Biological 3D printing tissue engineering rhinoplasty-like structural member and preparation and application thereof

Abstract

The invention relates to the technical field of biomedical tissue engineering, in particular to a biological 3D printing tissue engineering rhinoplasty-like structural member and preparation and application thereof. The preparation method comprises the following steps: firstly, wrapping cartilage cells with cartilage tissue specific hydrogel to prepare cartilage specific bio-ink; then uniformly mixing polycaprolactone with benzophenone, so as to obtain a polycaprolactone mixture; and finally, alternately printing by using the cartilage specific bio-ink and polycaprolactone as raw materials by using a double-needle extrusion type biological 3D printing technology to obtain the rhinoplasty structural member. Furthermore, the rhinoplasty-like structural member can be cultured to obtain the rhinoplasty cartilage, the rhinoplasty cartilage prepared by the method can achieve satisfactory cartilage formation without a long-term in-vitro culture process, and the rhinoplasty cartilage can be further applied to cartilage defect repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical tissue engineering, and in particular to a biological 3D printed tissue engineering rhinoplasty-like structural component and its preparation and application. Background Art

[0002] Rhinoplasty is a necessary means to solve the aesthetic and functional disorders of patients. At present, cartilage tissue engineering strategies can provide living cartilage donors for nasal functional reconstruction, but there are great challenges in constructing nasal structures and maintaining long-term structural fidelity. Alternatively, dual-nozzle 3D bioprinting technology combined with tissue engineering strategies has shown good potential in nose reconstruction. However, due to the lack of ideal bio-ink, suboptimal mass transfer, and poor interface integration between hard polymers and soft hydrogels, breakthroughs in 3D bioprinting nasal cartilage regeneration are very limited.

[0003] Therefore, it is crucial to provide a bio-3D printed tissue engineering rhinoplasty-like structure that can solve the above problems. Summary of the invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a biological 3D printed tissue engineering rhinoplasty-like structure and its preparation and application.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first object of the present invention is to provide a method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure, comprising the following steps:

[0007] (S1) using cartilage tissue-specific hydrogel to encapsulate chondrocytes to prepare cartilage-specific bio-ink;

[0008] (S2) mixing polycaprolactone and benzophenone to obtain a polycaprolactone mixture;

[0009] (S3) using double-needle extrusion biological 3D printing technology, using the cartilage-specific bio-ink prepared in step (S1) and the polycaprolactone prepared in step (S2) as raw materials, and alternately printing to obtain a rhinoplasty-like structural component;

[0010] Wherein, the rhinoplasty-like structural member is an assembly structure of a first structural member and a second structural member;

[0011] The first structural member includes an integrated semicircular ring structure and a column structure, wherein the column structure is located at the center of the opening side of the semicircular ring structure;

[0012] The second structural member is an annular fan-shaped structure, which is located on the upper surface of the semicircular ring structure, and the annular fan-shaped structure and the semicircular ring structure have the same opening direction and the same central axis;

[0013] Among them, polycaprolactone is used to provide support;

[0014] Benzophenone was used to achieve bonding between polycaprolactone and cartilage-specific bioink, providing adhesion.

[0015] In one embodiment of the present invention, the inner diameter of the semicircular ring structure is 9 to 11 mm, the outer diameter is 13 to 17 mm, and the thickness is 1 to 3 mm;

[0016] Preferably, the inner diameter of the semicircular ring structure is 10 mm, the outer diameter is 15 mm, and the thickness is 2 mm;

[0017] The bottom edge of the column structure is 1 to 3 mm, and the height is 9 to 11 mm; the inner diameter of the annular fan-shaped structure is 8 to 10 mm, the outer diameter is 13 to 17 mm, the angle is 50 to 70°, and the thickness is 1 to 3 mm;

[0018] Preferably, the bottom edge of the column structure is 2 mm and the height is 10 mm; the inner diameter of the annular fan-shaped structure is 9 mm, the outer diameter is 15 mm, the angle is 60°, and the thickness is 2 mm.

[0019] In one embodiment of the present invention, in step (S1), the density of chondrocytes in the cartilage-specific bio-ink is 10 4 ~10 10 / mL;

[0020] Preferably, in the cartilage-specific bio-ink, the density of chondrocytes is 10 7 ~10 8 / mL.

[0021] In one embodiment of the present invention, the cartilage tissue-specific hydrogel is prepared by the following method:

[0022] The water-soluble polymer is dissolved in a biocompatible medium and then cross-linked with cartilage tissue-specific components to prepare it.

[0023] In one embodiment of the present invention, after the water-soluble polymer is dissolved in a biocompatible medium, the concentration of the water-soluble polymer is 0.1% w / v to 60% w / v; the specific concentration is 6% w / v GelMA (methacrylic anhydride gelatin), 1% w / v CSMA (methacrylic anhydride chondroitin sulfate), 0.5% w / v PEO (polyethylene oxide);

[0024] In the cartilage tissue-specific hydrogel, the mass percentage of the cartilage tissue-specific component is 10 to 20%.

[0025] In one embodiment of the present invention, the water-soluble polymer is selected from one or more of natural polymer materials or synthetic polymer materials;

[0026] The biocompatible medium is selected from one or more of distilled water, physiological saline, buffer or cell culture medium solution;

[0027] The cartilage tissue-specific component includes chondroitin sulfate, a chondrocyte matrix.

[0028] In one embodiment of the present invention, the natural polymer material is selected from one of natural polysaccharides, natural polysaccharide modifications, natural polysaccharide degradation products, proteins, protein modifications or protein degradation products;

[0029] The synthetic polymer material is selected from polycaprolactone, dichloromethane and polyethylene oxide.

[0030] In one embodiment of the present invention, the natural polysaccharide material is selected from one or more of carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, and chondroitin sulfate;

[0031] The protein is selected from one or more of hydrophilic or water-soluble animal and plant proteins, collagen, serum protein, and elastin.

[0032] In one embodiment of the present invention, the protein degradation product is selected from one of gelatin and polypeptide.

[0033] In one embodiment of the present invention, the cartilage decellularized matrix is ​​a tissue containing cartilage (taken from an animal, wherein the animal is selected from one of pigs, cattle, sheep, dogs, rabbits or mice, preferably, the animal is rabbits, pigs, cattle, sheep, dogs); preferably, ear cartilage, articular cartilage, meniscus cartilage, costal cartilage;

[0034] In one embodiment of the present invention, the crosslinking is selected from one or more of physical crosslinking, chemical crosslinking or photocrosslinking.

[0035] In one embodiment of the present invention, the photocrosslinking is to form covalent crosslinks by a photochemical reaction (reference Huitang Xia, Dandan Zhao, Hailin Zhu, et al. ACS Appl. Mater. Interfaces 2018, 10, 31704.).

[0036] In one embodiment of the present invention, the photo-crosslinked hydrogel material is prepared by a photo-initiated polymerization crosslinking reaction, that is, the free radicals generated by the photoinitiator under the irradiation of a light source initiate the polymerization reaction of the double bond functional groups on the polymer derivative containing a methacrylate group, thereby preparing a photo-crosslinked hydrogel.

[0037] In one embodiment of the present invention, the photo-crosslinked hydrogel can be realized by dissolving a methacrylate-containing polymer derivative and a photoinitiator in a biocompatible medium to prepare a hydrogel precursor solution of a certain concentration, and irradiating the solution with a light source of a wavelength of 254nm to 450nm (preferably 365nm or 405nm) to obtain a photo-crosslinked hydrogel.

[0038] In one embodiment of the present invention, in step (S2), the mass percentage of polycaprolactone to benzophenone is 99-99.5:0.5-1.

[0039] The second object of the present invention is to provide a biological 3D printed tissue engineering rhinoplasty-like structure, which is prepared by the above method;

[0040] The interfacial adhesion between the cartilage-specific bio-ink and polycaprolactone is 1 kPa to 10 MPa, preferably, the interfacial adhesion is 100 kPa to 1 MPa.

[0041] The third object of the present invention is to provide a bio-3D printed tissue engineering rhinoplasty-like structure for use in the preparation of rhinoplasty cartilage.

[0042] The rhinoplasty cartilage can be further used to prepare cartilage defect repair products.

[0043] Rhinoplasty cartilage is prepared by culturing in vitro or in vivo using a rhinoplasty-like structure;

[0044] Wherein, the in vitro culture is selected from static culture or dynamic culture.

[0045] The static culture method is to place the 3D printed tissue engineering cartilage (i.e. the above-mentioned "rhinoplasty cartilage") in a culture dish for culture, and the dynamic culture method is to place the 3D printed tissue engineering cartilage in a bioreactor for stirring or pressurized culture to promote the exchange of nutrients in the hydrogel;

[0046] The in vivo subcutaneous implantation culture is to implant the tissue engineering rhinoplasty-like structure without or after in vitro culture into the nose of an animal for culture.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The natural hydrogel material used in this application has ideal biocompatibility and tissue-specific microenvironment, and can effectively promote specific tissue regeneration;

[0049] (2) 3D printed tissue-engineered rhinoplasty cartilage does not require a long in vitro culture process to achieve satisfactory cartilage formation and perform in vivo rhinoplasty surgery;

[0050] (3) The structures prepared by combining biological 3D printing technology can achieve precise control of cell arrangement and alternating structure;

[0051] (4) The interface integration of cartilage and polycaprolactone can be achieved through 3D printing and is not easy to fall off. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the preparation of bio-3D printed tissue engineering rhinoplasty-like structures;

[0053] Figure 2 It is a visual diagram of the effect of the biological 3D dual-phase printing process;

[0054] Figure 3 This is a general view of the 3D printed rhinoplasty-like structure;

[0055] Figure 4 The gross view and imaging (MRI) diagram of in vivo regeneration of rhinoplasty in rabbits with cell-based dual-phase 3D printed tissue engineering cartilage;

[0056] Figure 5 Schematic diagram of the in vivo regeneration ability evaluation of rhinoplasty in rabbits with cell-based dual-phase printed tissue engineering cartilage;

[0057] Figure 6 EPR spectra before and after UV irradiation (A and B) and mechanical compression measurement results (C). DETAILED DESCRIPTION

[0058] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0059] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0060] Example 1

[0061] This embodiment provides a method for preparing a bio-3D printed tissue engineering rhinoplasty-like structure and rhinoplasty cartilage (eg Figure 1 and Figure 2 As shown), comprising the following steps:

[0062] (S1) GelMA and PEO were dissolved in saline and then photocrosslinked with CSMA (365 nm, 20 mW / cm 2 , 10 min), and cartilage tissue-specific hydrogel (pGC: 6% w / v GelMA, 1% w / v CSMA, 0.5% w / v PEO) was prepared;

[0063] (S2) The cartilage tissue-specific hydrogel prepared in step (S1) was wrapped around the auricular chondrocytes to prepare cartilage-specific bio-ink (6×10 7 cells / mL);

[0064] Among them, GelMA and CSMA are components that mimic cartilage ECM, and PEO can form a porous structure based on the principle of microphase separation;

[0065] (S3) mixing polycaprolactone (PCL) and benzophenone (mass ratio of 99.2:0.8) to obtain a polycaprolactone mixture (mPCL);

[0066] (S4) Using double needle extrusion bio-3D printing technology ( Figure 1 and Figure 2 ), using the cartilage-specific bio-ink prepared in step (S2) and the polycaprolactone prepared in step (S3) as raw materials, alternately printing to obtain a rhinoplasty-like structure (P-pGC / mPCL), then culturing the rhinoplasty-like structure for 24 hours, removing the PEO component, and obtaining rhinoplasty cartilage ( Figure 3 );

[0067] Wherein, the rhinoplasty-like structure is an assembly structure of a first structural member and a second structural member;

[0068] The first structural member includes an integrated semicircular ring structure and a column structure, wherein the column structure is located at the center of the opening side of the semicircular ring structure;

[0069] The second structural member is an annular fan-shaped structure, which is located on the upper surface of the semicircular ring structure, and the annular fan-shaped structure and the semicircular ring structure have the same opening direction and the same central axis;

[0070] The inner diameter of the semicircular ring structure is 10 mm, the outer diameter is 15 mm, and the thickness is 2 mm;

[0071] The bottom edge of the column structure is 2mm and the height is 10mm; the inner diameter of the annular fan-shaped structure is 9mm, the outer diameter is 15mm, the angle is 60°; and the thickness is 2mm.

[0072] Among them, the interfacial adhesion between cartilage-specific bio-ink and polycaprolactone is 8Mpa.

[0073] The soft tissue-specific hydrogel of this embodiment not only promotes nutrient exchange and cell proliferation through the double-pore transport channel, but also maintains the cartilage phenotype and promotes cartilage-specific ECM secretion; through the physical entanglement of the polycaprolactone framework and the chemical bond bonding through interfacial photoinitiated polymerization, the 3D bioprinting of the hard polycaprolactone phase and the soft hydrogel phase is ensured, thereby improving the mechanical strength and structural fidelity, and having satisfactory cartilage regeneration quality.

[0074] It should be noted that in the embodiment, the 3D bioprinting process was completed using a 3D BioArchitect workstation (Regenovo), as follows: a low-temperature nozzle was used to extrude cartilage-specific bio-ink at 15°C on a 20°C printing platform, while another high-temperature nozzle was used to melt-deposit a polycaprolactone mixture at 80°C. The printing process was performed alternately on each layer, and the platform temperature was controlled at 10°C; photo-crosslinking of the hydrogel was applied after 3D bioprinting (printing parameters are shown in Table 1).

[0075] Table 1. Printing parameters

[0076]

[0077]

[0078] Performance Analysis:

[0079] The rhinoplasty cartilage prepared in this example was implanted into the nasal cartilage of rabbits, and the samples were collected 12 weeks later ( Figure 4 As shown in FIG. 1 , the product prepared using only polycaprolactone (labeled as “pure PCL”) was used as the control group, and hematoxylin-eosin and safranin O were used to perform histological evaluation on the rhinoplasty of rabbits. Figure 5 shown.

[0080] pass Figure 4 and Figure 5 It can be found that pure PCL can maintain a better tissue morphology, while bidirectional printing (i.e., printing method with added cell bio-ink) can maintain a better morphology while maintaining cell activity and regenerating specific cartilage tissue.

[0081] 530 mg of pGC was dissolved in 1 mL of deionized water; subsequently, 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added and mixed well to obtain a mixture; thereafter, 172 mg of mPCL was added to the above mixture to obtain P-pGC / mPCL.

[0082] Electron paramagnetic resonance (EPR) spectra were recorded using a CIQTEK EPR200-Plus spectrometer before and after 5 min UV irradiation. The results showed that the presence of superoxide radicals and hydroxyl radicals generated by mPCL ( Figure 6 A and Figure 6 B), thus further initiating the free radical polymerization of pGC at the mPCL interface.

[0083] Mechanical testing: For mechanical testing, P-pGC / mPCL and P-pGC / PCL (530 mg of pGC was dissolved in 1 mL of deionized water; subsequently, 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added and mixed thoroughly to obtain a mixture; thereafter, 172 mg of PCL was added to the above mixture to obtain P-pGC / PCL) were prepared into cylindrical shapes (10 mm in diameter and 2 mm in height).

[0084] The compression test was performed using a mechanical analyzer (Instron-5542) with a capacity of 500 N. Specifically, under light irradiation (365 nm LED, 20 mw / cm 2 ) After inducing complete gelation for 30 seconds, the prepared hydrogel was subjected to compression testing. During the compression test, the test speed was set to 1 mm / min and the maximum deformation was 95%. The compression modulus was calculated as the slope of the linear region (15-25% strain), and the point corresponding to the maximum compressive strength was determined at the same time. The cyclic compression test was carried out at a continuous plane unconfined strain rate of 1 mm / min, with a maximum deformation of 40% or 50%. The fracture energy, denoted as W, represents the energy required for the specimen to withstand tensile loads and undergo crack propagation per unit area. It is calculated by multiplying the area under the stress-separation displacement curve by the characteristic length (d) of the specimen (the fracture energy range is 0-25% displacement).

[0085] Mechanical compression measurements were performed to evaluate the interfacial bonding strength of cartilage-specific bioink and polycaprolactone, and the results were shown in Figure 6 As shown in Figure C, P-pGC / mPCL showed strong interfacial integration under external compressive stress in the strain range of about 60%, with no separation between the PCL phase and the pGC phase, thus effectively maintaining the structural integrity. In contrast, P-pGC / PCL showed easy interfacial separation and structural decomposition, thus limiting the long-term structural stability for tissue regeneration.

[0086] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure, characterized in that: The following steps are involved: (S1) using cartilage tissue-specific hydrogel to encapsulate chondrocytes to prepare cartilage-specific bio-ink; (S2) mixing polycaprolactone and benzophenone to obtain a polycaprolactone mixture; (S3) using double-needle extrusion bio-3D printing technology, using the cartilage-specific bio-ink prepared in step (S1) and the polycaprolactone mixture prepared in step (S2) as raw materials, and alternately printing to obtain a rhinoplasty-like structure; Wherein, the rhinoplasty-like structural member is an assembly structure of a first structural member and a second structural member; The first structural member includes an integrated semicircular ring structure and a column structure, wherein the column structure is located at the center of the opening side of the semicircular ring structure; The second structural member is an annular fan-shaped structure, which is located on the upper surface of the semicircular ring structure, and the annular fan-shaped structure and the semicircular ring structure have the same opening direction and the same central axis; Among them, polycaprolactone is used to provide support; Benzophenone was used to achieve bonding between polycaprolactone and cartilage-specific bioink, providing adhesion.

2. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 1, characterized in that: The inner diameter of the semicircular ring structure is 9 to 11 mm, the outer diameter is 13 to 17 mm, and the thickness is 1 to 3 mm; The bottom side of the column structure is 1-3 mm and the height is 9-11 mm; the inner diameter of the annular fan-shaped structure is 8-10 mm, the outer diameter is 13-17 mm, the angle is 50-70°, and the thickness is 1-3 mm.

3. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 1, characterized in that: In step (S1), the density of chondrocytes in the cartilage-specific bio-ink is 10 4 ~10 10 / mL.

4. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 3, characterized in that: The cartilage tissue-specific hydrogel is prepared by the following method: The water-soluble polymer is dissolved in a biocompatible medium and then cross-linked with cartilage tissue-specific components to prepare it.

5. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 4, characterized in that: After the water-soluble polymer is dissolved in a biocompatible medium, the concentration of the water-soluble polymer is 0.1% w / v-60% w / v; In the cartilage tissue-specific hydrogel, the mass percentage of the cartilage tissue-specific component is 10 to 20%.

6. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 3, characterized in that: The crosslinking is selected from one or more of physical crosslinking, chemical crosslinking or photocrosslinking.

7. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 6, characterized in that: The crosslinking is photo-crosslinking; During the photocrosslinking process, the wavelength is 254nm to 450nm; preferably 365nm, 20mW / cm 2 .

8. The method for preparing a biological 3D printed tissue engineering rhinoplasty-like structure according to claim 1, characterized in that: In step (S2), the mass percentage of polycaprolactone to benzophenone is 99-99.5:0.5-1.

9. A biological 3D printed tissue engineering rhinoplasty-like structure, characterized in that: Prepared by the method according to any one of claims 1 to 8; Wherein, the interfacial adhesion between the cartilage-specific bio-ink and polycaprolactone is 1 kPa to 10 MPa.

10. Use of the biological 3D printed tissue engineering rhinoplasty-like structure as claimed in claim 9 in preparing rhinoplasty cartilage.

Images