.5-1.6mm.

[0044] Example 3

[0045] A double-layer membrane for guiding oral gingival tissue regeneration and repair comprises a PCL dense layer and a recombinant collagen / polyhydroxyalkanoate blended loose layer. The preparation method comprises the following steps:

[0046] S1 PCL dense layer preparation,

[0047] 1) Preparation of PCL spinning solution: A certain amount of polycaprolactone was added to an organic solvent and completely dissolved to obtain a 12 wt % polycaprolactone solution as the PCL spinning solution; the intrinsic viscosity of polycaprolactone was 0.9 dL / g (0.1% g / mL, chloroform, 25° C.).

[0048] 2) Spinning: Add the obtained PCL spinning solution into the syringe pump of the electrospinning equipment, install the needle, set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, and control the spinning temperature to 20-45°C and the humidity to 20-45% during the spinning process. Repeat the operation to obtain a dense layer with a fiber diameter of 2-3μm and a thickness of 0.2-0.4mm.

[0049] Preparation of S2 recombinant collagen / polyhydroxyalkanoate blended loose layer,

[0050] 1) Preparing a recombinant collagen spinning solution: adding a certain amount of recombinant collagen into an organic solvent and completely dissolving it to obtain a 3 wt % recombinant collagen solution as the recombinant collagen spinning solution; the recombinant collagen is preferably type I recombinant collagen.

[0051] 2) preparing a polyhydroxyalkanoate spinning solution: adding a certain amount of polyhydroxyalkanoate into an organic solvent and dissolving it completely to obtain a 12 wt % polyhydroxyalkanoate solution as the polyhydroxyalkanoate spinning solution, wherein the weight average molecular weight of the polyhydroxyalkanoate is 30w Daltons;

[0052] 3) Spinning: Inject equal volumes of collagen spinning solution and polyhydroxyalkanoate spinning solution into the syringe pump of the double-nozzle electrospinning machine respectively. On the basis of the dense layer, carry out the second spinning process: blending. Set the electrospinning voltage to 15V, the liquid feeding speed to 2mL / h, the roller speed to 1100rpm, control the spinning temperature to 20-45°C, and the humidity to 20-45% during the spinning process to obtain a loose layer with a fiber diameter of 500-800μm and a thickness of 1-1.2mm.

[0053] Comparative Example 1

[0054] Commercially available acellular dermal matrix as a repair membrane for oral gingival regeneration.

[0055] Comparative Example 2

[0056] Compared with Example 1, the difference is that the loose layer is electrospun using only recombinant collagen, the dense layer is the same, and the spinning process is also the same.

[0057] Comparative Example 3

[0058] Compared with Example 1, the difference is that the loose layer is electrospun using only polyhydroxyalkanoate, the dense layer is the same, and the spinning process is also the same.

[0059] Comparative Example 4

[0060] Compared with Example 1, the difference is that the amount of collagen and polyhydroxyalkanoate in the loose layer is different from that in the example. The recombinant collagen is a 15wt% recombinant collagen solution, and the polyhydroxyalkanoate is a 5wt% polyhydroxyalkanoate solution. The dense layer is the same, and the spinning process is also the same.

[0061] Testing and analysis:

[0062] 1. Hydrophilicity and hydrophobicity contact angle test

[0063] (1) Experimental methods

[0064] Connect the contact angle tester to the computer terminal; turn on the light source and adjust the lens clarity to see the clearest water drop; place a clean glass slide on the test bench, adjust the upper lifting platform to place the needle within the lens test range; then fix the loose layers of the embodiment and comparative samples respectively for testing; rotate the syringe plunger to empty the bubbles in the syringe, and squeeze out 2 μL of droplet according to the standard during the test; rotate the lifting platform to receive the droplet, press the stopwatch immediately after the droplet leaves the syringe, take a screenshot of the reading immediately after 10 seconds, and finally repeat the above experiment three times to take the average value.

[0065] (2) Results Analysis

[0066] The results are shown in Table 1. As shown in Table 1, the recombinant collagen / polyhydroxyalkanoate blended loose layer has suitable hydrophobicity when the recombinant collagen:polyhydroxyalkanoate=2-5:8-15.

[0067] 2. Tensile strength test

[0068] (1) Test method

[0069] The tensile strength test of the dry film was carried out according to the method specified in GB1030.3-2006. The results are shown in Table 1.

[0070] (2) Results Analysis

[0071] The results are shown in Table 1. As can be seen from Table 1, Examples 1-3 significantly improve the tensile strength of the membrane relative to Comparative Example 1, Comparative Example 2, and Comparative Example 4. The appropriate relative strength can enable the membrane to be smoothly placed vertically in the filling area and facilitate suturing.

[0072] Table 1 Contact angle test results

[0073] serial number Sample name Static contact angle Tensile Strength 1 Example 1 74.7° 15.61MPa 2 Example 2 67.6° 20.24MPa 3 Example 3 71.5° 13.4MPa 4 Comparative Example 1 42.8° 8.95MPa 5 Comparative Example 2 25.3° 2.67MPa 6 Comparative Example 3 115.6° 50.21MPa 7 Comparative Example 4 34.5° 5.76MPa

[0074] 3. Fibroblast proliferation effect

[0075] (1) Experimental methods

[0076] Gingival fibroblasts (hGFs) isolated from gingival tissue were inoculated in a sterile culture dish with a diameter of 10 cm, and cultured in a 37°C 5% CO2 incubator using RPMI1640 medium containing 10% fetal bovine serum. The medium was replaced every 3 days. When the cells grew and proliferated to a state close to confluence, 0.05% trypsin was routinely used for digestion and passage. Fibroblasts of the 4th to 7th generations were selected for the experiment. Then, 1×10 human gingival fibroblasts (hGFs) were selected and cultured. 4 Cells were inoculated in the culture well plates of Example 1 and Comparative Examples 1-3, with the loose layer facing upward, and cultured. Cell proliferation / survival rate was measured after 3, 7, and 14 days of culture. 20 μL of CCK-8 reagent was added to each culture well plate, and the culture well plates were incubated at 37°C and 5% CO2 for 2 hours. Thereafter, 100 μL of each culture was transferred to a separate 96-well plate, and the density (OD) value was measured at 450 nm using a microplate reader. The measurement was repeated 5 times to obtain the average value. The results are shown in FIG. Figure 1 As shown:

[0077] (2) Results Analysis

[0078] from Figure 1 The results show that the embodiment has a significant effect on promoting the growth and proliferation of human gingival fibroblasts compared with the comparative example. After 7 days of culture, the proliferation effect of human gingival fibroblasts is 4-7 times that of comparative example 1, comparative example 2, and comparative example 3, showing significant clinical application potential.

[0079] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair, characterized in that: include: A dense layer and a loose layer, wherein the dense layer is formed by PCL fibers, and the loose layer is formed by recombinant collagen / polyhydroxyalkanoate blended fibers, and the mass ratio of recombinant collagen to polyhydroxyalkanoate is 2-5:8-15.

2. The absorbable double-layer membrane according to claim 1, characterized in that: The intrinsic viscosity of the PCL is 0.8-1.0 dL / g, and the test conditions are: 0.1% g / mL, chloroform, 25°C.

3. The absorbable double-layer membrane according to claim 1, characterized in that: The recombinant collagen is type I recombinant collagen.

4. The absorbable double-layer membrane according to claim 1, characterized in that: The polyhydroxyalkanoate is selected from medical grade polyhydroxyalkanoate powder with a purity greater than 99% and a weight average molecular weight of 15-30w Daltons.

5. The absorbable double-layer membrane according to claim 1, characterized in that: The fiber diameter of the dense layer is 1-3 μm, and the thickness is 0.2-0.8 mm.

6. The absorbable double-layer membrane according to claim 5, characterized in that: The thickness of the dense layer is 0.3-0.4 mm.

7. The absorbable double-layer membrane according to claim 1, characterized in that: The fiber diameter of the loose layer is 200-800 μm, and the thickness is 1-1.6 mm.

8. The absorbable double-layer membrane according to claim 7, characterized in that: The fiber diameter of the loose layer is 400-600 μm, and the thickness is 1.4-1.5 mm.

9. The method for preparing the absorbable double-layer membrane for guiding oral gingival tissue regeneration and repair according to any one of claims 1 to 8, characterized in that: Includes steps: Preparing a PCL spinning solution and spinning to obtain the dense layer; Recombinant collagen spinning solution and polyhydroxyalkanoate spinning solution are prepared respectively, and on the basis of the dense layer, the loose layer is obtained by blending.

10. The preparation method according to claim 9, characterized in that: In the PCL spinning solution, the PCL content is 8-15wt%; in the recombinant collagen spinning solution, the recombinant collagen content is 2-5wt%; in the polyhydroxyalkanoate spinning solution, the polyhydroxyalkanoate content is 8-15wt%.

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