Method for preparing a functionally gradient material for guided periodontal hard and soft tissue regeneration

Abstract

A functionally gradient material for guided periodontal hard and soft tissue regeneration includes a 3D printed scaffold layer and an electrospun fibrous membrane layer. The content of hydroxyapatite in the 3D printed scaffold layer is higher than the content of hydroxyapatite in the electrospun fibrous membrane layer. The pore size of the 3D printed scaffold layer is larger than the pore size of the electrospun fibrous membrane layer. The pore size of the 3D printed scaffold layer is 100-1000 μm, and the fiber diameter of the electrospun fibrous membrane layer is 300-5000 nm. The electrospun fibrous membrane layer is in a random distribution or an oriented arrangement or has a mesh structure. The thickness of the electrospun fibrous membrane layer is 0.08-1 mm.

Claims

1. A method for preparing a functionally gradient material for a guided periodontal hard and soft tissue regeneration, the functionally gradient material comprising a 3D printed scaffold layer and an electrospun fibrous membrane layer, wherein a content of hydroxyapatite in the 3D printed scaffold layer is higher than a content of hydroxyapatite in the electrospun fibrous membrane layer; a pore size of the 3D printed scaffold layer is larger than a pore size of the electrospun fibrous membrane layer; the pore size of the 3D printed scaffold layer is 100 μm-1000 μm; a fiber diameter of the electrospun fibrous membrane layer is 300 nm-5000 nm; the electrospun fibrous membrane layer is in a random distribution or an oriented arrangement or has a mesh structure; and a thickness of the electrospun fibrous membrane layer is 0.08 mm-1 mm, the method comprising the following steps: S1, ultrasonically dispersing the hydroxyapatite in a solvent for 1 h-2 h to obtain a dispersion solution, then adding fish collagen and poly (lactic-co-glycolic acid) to the dispersion solution to obtain a mixture, shaking well the mixture for 1.5 h-3 h, and then ultrasonically dispersing the mixture for 0.5 h-1 h to obtain a spinning solution; S2, stirring the spinning solution obtained in step S1 to evaporate the solvent to obtain a bio-ink; S3, preparing the electrospun fibrous membrane layer by using the spinning solution obtained in step S1 via an electrospinning; and S4, placing the electrospun fibrous membrane layer obtained in step S3 on a platform of a 3D bioprinter, and printing on the electrospun fibrous membrane layer by the 3D bioprinter using the bio-ink obtained in step S2 to construct the functionally gradient material compounded by the electrospun fibrous membrane layer with the 3D printed scaffold layer.

2. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the content of the hydroxyapatite in the electrospun fibrous membrane layer is 5 wt %-40 wt %, and a content of the fish collagen in the electrospun fibrous membrane layer is 1 wt %-30 wt %.

3. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the content of the hydroxyapatite in the 3D printed scaffold layer is 10 wt %-70 wt %.

4. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the hydroxyapatite in step S1 comprises short rod-shaped hydroxyapatite, needle-shaped hydroxyapatite, microspheric hydroxyapatite, and mesoporous hydroxyapatite.

5. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the poly (lactic-co-glycolic acid) in step S1 is replaced with one selected from the group consisting of polycaprolactone, polylactic acid, polyurethane, and chitosan.

6. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the fish collagen in step S1 is derived from a fish skin or a fish scale of a fish, and the fish is one selected from the group consisting of a cod, a tilapia, a grass carp and a silver carp.

7. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the solvent in step S1 is one selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, dichloromethane, acetone, N, N-dimethylformamide, a mixed solution of the trifluoroethanol and the N, N-dimethylformamide in a volume ratio of 7-9:1-3, and a mixed solution of the acetone and the N, N-dimethylformamide in a volume ratio of 2-4:1.

8. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, step S3 specifically comprises: collecting randomly distributed fibrous membranes, orientedly arranged fibrous membranes and mesh fibrous membranes by using a flat plate collector, an oriented collector and a mesh collector, respectively, and the electrospinning is performed under process parameters comprising an applied voltage of 7 kV-12 kV, a receiving distance of 12-18 cm and an injection rate of 0.3 mL/h-0.6 mL/h; wherein a rotational speed of a roller of the oriented collector is 2000 r/min-4000 r/min, and a mesh aperture size of the mesh collector is 400 μm-800 μm.

9. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, an extruded filament of the 3D printed scaffold layer in step S4 has a diameter of 0.1 mm-0.4 mm.

10. The method for preparing the functionally gradient material for the guided periodontal hard and soft tissue regeneration according to claim 1, wherein, the hydroxyapatite is replaced with calcium phosphate or calcium silicate.

11. A method for preparing a functionally gradient material for a guided periodontal hard and soft tissue regeneration, the functionally gradient material comprising a 3D printed scaffold layer and an electrospun fibrous membrane layer, wherein a content of hydroxyapatite in the 3D printed scaffold layer is higher than a content of hydroxyapatite in the electrospun fibrous membrane layer; a pore size of the 3D printed scaffold layer is larger than a pore size of the electrospun fibrous membrane layer; the pore size of the 3D printed scaffold layer is 100 μm-1000 μm; a fiber diameter of the electrospun fibrous membrane layer is 300 nm-5000 nm; the electrospun fibrous membrane layer is in a random distribution or an oriented arrangement or has a mesh structure; and a thickness of the electrospun fibrous membrane layer is 0.08 mm-1 mm, the method comprising the following steps: S1, ultrasonically dispersing the hydroxyapatite in a solvent for 1 h-2 h to obtain a dispersion solution, then adding fish collagen and a reagent to the dispersion solution to obtain a mixture, shaking well the mixture for 1.5 h-3 h, and then ultrasonically dispersing the mixture for 0.5 h-1 h to obtain a spinning solution, wherein the reagent is selected from the group consisting of polycaprolactone, polylactic acid, polyurethane, and chitosan; S2, stirring the spinning solution obtained in step S1 to evaporate the solvent to obtain a bio-ink; S3, preparing the electrospun fibrous membrane layer by using the spinning solution obtained in step S1 via an electrospinning; and S4, placing the electrospun fibrous membrane layer obtained in step S3 on a platform of a 3D bioprinter, and printing on the electrospun fibrous membrane layer by the 3D bioprinter using the bio-ink obtained in step S2 to construct the functionally gradient material compounded by the electrospun fibrous membrane layer with the 3D printed scaffold layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced hereinafter. It should be understood that the drawings only show some embodiments of the present invention and thus should not be construed as a limitation on the scope. Those having ordinary skill in the art can also obtain other relevant drawings according to these drawings without creative efforts.

(2) FIG. 1 is an image showing the morphology of the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane;

(3) FIG. 2 is a graph showing the fiber diameter distribution of the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane;

(4) FIG. 3 is an image showing the morphology of the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane after being degraded in a phosphate buffer solution for 8 weeks;

(5) FIG. 4 is an image showing the repair effect in the defect site reconstructed by micro computed tomography (Micro-CT) 4 weeks after the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane (PFC5H15) is implanted in a rat model with bilateral cranial defects;

(6) FIG. 5 is an image showing the morphology of the mesh fibrous membrane;

(7) FIG. 6 shows stress-strain curves; and

(8) FIG. 7 is an image showing the functionally gradient material compounded by the fibrous membrane with 3D printed scaffolds with different pore structures.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) In order to more clearly describe the objectives, technical solutions and advantages of the present invention, hereinafter, the present invention is further described in detail with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only intended to illustrate the present invention rather than to limit the present invention, namely, the described embodiments are only a part of the embodiments of the present invention rather than all the embodiments. The components of the embodiments of the present invention described and illustrated in the drawings herein can generally be arranged and designed in various configurations.

(10) Therefore, the following detailed description of the embodiments of the present invention and the drawings are only intended to illustrate the preferred embodiments of the present invention rather than to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

(11) It should be noted that the terminologies such as “first”, “second”, and the like are only used to distinguish one entity or operation from another entity or operation without necessarily requiring or implying any such actual relationships or sequences between these entities or operations. Moreover, the terminologies “include”, “comprise” or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also includes other elements that are not explicitly listed, or elements inherent to such a process, method, article or device. Without further restrictions, elements defined by the statement “include one . . . ” do not exclude the presence of other additional identical elements in the process, method, article or device that includes these elements.

(12) The features and performance of the present invention are further described in detail hereinafter with reference to the embodiments.

Embodiment 1

(13) The preferred embodiment of the present invention provides a method for preparing a functionally gradient material for guided periodontal hard and soft tissue regeneration. The raw materials include: fish collagen purchased from Sangon Biotech (Shanghai) Co., Ltd., hexafluoroisopropanol purchased from Shanghai Aladdin Bio-Chem Co., Ltd., and poly (lactic-co-glycolic acid) purchased from Jinan Daigang Biomaterial Co., Ltd. The specific steps are as follows.

(14) Step 1: 0.06 g of nano-hydroxyapatite is ultrasonically dispersed in 2 mL of hexafluoroisopropanol for 1 h via an ultrasonic cell disruptor.

(15) Step 2: 0.02 g of fish collagen is added into the dispersion solution obtained in step 1 to obtain a mixture, and the mixture is well shaken for 10 min via a thermostatic oscillator;

(16) Step 3: 0.4 g of poly (lactic-co-glycolic acid) is added to the mixed solution obtained in step 2 to obtain a mixture, and the mixture is well shaken for 2 h at 25° C. in the thermostatic oscillator to obtain a spinning solution.

(17) Step 4: The spinning solution obtained in step 3 is ultrasonically dispersed again for 30 min via the ultrasonic cell disruptor.

(18) Step 5: The spinning solution obtained in step 4 is used to prepare the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by an electrospinning machine using a 23 G flat-head needle and a flat plate collector. The electrospinning is performed under parameters including an applied voltage of 8 kV, an injection rate of 0.5 mL/h, and a receiving distance of 16 cm.

(19) Step 6: After being continuously collected for 1 h using a mesh collector in step 5, the fibrous membrane is removed, and then is dried for 3 days in a vacuum drying oven to obtain the electrospun fibrous membrane layer.

(20) Step 7: 0.6 g of nano-hydroxyapatite is ultrasonically pre-dispersed in 10 mL of dichloromethane for 20 min via an ultrasonic cleaner.

(21) Step 8: The dispersion solution obtained in step 7 is ultrasonically dispersed for 30 min by the ultrasonic cell disruptor.

(22) Step 9: 0.2 g of fish collagen is added into the dispersion solution obtained in step 8 to obtain a mixture, and the mixture is well shaken for 20 min via the thermostatic oscillator to obtain a mixed suspension containing the fish collagen homogeneously dispersed in the mixed suspension.

(23) Step 10: 4 g of poly (lactic-co-glycolic acid) is added to the mixed suspension obtained in step 9 to obtain a mixed solution, and the mixed solution is well shaken at 25° C. for 2 h in the thermostatic oscillator.

(24) Step 11: The mixed solution obtained in step 10 is stirred in a fume hood to obtain the bio-ink, wherein the viscosity of the mixed solution is measured to be 40±10 mPa.Math.s.

(25) Step 12: The electrospun fibrous membrane layer obtained in step 6 is placed on the platform of the 3D bioprinter, and the bio-ink obtained in step 11 is used for printing on the composite fibrous membrane by the 3D bioprinter using a conical needle with an inner diameter of 0.16-0.41 mm to obtain the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) scaffold. The 3D printing is performed under parameters including a scaffold size of 10×10×5 mm.sup.3, an extrusion pressure of 5 bar, a needle temperature of 30° C., a receiving platform temperature of 20° C., a needle velocity of 4 mm/s, an initial needle-to-platform distance of 0.208 mm, a layer spacing of 0.208 mm, an initial needle tip-to-platform distance 0.208 mm, and a printing line spacing of 0.3-0.8 mm.

(26) Step 13: The printed scaffold is dried for 3 days in the vacuum drying oven to obtain the functionally gradient material.

Embodiment 2

(27) The preferred embodiment of the present invention provides a method for preparing a functionally gradient material for guided periodontal hard and soft tissue regeneration. The raw materials include: fish collagen purchased from Sangon Biotech (Shanghai) Co., Ltd., hexafluoroisopropanol purchased from Shanghai Aladdin Bio-Chem Co., Ltd., and poly (lactic-co-glycolic acid) purchased from Jinan Daigang Biomaterial Co., Ltd. The specific steps are as follows.

(28) Step 1: 0.06 g of nano-hydroxyapatite is ultrasonically dispersed in 2 mL of hexafluoroisopropanol for 1 h via an ultrasonic cell disruptor.

(29) Step 2: 0.02 g of fish collagen is added into the dispersion solution obtained in step 1 to obtain a mixture, and the mixture is well shaken for 10 min via a thermostatic oscillator.

(30) Step 3: 0.4 g of poly (lactic-co-glycolic acid) is added to the mixed solution obtained in step 2 to obtain a mixture, and the mixture is well shaken for 2 h at 25° C. in the thermostatic oscillator to obtain a spinning solution.

(31) Step 4: The spinning solution obtained in step 3 is ultrasonically dispersed again for 30 min via the ultrasonic cell disruptor.

(32) Step 5: The spinning solution obtained in step 4 is used to prepare the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by an electrospinning machine using a 23 G flat-head needle and a flat plate collector. The electrospinning is performed under parameters including an applied voltage of 8 kV, an injection rate of 0.5 mL/h, and a receiving distance of 16 cm.

(33) Step 6: After being continuously collected for 1 h using a mesh collector in step 5, the fibrous membrane is removed, and then is dried for 3 days in a vacuum drying oven to obtain the electrospun fibrous membrane layer.

(34) Step 7: 0.6 g of nano-hydroxyapatite is ultrasonically pre-dispersed in 10 mL of dichloromethane for 20 min via an ultrasonic cleaner.

(35) Step 8: The dispersion solution obtained in step 7 is ultrasonically dispersed for 30 min by the ultrasonic cell disruptor.

(36) Step 9: 0.2 g of fish collagen is added into the dispersion solution obtained in step 8 to obtain a mixture, and the mixture is well shaken for 20 min via the thermostatic oscillator to obtain a mixed suspension containing the fish collagen homogeneously dispersed in the mixed suspension.

(37) Step 10: 4 g of poly (lactic-co-glycolic acid) is added to the mixed suspension obtained in step 9 for well shaking at 25° C. for 2 h in the thermostatic oscillator.

(38) Step 11: The mixed solution obtained in step 10 is stirred in a fume hood to obtain the bio-ink, wherein the viscosity of the mixed solution is measured to be 40±10 mPa.Math.s.

(39) Step 12: The electrospun fibrous membrane layer obtained in step 6 is placed on the platform of the 3D bioprinter, and the bio-ink obtained in step 11 is used for printing on the composite fibrous membrane by the 3D bioprinter. After the printing on one layer is ended, the printing is paused to place the electrospun fibrous membrane layer obtained in step 6 on the first 3D printed layer. The above-mentioned operation is repeated for more than 10 times to prepare the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) gradient scaffold material formed by the electrospun fibrous membrane and the 3D printed scaffold alternately. A conical needle with an inner diameter of 0.16-0.41 mm is used. The 3D printing is performed under parameters including a scaffold size of 10×10×5 mm.sup.3, an extrusion pressure of 5 bar, a needle temperature of 30° C., a receiving platform temperature of 20° C., a needle velocity of 4 mm/s, an initial needle-to-platform distance of 0.208 mm, a layer spacing of 0.2-0.3 mm, an initial needle tip-to-platform distance 0.208 mm, and a printing line spacing of 0.3-0.8 mm.

(40) Step 13: The printed scaffold is dried for 3 days in the vacuum drying oven to obtain the functionally gradient material.

Experimental Example 1

(41) In the experiment, the steps of preparing a composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by electrospinning specifically include:

(42) Step 1: 0.06 g of nano-hydroxyapatite is ultrasonically dispersed in 2 mL of hexafluoroisopropanol for 1 h via an ultrasonic cell disruptor.

(43) Step 2: 0.02 g of fish collagen is added into the dispersion solution obtained in step 1 to obtain a mixture. The mixture is shaken well for 10 min via the thermostatic oscillator.

(44) Step 3: 0.4 g of poly (lactic-co-glycolic acid) is added to the mixed solution obtained in step 2 to obtain a mixture. The mixture is well shaken at 25° C. for 2 h in the thermostatic oscillator to obtain a spinning solution.

(45) Step 4: The spinning solution obtained in step 3 is ultrasonically dispersed again for 30 min by the ultrasonic cell disruptor.

(46) Step 5: The spinning solution obtained in step 4 is used to prepare the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by the electrospinning machine using a flat-head needle with a diameter of 23 G and a flat plate collector. The electrospinning is performed under parameters including an applied voltage of 8 kV, an injection rate of 0.5 mL/h, and a receiving distance of 16 cm;

(47) Step 6: After being continuously collected for 2 h, the fibrous membrane is removed from the collector and dried in a vacuum drying oven for 3 days to fully evaporate the solvent.

(48) Step 7: The nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane is prepared by electrospinning.

(49) FIG. 1 shows the observed result of the morphology of the nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane via a scanning electron microscope, and no beads are observed on the smooth fibers. FIG. 2 shows the result of a measurement of the diameter distribution, and the average diameter of the fibers is 486±64 nm. The uniform distribution of nano-hydroxyapatite in the fibers is achieved by the two-step ultrasonic method, and the fibers exhibit excellent morphology.

(50) FIG. 3 shows the observed result of the morphology of the composite fibrous membrane after 8 weeks of in vitro degradation experiment by the scanning electron microscope. FIG. 3 shows that the introduction of fish collagen changes the main degradation behavior of the fibers. In addition to fiber breakage, fiber swelling and fiber corrosion, porous degradation occurs at the same time, which significantly accelerates the degradation of the fibrous membrane.

(51) FIG. 4 shows the result of the in vivo bone repair effect of the composite fibrous membrane evaluated by the rat model with bilateral cranial defects. Visibly, new bone tissue is formed in the cranial defect site, indicating that the composite fibrous membrane has promising application prospects in the field of guided tissue regeneration.

Experimental Example 2

(52) In the experiment, the steps of preparing a composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by electrospinning specifically include:

(53) Step 1: 0.06 g of nano-hydroxyapatite is ultrasonically dispersed in 2 mL of hexafluoroisopropanol for 1 h via an ultrasonic cell disruptor to obtain a dispersion solution.

(54) Step 2: 0.02 g of fish collagen is added into the dispersion solution obtained in step 1 to obtain a mixture. The mixture is well shaken via a thermostatic oscillator for 10 min.

(55) Step 3: 0.4 g of poly (lactic-co-glycolic acid) is added to the mixed solution obtained in step 2 to obtain a mixture. The mixture is well shaken at 25° C. for 2 h in the thermostatic oscillator to obtain a spinning solution.

(56) Step 4: The spinning solution obtained in step 3 is ultrasonically dispersed again for 30 min by the ultrasonic cell disruptor.

(57) Step 5: The spinning solution obtained in step 4 is used to prepare the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane by the electrospinning machine using a 23 G flat-head needle and a flat plate collector. The electrospinning is performed under parameters including an applied voltage of 8 kV, an injection rate of 0.5 mL/h, and a receiving distance of 16 cm.

(58) Step 6: After being continuously collected for 1 h using a mesh collector in step 5, the fibrous membrane is removed, and then is dried for 3 days in a vacuum drying oven.

(59) FIG. 5 shows the observed result of the morphology of the mesh fibrous membrane by the scanning electron microscope. The fibrous membrane forms uniformly arranged mesh repeating units with a mesh size of around 500 μm.

(60) Experimental Example 3

(61) The poly (lactic-co-glycolic acid) fibrous membrane (P), the fish collagen/poly (lactic-co-glycolic acid) fibrous membrane (PFC5) and the composite nano-hydroxyapatite/fish collagen/poly (lactic-co-glycolic acid) fibrous membrane (PFC5H15) are respectively prepared for tensile strength testing. The results thereof are shown in FIG. 6 and Table 1 below. It can be seen that the introduction of fish collagen significantly increases the tensile strength of the fibrous membrane.

(62) TABLE-US-00001 TABLE 1 Mechanical properties of the fibrous membranes Tensile Elastic Elongation at strength modulus break Sample (Mpa) (Mpa) (%) P 1.5 ± 0.11 31.7 ± 4.4 232.6 ± 10.9 PFC5 6.5 ± 0.13 104.8 ± 6.7  122.8 ± 10.5 PFC5H15 5.2 ± 0.03 124.3 ± 22.7 127.4 ± 7.5 

(63) The above-mentioned description is only the preferred embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall fall within the scope of protection of the present invention.