CN102166378B - Tissue regeneration guiding membrane and preparation method thereof - Google Patents

Abstract

The invention relates to a guided tissue regeneration membrane and a preparation method thereof, which solves the technical problems of limited membrane thickness, poor cell growth effect and limited strength in the prior art. The guided tissue regeneration membrane provided by the invention , has a layered structure, and its arrangement sequence is as follows: randomly arranged fiber layer with random fiber arrangement, parallel fiber layer with parallel fiber arrangement and grid arrangement fiber layer with grid fiber arrangement Layer, the fiber layer above all contains aliphatic polyester, and the guided tissue regeneration membrane provided by the present invention can better meet the clinical needs of guided tissue regeneration and repair.

Description

Guided tissue regeneration film and preparation method thereof

technical field

The invention relates to a medical device and a preparation method thereof, in particular to a tissue regeneration guiding membrane and a preparation method thereof.

Background technique

The guided tissue regeneration technique was first used to regenerate periodontal tissue lost in the development of periodontal disease and to generate new periodontal attachments. The principle is to cover the loss of periodontal bone and the exposed root with the diaphragm, and use the physical barrier function of the diaphragm to prevent the gingival epithelial cells and connective tissue from intruding into the root and create space for the regeneration of periodontal tissue.

Nonabsorbable and bioabsorbable guided tissue regeneration membranes have similar histological and clinical repair effects as mechanical isolation barriers. However, the non-absorbable guided tissue regeneration membrane represented by polytetrafluoroethylene cannot be degraded in the body, so it needs to be removed by a second operation, which not only increases the pain of the patient, but also increases the possibility of postoperative infection. Moreover, since it is difficult to compound biologically active substances and antibiotics, the non-absorbable film can only play a simple barrier role.

The development of bioabsorbable guided tissue regeneration membrane has comprehensively improved the above-mentioned deficiencies. Natural biodegradable materials with excellent biological properties such as collagen, gelatin, chitosan, etc., synthetic biodegradable materials with good mechanical properties such as polylactic acid, polylactic acid-glycolic acid, polyamide, etc., and osteoinductive growth Capable inorganic components, such as nano-hydroxyapatite, have been used in the preparation of bioabsorbable guided tissue regeneration membranes. However, single-component, single-layer membrane materials cannot meet the requirements of biocompatibility, mechanical stability, osteoinductive generation ability, epithelial cells and connective tissue barrier properties for guided tissue regeneration membranes. The guided tissue regeneration membrane with gradient functional changes in structure and composition can better meet clinical requirements.

Chinese invention patent CN1216653C discloses a method for preparing a nanocrystalline hydroxyapatite/collagen/polylactic acid-glycolic acid composite membrane material for guiding tissue regeneration. The prepared membrane has a rough structure on one side and a smooth surface on the other.

Chinese invention patent CN1319604C discloses a double-layer composite collagen-based guided tissue regeneration material and a preparation method thereof. A double-layer composite material with a dense layer and a loose layer structure is prepared from collagen and hyaluronic acid or its sodium salt.

Chinese invention patent CN100408115C discloses a biomaterial membrane with a porous structure and its preparation method, which is characterized in that the pore diameter gradually changes between the two surfaces of the medical polyamide component or the medical polyamide/nano-like bone apatite composite component membrane. Interpenetrating pores are distributed, one side of the membrane is a dense surface with small pores with a pore diameter of 0.01-30um, and the other side is a loose surface with large pores with a pore diameter of 30-300um.

The loose and porous side of these membrane materials can face the defect area, which is conducive to tissue regeneration, and the dense side can face the surrounding tissue, preventing epithelial cells and connective tissue from entering the defect area. Although such a membrane structure can meet the clinical requirements of guided tissue regeneration membranes, based on the fiber network characteristics of natural extracellular matrix, from the perspective of bionics, electrospinning technology has been applied in the preparation of multilayer guided tissue regeneration membranes. .

In the Chinese invention patent publication CN101584885A, a certain proportion of natural biomaterials, degradable polymers, and nano-hydroxyapatite mixtures are dissolved in a specific solvent, and a three-layer method is prepared by layer-by-layer electrospinning and using aluminum foil as a receiver. Gradient composite membrane, one side of the membrane material is rough and loose, and the other side is smooth and dense. However, in this invention patent publication, due to the change of the electric field strength, the thickness of the membrane prepared by layer-by-layer electrospinning will be limited; in addition, the non-woven fiber membrane collected by aluminum foil as the receiver, although the porosity is high, but Often due to the high fiber packing density, the effect of cell growth is inhibited, and the strength is also very limited.

Contents of the invention

The purpose of the present invention is to solve the technical problems of limited membrane thickness, poor effect of cell growth and limited strength in the prior art, and provide a membrane for guiding tissue regeneration and a preparation method thereof.

To this end, the present invention provides a guiding tissue regeneration membrane, which has a layered structure, and its arrangement order is as follows: a randomly arranged fiber layer with randomly arranged fibers, a parallel arranged fiber layer with parallel arranged fibers and a grid-arranged fiber layer in which the fibers are arranged in a grid, and the above-mentioned fiber layers all contain aliphatic polyester.

The preferred technical scheme of the present invention is that the randomly arranged fiber layer also contains natural polymers; the grid arranged fiber layer also contains natural polymers; the grid arranged fiber layer also contains nano-hydroxyapatite; the film thickness is 0.05- 2 mm, and more preferably a thickness of 0.1 to 0.5 mm; the aliphatic polyester is poly-L-lactic acid, polycaprolactone, polylactic acid-glycolic acid, polylactic acid-caprolactone, polylactic acid-glycolic acid-caprolactone One; the natural polymer is a kind of collagen or gelatin; the number n of fiber layers satisfies 3≤n≤30, the number x of randomly arranged fiber layers, the number y of parallel arranged fiber layers and the grid arrangement The number z of cloth fiber layers satisfies 1≦x, y, z≦10; the laying angle of multiple parallel fiber layers is 0-180°.

The present invention simultaneously provides a method for guiding tissue regeneration membrane, which comprises the following steps:

(1) Dissolve aliphatic polyester in a solvent, stir at room temperature for 6 to 24 hours to obtain a solution A with a concentration of 0.05 to 0.4 g/ml, carry out electrospinning of the above solution A, use a stainless steel drum as a receiving device, and receive parallel arrangement of fiber layers;

(2) Dissolve the natural polymer in a solvent, stir at room temperature for 6 to 24 hours to obtain a solution B with a concentration of 0.05 to 0.4 g/ml, mix the above solution B with solution A, and obtain a total solution concentration of 0.05 to 0.4 g/ml solution C, the weight ratio of aliphatic polyester and natural polymer in the solution C is 90/10～10/90, the solution C is subjected to electrospinning, with aluminum foil and copper mesh as the receiving device respectively, receiving a randomly arranged fiber layer and a grid arranged fiber layer;

(3) Disperse nano-hydroxyapatite in a solvent, obtain a suspension with a content of 0.1 to 0.5 g/ml after ultrasonic dispersion, take the above-mentioned suspension and add solution A or solution C to obtain polymer and nano-hydroxyapatite A solution with a weight ratio of 100/0 to 70/30, the above solution is electrospun, and the copper mesh is used as the receiving device to receive the grid-arranged fiber layer;

(4) stretching the fiber film obtained in step (1) along the direction of fiber arrangement, the drafting force is 200-500g, the drafting temperature is 50-100°C, and the drafting rate is 100-300%;

(5) Place the fiber membranes obtained in steps (2), (3) and (4) in a vacuum drying oven at a constant temperature of 25-35° C., and dry for 12-48 hours at a vacuum degree lower than 40 Pa;

(6) The fiber layers obtained in step (5) are superimposed in the order of parallel fiber layers in the middle, random fiber layers and grid fiber layers on both sides, and then use a concentration of 0.001- After infiltration with 0.02g/ml natural polymer aqueous solution, freeze-dry to obtain the guided tissue regeneration membrane.

The preferred technical solution of the present invention is that the solvent is one of trifluoroethanol or hexafluoroisopropanol; the aliphatic polyester is poly-L-lactic acid, polycaprolactone, polylactic acid-glycolic acid, polylactic acid-caprolactone 1. One of polylactic acid-glycolic acid-caprolactone; the natural polymer is one of collagen or gelatin; the copper mesh used in steps (2) and (3) has an aperture diameter of 100-550 μm.

The guided tissue regeneration membrane provided by the present invention, from the side facing the surrounding tissue to the side facing the defect area, is: randomly arranged fiber layers, which can face the surrounding tissue, and high-density fiber accumulation prevents the entry of epithelial or connective tissue The growth can be prevented, and at the same time, it is beneficial to the attachment and growth of epithelial or fibroblasts; the parallel arrangement of fiber layers can ensure the mechanical strength of the membrane, and at the same time prevent the growth of epithelial or fibroblasts into the defect; The fiber layer is arranged in grids, which can face the defect area, and the loose fiber arrangement area between the grids is conducive to the attachment and growth of bone or periodontal ligament cells. The details are shown in Table 1 and accompanying drawings 4, 5, 6, and 7.

Table 1: Comparison of mechanical properties of poly-L-lactic acid fiber membranes with different fiber arrangements (n=10, p<0.05)

The invention adopts composite biodegradable aliphatic polyester and natural polymer as the main components on both sides of the guided tissue regeneration membrane of gradient functional structure, and the hydrophobic and degradable aliphatic polyester component is beneficial to the fiber membrane after being exposed to water and heat. The structure maintenance, hydrophilic natural polymer components are beneficial to improve biocompatibility and cell affinity.

In the grid arrangement fiber layer of the present invention, the addition of the nano-hydroxyapatite component improves the induction of bone regeneration.

The present invention can achieve independent control of a single functional layer through the combination of different fiber layers, and can achieve multi-angle (0-180°) layering of biodegradable aliphatic polyester fiber membranes with multiple layers of fibers arranged in parallel, Make the guided tissue regeneration membrane has isotropic high mechanical strength.

In the invention, the multiple fiber layers of the lay-up are compounded through the natural polymer aqueous solution, so that the interlayers are well bonded and biocompatibility is increased at the same time.

In the invention, by selecting biodegradable aliphatic polyesters with different compositions, the gradient functional structure is regulated to guide the degradation rate and in vivo absorption rate of the tissue regeneration membrane.

The present invention uses electrospinning technology to prepare fibrous membranes with differences in composition and arrangement, and then laminates and composites, which not only makes it easier to realize the gradient functional structure of the guiding tissue regeneration membrane, but also makes the guiding tissue regeneration membrane The thickness and mechanical properties are easier to control, and the introduction and controlled release of growth factors and antibiotics are easier to achieve, which can better meet the clinical needs of guiding tissue regeneration and repair.

Below in conjunction with the accompanying drawings, the content of the present invention will be described in detail with specific embodiments, but the present invention is not limited to the following examples, without departing from the above-mentioned technical ideas of the present invention, made according to common technical knowledge and conventional means in the art Various substitutions and changes should be included within the scope of the present invention.

Description of drawings

Fig. 1 is the scanning electron micrograph (JSM 4700 type scanning electron microscope, Japan Electronics) of the poly-L-lactic acid/gelatin (50/50, w/w) composite fiber film of the fiber random arrangement that electrospinning of the present invention prepares;

Fig. 2 is the scanning electron micrograph (JSM 4700 type scanning electron microscope, Japan Electronics) of the poly-L-lactic acid fiber film that the fiber that electrospinning of the present invention prepares is arranged in parallel;

Fig. 3 is the scanning electron microscope photo (JSM 4700 type scanning electron microscope, Japan Electronics) of the poly-L-lactic acid/nanometer hydroxyapatite composite fiber film that the fiber that electrospinning of the present invention prepares is grid arrangement;

Figure 4 is a laser confocal microscope observation of cell stress fibers after periodontal ligament cells were cultured on the poly-L-lactic acid fiber layer with randomly arranged fibers (from left to right: from the surface to the inside of the fiber layer, the thickness of the slice is 1.18 μm );

Figure 5 is a laser confocal microscope observation of cell stress fibers after periodontal ligament cells were cultured on a 100% hot-stretched parallel poly-L-lactic acid fiber layer for 7 days (from left to right: from the surface of the fiber membrane to the inside, slice thickness 1.17 μm);

Figure 6 is the SEM morphological observation photos of periodontal ligament cells cultured on the grid-type poly-L-lactic acid fiber layer for 3 days (a: x 200; b: x 500; c: x 2000);

Figure 7 is a laser confocal microscope observation of cell stress fibers in the non-woven region after periodontal ligament cells were cultured on the grid-type poly-L-lactic acid nanofibrous membrane for 7 days (from left to right: from the surface of the fiber membrane to the inside, section Thickness 5μm).

Detailed ways

Example 1

1. Dissolve poly-L-lactic acid (produced by Shandong Medical Device Research Institute, the same below) in trifluoroethanol (produced by Shanghai Chemical Reagent Co., Ltd., analytically pure, the same below), and stir at room temperature for 12 hours to obtain a concentration of 10% (g/ ml) of solution A;

2. Dissolve gelatin (manufactured by Sigma-Aldrich, the same below) in trifluoroethanol, and stir at room temperature for 12 hours to obtain solution B with a concentration of 10% (g/ml);

3. Mix solutions A and B in equal proportions to obtain solution C, the weight ratio of poly-L-lactic acid to gelatin is 1/1, and the total polymer concentration is 10% (g/ml);

4. Take solution A for electrospinning, use a stainless steel drum as the receiving device, the drum winding speed is 12m/s, the voltage is 12kV, the receiving distance is 15cm, and the flow rate is 0.8mL/h to obtain a poly-L-lactic acid fiber membrane with fibers arranged in parallel. Silk 10h, thickness about 100μm;

5. Take solution C for electrospinning, using aluminum foil as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, to obtain a poly-L-lactic acid/gelatin composite fiber membrane with randomly arranged fibers, spinning for 10 hours, with a thickness of about 50μm;

6. Take solution C for electrospinning, use copper mesh with 400 μm mesh as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, and obtain poly-L-lactic acid/gelatin composite fiber membrane with fibers arranged in a grid , spinning for 10 hours, with a thickness of about 50 μm;

7. Place the fiber membranes obtained in steps 4, 5, and 6 in a vacuum drying oven at a constant temperature of 30°C and a vacuum of less than 12 Pa, and dry for 24 hours;

8. Stretching the poly-L-lactic acid fiber film obtained in step 4 with fibers arranged in parallel along the direction of fiber arrangement, the drafting force is 300g, the drafting temperature is 100°C, and the drafting rate is 100%;

9. Dry the fiber membranes obtained in steps 5 and 6 and stack them successively with the fiber membranes stretched in step 8, impregnate them with 0.1% (g/ml) gelatin aqueous solution, and freeze-dry to obtain a gradient functional composite fiber membrane , the film thickness is about 200 μm.

The used accessories of the electrospinning device used in the present invention are as follows (the following examples are the same): the Baoding Lange constant current pump whose model is TS2-60 is selected for use in the medical injection pump; 30kV, 3mA products; the high-speed drum receiving device is self-made, and the specification is 0-7000rpm.

Example 2

1. Dissolve polylactic acid-glycolic acid (produced by Shandong Medical Device Research Institute, the same below) in hexafluoroisopropanol, and stir at room temperature for 24 hours to obtain a solution A with a concentration of 40% (g/ml);

2. Dissolve gelatin in hexafluoroisopropanol and stir at room temperature for 24 hours to obtain solution B with a concentration of 40% (g/ml);

3. Mix 3ml of solution A and 1ml of solution B to obtain solution C, the weight ratio of polylactic acid-glycolic acid to gelatin is 3/1, and the total polymer concentration is 40% (g/ml);

4. Disperse a certain amount of nano-hydroxyapatite in hexafluoroisopropanol, disperse with 400W ultrasonic for 5 minutes to obtain a paste suspension D, and the content of nano-hydroxyapatite is 50% (g/ml);

5. Take 10ml of solution A and 2ml of pasty suspension D and mix to obtain solution E. The weight ratio of polymer to nano-hydroxyapatite is 4/1;

6. Take solution A for electrospinning, using a stainless steel drum as the receiving device, the drum winding speed is 12m/s, the voltage is 12kV, the receiving distance is 15cm, and the flow rate is 0.5mL/h to obtain a polylactic acid-glycolic acid fiber membrane with parallel fibers , spinning for 10 hours, with a thickness of about 400 μm;

7. Take solution C for electrospinning, using aluminum foil as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.5mL/h, to obtain polylactic acid-glycolic acid/gelatin composite fiber membrane with random fiber arrangement, spinning for 10h, The thickness is about 400μm;

8. Take solution E for electrospinning, using copper mesh with 400 μm mesh as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.5mL/h, to obtain polylactic acid-glycolic acid/nano-hydroxy Apatite composite fiber membrane, spinning for 10 hours, with a thickness of about 400 μm;

9. Place the fiber membranes obtained in steps 6, 7, and 8 in a vacuum drying oven at a constant temperature of 35°C and a vacuum degree lower than 12 Pa, and dry for 48 hours;

10. Stretch the polylactic acid-glycolic acid fiber film obtained in step 6 with the fibers arranged in parallel along the fiber arrangement direction, with a drafting force of 500 g, a drafting temperature of 70° C., and a drafting rate of 200%;

11. The fiber membranes obtained in steps 5 and 6 are dried and stacked successively with the fiber membranes stretched in step 8, impregnated with 2% (g/ml) gelatin aqueous solution, and freeze-dried to obtain a gradient functionalized composite fiber membrane. The film thickness is 1mm.

Example 3

1. Dissolve polycaprolactone (produced by Shandong Institute of Medical Devices, the same below) in trifluoroethanol, and stir at room temperature for 6 hours to obtain solution A with a concentration of 5% (g/ml);

2. Dissolve gelatin in trifluoroethanol and stir at room temperature for 6 hours to obtain solution B with a concentration of 5% (g/ml);

3. Mix solutions A and B in equal proportions to obtain solution C, the weight ratio of polycaprolactone to gelatin is 1/1, and the total polymer concentration is 5% (g/ml);

4. Disperse a certain amount of nano-hydroxyapatite in trifluoroethanol, and disperse with 400W ultrasonic for 5 minutes to obtain paste suspension D, the content of nano-hydroxyapatite is 50% (g/ml);

5. Take 10ml of solution C and 0.2ml of pasty suspension D and mix to obtain solution F. The weight ratio of polymer to nano-hydroxyapatite is 5/1;

6. Take solution A and carry out electrospinning, with a stainless steel drum as the receiving device, the drum winding speed is 12m/s, the voltage is 12kV, the receiving distance is 15cm, and the flow rate is 0.4mL/h to obtain a polycaprolactone fiber membrane with parallel fibers. Spinning for 10 hours, the thickness is about 20 μm;

7. Take solution C for electrospinning, using aluminum foil as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, to obtain polycaprolactone/gelatin composite fiber membrane with randomly arranged fibers, spinning for 10h, thickness About 20 μm;

8. Take solution F for electrospinning, use copper mesh with 100 μm mesh as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, and obtain polycaprolactone/gelatin/nano Hydroxyapatite composite fiber membrane, spinning for 10 hours, with a thickness of about 20 μm;

9. Place the fiber membranes obtained in steps 6, 7, and 8 in a vacuum drying oven at a constant temperature of 25°C and a vacuum degree lower than 40Pa, and dry for 12 hours;

10. Stretch the polycaprolactone fiber film obtained in step 6 with the fibers arranged in parallel along the fiber arrangement direction, with a drafting force of 200 g, a drafting temperature of 50° C., and a drafting rate of 300%;

11. Dry the fiber membranes obtained in steps 5 and 6 and stack them successively with the fiber membranes stretched in step 8, impregnate them with 0.5% (g/ml) gelatin aqueous solution, and freeze-dry to obtain a gradient functionalized composite fiber membrane. The film thickness is 0.05mm.

Example 4

1. Dissolve polylactic acid-glycolic acid-caprolactone (produced by Shandong Medical Device Research Institute, the same below) in trifluoroethanol, and stir at room temperature for 24 hours to obtain a solution A with a concentration of 30% (g/ml);

2. Dissolve gelatin in trifluoroethanol and stir at room temperature for 12 hours to obtain solution B with a concentration of 20% (g/ml);

3. Mix 2ml of solution A with 3ml of solution B to obtain solution C. The weight ratio of poly(lactic acid-glycolic acid-caprolactone) to gelatin is 1/1, and the total polymer concentration is 24% (g/ml);

4. Disperse a certain amount of nano-hydroxyapatite in trifluoroethanol, and disperse with 400W ultrasonic for 5 minutes to obtain paste suspension D, the content of nano-hydroxyapatite is 50% (g/ml);

5. Take 10ml of solution C and 0.5ml of pasty suspension D and mix to obtain solution F. The weight ratio of polymer to nano-hydroxyapatite is 10/1;

6. Take solution A for electrospinning, using a stainless steel drum as the receiving device, the drum winding speed is 12m/s, the voltage is 15kV, the receiving distance is 18cm, and the flow rate is 0.8mL/h to obtain polylactic acid-glycolic acid-hexyl Lactone fiber membrane, spinning for 10 hours, with a thickness of about 250 μm;

7. Take solution C for electrospinning, using aluminum foil as the receiving device, voltage 15kV, receiving distance 20cm, flow rate 0.4mL/h, to obtain polylactic acid-glycolic acid-caprolactone/gelatin composite fiber membrane with random fiber arrangement, Spinning 20h, the thickness is about 150μm;

8. Take solution F for electrospinning, use copper mesh with 400 μm mesh as the receiving device, voltage 15kV, receiving distance 20cm, flow rate 0.4mL/h, and obtain polylactic acid-glycolic acid-hexanol with fibers arranged in a grid Ester/gelatin/nano-hydroxyapatite composite fiber membrane, spinning for 20 hours, with a thickness of about 150 μm;

9. Place the fiber membranes obtained in steps 6, 7, and 8 in a vacuum drying oven at a constant temperature of 30°C and a vacuum degree lower than 12 Pa, and dry for 24 hours;

10. Stretch the polylactic acid-glycolic acid-caprolactone fiber film obtained in step 6 with the fibers arranged in parallel along the direction of fiber arrangement, with a drafting force of 200 g, a drafting temperature of 70° C., and a drafting rate of 100%;

11. Dry the fiber membranes obtained in steps 5 and 6 and stack them successively with the fiber membranes stretched in step 8, impregnate them with 0.5% (g/ml) gelatin aqueous solution, and freeze-dry to obtain a gradient functionalized composite fiber membrane. The film thickness is 650 μm.

Example 5

1. Dissolve polylactic acid-caprolactone in trifluoroethanol and stir at room temperature for 12 hours to obtain solution A with a concentration of 10% (g/ml);

2. Dissolve collagen in trifluoroethanol and stir at room temperature for 12 hours to obtain a solution B with a concentration of 10% (g/ml);

3. Mix solutions A and B in equal proportions to obtain solution C, the weight ratio of polylactic acid-caprolactone to collagen is 1/1, and the total polymer concentration is 10% (g/ml);

4. Disperse a certain amount of nano-hydroxyapatite in trifluoroethanol, and disperse with 400W ultrasonic for 5 minutes to obtain paste suspension D, the content of nano-hydroxyapatite is 50% (g/ml);

5. Take 10ml of solution C and 0.2ml of pasty suspension D and mix to obtain solution F. The weight ratio of polymer to nano-hydroxyapatite is 10/1;

6. Take solution A for electrospinning, use a stainless steel drum as the receiving device, the drum winding speed is 12m/s, the voltage is 12kV, the receiving distance is 15cm, and the flow rate is 0.8mL/h to obtain polylactic acid-glycolic acid-hexyl Lactone fiber membrane, spinning for 10 hours, with a thickness of about 100 μm;

7. Take solution C for electrospinning, using aluminum foil as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, to obtain polylactic acid-caprolactone/collagen composite fiber membrane with random fiber arrangement, spinning for 10h , with a thickness of about 50 μm;

8. Take solution F for electrospinning, use copper mesh with 400 μm mesh as the receiving device, voltage 12kV, receiving distance 20cm, flow rate 0.4mL/h, and obtain polylactic acid-caprolactone/collagen with fibers arranged in a grid / Nano-hydroxyapatite composite fiber membrane, spinning for 10 hours, with a thickness of about 50 μm;

9. Place the fiber membranes obtained in steps 6, 7, and 8 in a vacuum drying oven at a constant temperature of 30°C and a vacuum degree lower than 12 Pa, and dry for 24 hours;

10. Stretching the polylactic acid-caprolactone fiber film obtained in step 6 with the fibers arranged in parallel along the fiber arrangement direction, the drafting force is 300g, the drafting temperature is 100°C, and the drafting rate is 100%;

11. Dry the fibrous membranes obtained in steps 5 and 6 and stack them with the stretched fibrous membranes in step 8 in sequence, impregnate them with 0.5% (g/ml) collagen aqueous solution, and freeze-dry to obtain a gradient functionalized composite fibrous membrane , film thickness 200 μm.

Example 6

1. The preparation method of the polylactic acid-glycolic acid-caprolactone/gelatin composite fiber membrane with randomly arranged fibers is the same as in Example 4;

2. The preparation method of the poly(lactic acid-glycolic acid-caprolactone) fiber membrane with fibers arranged in parallel is the same as in Example 4;

3. The polylactic acid-glycolic acid-caprolactone/gelatin/nano-hydroxyapatite composite fiber membrane with fibers arranged in a grid is the same as in Example 4;

4. The polylactic acid-glycolic acid-caprolactone/gelatin composite fiber membrane with 4 layers of fibers arranged randomly, the polylactic acid-glycolic acid-caprolactone fiber membrane with 4 layers of fibers arranged in parallel, and the 4 layers of fibers are arranged in a grid The polylactic acid-glycolic acid-caprolactone/gelatin/nano-hydroxyapatite composite fiber membranes of the cloth are stacked in sequence, and the four layers of parallel fibers , 135 ° four directions for lamination, impregnated with 0.5% (g/ml) gelatin aqueous solution, and freeze-dried to obtain a gradient functional composite fiber membrane with a thickness of about 2 mm.

Example 7

1. The preparation method of the poly-L-lactic acid/gelatin composite fiber membrane with randomly arranged fibers is the same as in Example 1;

2. The preparation method of the poly-L-lactic acid fiber membrane with fibers arranged in parallel is the same as in Example 1;

3. The poly-L-lactic acid/gelatin composite fiber membrane in which the fibers are arranged in a grid is the same as in Example 1;

4. Stack 10 layers of polylactic acid/gelatin composite fiber membranes with randomly arranged fibers, 10 layers of polylactic acid fiber membranes with fibers arranged in parallel, and 10 layers of polylactic acid/gelatin composite fiber membranes with fibers arranged in a grid. Layers of polylactic acid fiber membranes with fibers arranged in parallel are laminated in four directions of 0, 45, 90, and 135°, impregnated with 0.5% (g/ml) gelatin aqueous solution, and freeze-dried to obtain a gradient functional composite fiber membrane. 2mm thick.

Claims (5)

1. the preparation method of a guide tissue regeneration film, described guide tissue regeneration film has layer structure, it puts in order and is followed successively by: fiber is parallel arrangement fibrous layer that the random random fibrous layer of arranging of arranging, fiber be parallel arrangement and fiber and is grid that grid the arranges fibrous layer of arranging, all contain aliphatic polyester in the above-mentioned fibrous layer, it is characterized in that containing and have the following steps:

(1) aliphatic polyester being dissolved in solvent, stirring under the room temperature and obtained the solution A that concentration is 0.05～0.4g/mL in 6～24 hours, above-mentioned solution A is carried out electrostatic spinning, is receiving system with the stainless steel drum, receives the parallel arrangement fibrous layer;

(2) natural polymer is dissolved in solvent, stirred under the room temperature 6～24 hours, obtain the solution B that concentration is 0.05～0.4g/mL, above-mentioned solution B is mixed with solution A, obtain the solution C that the solution total concentration is 0.05～0.4g/mL, the weight ratio of aliphatic polyester and natural polymer is 90/10～10/90 in this solution C, and this solution C is carried out electrostatic spinning, be receiving system with aluminium foil and copper mesh respectively, receive random arrange fibrous layer and the grid fibrous layer of arranging;

(3) nanometer hydroxyapatite is scattered in solvent, obtaining nanometer hydroxyapatite content behind the ultra-sonic dispersion is 0.1～0.5g/mL suspension, get above-mentioned suspension and add solution A or solution C, the weight ratio that obtains polymer and nanometer hydroxyapatite is 100/0～70/30 solution, above-mentioned solution is carried out electrostatic spinning, be receiving system with the copper mesh, receive the grid fibrous layer of arranging;

(4) fibrous membrane that step (1) is obtained carries out drawing-off along the fiber alignment direction, drafting force 200～500g, 50～100 ℃ of drawing temperatures, degree of draft 100～300%;

(5) fibrous membrane that step (2), (3), (4) are obtained places vacuum drying oven, 25～35 ℃ of constant temperature, and vacuum is lower than under the 40Pa, dry 12～48h;

(6) with the fibrous layer that obtains in the step (5) by the parallel arrangement fibrous layer in the centre, random arrange fibrous layer and the grid order of fibrous layer in both sides of arranging superposes, after being the natural polymer solution immersion of 0.001～0.02g/mL with concentration then, lyophilization obtains guide tissue regeneration film.

2. the preparation method of guide tissue regeneration film according to claim 1 is characterized in that described solvent is trifluoroethanol or hexafluoroisopropanol.

3. the preparation method of guide tissue regeneration film according to claim 1 is characterized in that described aliphatic polyester is a kind of in Poly-L-lactic acid, polycaprolactone, polylactic acid-glycolic guanidine-acetic acid, polylactic acid-caprolactone, the polylactic acid-glycolic guanidine-acetic acid-caprolactone.

4. the preparation method of guide tissue regeneration film according to claim 1 is characterized in that natural polymer is a kind of in collagen or the gelatin.

5. the preparation method of guide tissue regeneration film according to claim 1 is characterized in that copper mesh aperture used in described step (2) and (3) is 100～550 μ m.

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