阅读说明：本技术 用于改良AMIC技术软骨修复的生物3d打印的活性生物膜及其制备方法 (Biological 3d printed active biofilm for improving AMIC technology cartilage repair and preparation method thereof ) 是由 桂鉴超 周杨 蒋逸秋 秦然 陈通 于 2020-03-11 设计创作，主要内容包括：一种用于软骨修复的改良AMIC的生物3d打印的活性生物膜,其特征在于：该生物膜以海藻酸钠/明胶/透明质酸为原料制备混合水凝胶,水凝胶中混入软骨前体细胞及纤连蛋白(fibronectin),利用沉积式生物3D打印技术构建出多孔水凝胶生物膜,通过氯化钙浸泡实现化学交联增强力学性能。本发明所用材料均为天然材料,免疫原性低,生物相容性好,来源广泛,同时具有一定的力学性能可为软骨再生提供良好的支撑。就制备方法而言,300-500微米的空隙可促进软骨再生,同时优秀的孔隙率结构等也可促进细胞间的物质交换、沟通,有利于细胞因子或细胞的粘附,有利于细胞在其内部生长和增殖。(An active biofilm for biological 3d printing of improved AMIC for cartilage repair, comprising: the biological membrane is prepared by taking sodium alginate/gelatin/hyaluronic acid as raw materials to prepare mixed hydrogel, cartilage precursor cells and fibronectin (fibronectin) are mixed in the hydrogel, a deposition type biological 3D printing technology is utilized to construct the porous hydrogel biological membrane, and chemical crosslinking is realized through calcium chloride soaking to enhance mechanical properties. The materials used in the invention are all natural materials, have low immunogenicity, good biocompatibility and wide sources, and simultaneously have certain mechanical properties and can provide good support for cartilage regeneration. In terms of the preparation method, the 300-500 micron gap can promote the regeneration of cartilage, and the excellent porosity structure can promote the exchange and communication of substances among cells, is beneficial to the adhesion of cytokines or cells, and is beneficial to the growth and proliferation of cells in the cell.)

1. An active biofilm for biological 3d printing of improved AMIC for cartilage repair, comprising: the biological membrane is prepared by taking sodium alginate/gelatin/hyaluronic acid as raw materials to prepare mixed hydrogel, cartilage precursor cells and fibronectin (fibronectin) are mixed in the hydrogel, a deposition type biological 3D printing technology is utilized to construct the porous hydrogel biological membrane, and chemical crosslinking is realized through calcium chloride soaking to enhance mechanical properties.

2. A method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair comprising the steps of:

step 1), preparing a printing material;

step 2), preparing printing hydrogel;

step 3) printing and post-processing of the hydrogel active biological membrane:

pretreating the biological ink in the step 2) for 10min to form a gel state suitable for jelly, transferring the printing needle cylinder filled with the biological ink into a 3d printer, and setting the temperature of a printer nozzle and the temperature of a printer platform. And starting air pressure, adjusting relevant parameters of the biological membrane, and starting printing after the nozzle stably extrudes the hydrogel microfilaments. And after printing, fully curing the hydrogel film scaffold by using a calcium chloride solution to finally obtain the cell-loaded hydrogel film scaffold.

3. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: the step 1) of preparing the printing material comprises the following steps:

step 1.1) sterile treatment: performing ultraviolet disinfection on gelatin, sodium alginate, hyaluronic acid and calcium chloride powder for 24 hours, and placing a magnetic heating stirrer on an ultra-clean bench to prepare the hydrogel pre-prepared liquid. All other operations are operated in a clean bench.

Step 1.2) preparation of gelatin solution: heating 2.5g gelatin in 20ml deionized water in 48 deg.C water bath, stirring with constant temperature magnetic stirrer at 300r/min, and dissolving completely to obtain gelatin solution.

Step 1.3) preparation of sodium alginate/gelatin solution: adding 20ml of deionized water and 1.25g of sodium alginate into the gelatin solution, heating in a water bath at 48 ℃ at the rotating speed of 300r/min, and fully dissolving the mixture into the sodium alginate/gelatin solution.

Step 1.4) preparation of sodium alginate/gelatin/hyaluronic acid mixed solution: adding 10ml of deionized water and 0.1g of hyaluronic acid into the sodium alginate/gelatin solution, heating in a water bath at 48 ℃ at the rotating speed of 300r/min to fully dissolve the sodium alginate/gelatin/hyaluronic acid solution, wherein the final sodium alginate concentration is 10% (w/v), the hyaluronic acid concentration is 0.1% (w/v) and the gelatin concentration is 5% (w/v); for storage at room temperature, ready for use.

Step 1.5) digesting the chondrocytes, adding the digested chondrocytes into a cartilage culture medium which normally contains 10% serum and 1% double antibody, adding the chondrocyte suspension into a 10cm culture dish coated with prepared fibronectin in advance, adsorbing for 20min, removing the culture medium, obtaining cartilage precursor cells adsorbed at the bottom of the culture dish, and normally placing the cartilage precursor cells in a cell culture box with the carbon dioxide concentration of 5% and the temperature of 37 ℃ for later use. The cartilage precursor cells described in the present invention are cartilage precursor cells obtained after 3 normal culture generations.

Step 1.6) the lyophilized fibronectin powder is diluted with a culture medium, sufficiently dissolved by vortex, prepared into a solution of 100 mu g/ml, and stored at-20 ℃ for later use.

4. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: the step 2) configuration of printing the hydrogel comprises the following steps:

step 2.1) mixing and stirring 2ml of fibrinectin solution with the concentration of 100 mu g/ml, 2ml of cell suspension with the cell concentration of 5x106cells/ml and 6ml of sodium alginate/gelatin/hyaluronic acid hydrogel pre-prepared solution uniformly, wherein the final concentration of sodium alginate is 2.5% (w/v), the concentration of hyaluronic acid is 0.1% (w/v), the concentration of gelatin is 5% (w/v), the cell concentration is 106cells/ml and the concentration of fibrinectin is 10ml of sodium alginate/gelatin/hyaluronic acid bio-ink with the concentration of 20 mu g/ml;

and 2.2) pouring the fully and uniformly dissolved sodium alginate/gelatin/hyaluronic acid solution into a printing needle cylinder for centrifugal defoaming.

5. A method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 3, wherein: in the step 1.2), the step 1.3) and the step 1.4), the dosage of the gelatin (sigma) is 0.5g, the dosage of the sodium alginate (alatin) is 0.25g, the dosage of the hyaluronic acid (Mecline) is 0.02g, and the ratio of the gelatin (sigma) to the sodium alginate to the hyaluronic acid (Mecline) is 100:25: 2.

6. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: in step 3), the concentration of the calcium chloride (Solarbio) solution is 4% (w/v), and the solid-phase crosslinking time is 40 s.

7. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: in the step 3), the temperature of the syringe is set to 35 ℃, and the temperature of the platform is set to 5 ℃.

8. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: in step 3), the shape and size of the active biofilm can be designed according to actual needs, and the internal structure of the stent is that the height of the printing stent is 0.11mm, the length and the width are 2.2mm and 2.2mm, and the height of each layer is 0.18 mm.

9. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: the extrusion pressure in step 3) was 8kpa, the nozzle was 0.2mm from the floor surface, the nozzle specification was 20G, and the moving speed was 360 mm/min.

10. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 2, wherein: the extrusion type biological 3D printer in the step 3) is EFL-BP 6601.

Technical Field

The invention relates to the technical field of biomedical materials, in particular to an active biomembrane of an improved AMIC for biological 3d printing of cartilage repair and a preparation method thereof.

Background

The hyaline cartilage of the joint is lack of the blood vessels, nerves and lymph, once the hyaline cartilage is damaged, the hyaline cartilage is difficult to self-heal and is easy to develop into degenerative diseases, and the clinical existing treatment technologies such as osteochondral transplantation, autologous chondrocyte transplantation, microfracture technology, AMIC technology and the like have limited repair effect and poor long-term treatment effect. In recent years, tissue engineering technology and biological 3d printing technology are becoming a new generation of cartilage repair technology. The 3d printing technology can accurately control the internal structure of the stent, construct a morphological structure similar to cartilage, and simultaneously control the internal pore size to realize customization according to cartilage defects. The biological 3d printing technology can also be used for experimental cell-containing printing or active cytokine printing, and provides a new direction for transplanting plants in the construction body and the like. The hydrogel used as the most applied material for deposition printing has many advantages such as a structure similar to human soft tissue, a large amount of water content, good biocompatibility and the like, and in recent years, many advances have been made in biological 3d printing based on hydrogel, but the selection of seed cells and growth factors, immune rejection after allogeneic cell transplantation and the like and whether long-term curative effect is still observed.

The microfracture technique is a technique for repairing cartilage by oozing bone marrow blood containing mesenchymal stem cells by drilling holes on the surface of a bone, generally removes the damaged part of the cartilage by using an arthroscope technique, and then drills a plurality of holes on the bone to enable bone marrow cells and the blood to be coagulated to form smooth and firm repair tissues, thereby replacing the function of the cartilage and being a safe and effective method for treating the full-thickness cartilage defect of the knee joint. However, since the regeneration is substantially fibrocartilage and the articular surface itself is hyaline cartilage, it is poor in older patients, patients with excessive weight, and patients with cartilage defects exceeding 2.5 cm, and lacks long-term efficacy. Therefore, the AMIC technology (autologous matrix induced cartilage regeneration) is proposed clinically as a microfracture improvement technology, and on the basis of microfracture, a collagen I/III membrane (Chondro-Gide; Geistlich Pharma AG) is adhered to the cartilage defect by using bioprotein gel glue (Tissucol; Baxter). Compared with the microfracture technology, the method can effectively and rapidly relieve pain, recover joint function, successfully recover motion function and have relatively long-term curative effect. However, the AMIC technology still cannot completely repair cartilage defects, and the generation of hyaline cartilage similar to the original cartilage is still a problem to be solved.

The sedimentary biological 3d printing technology can endow the scaffold with biological activity by realizing the joint printing of growth factors, cells and hydrogel, thereby preparing the scaffold with biological activity for cartilage repair. Sodium alginate is used as a natural polysaccharide, has wide sources and low price, has good histocompatibility and biodegradability, and can quickly generate ion exchange crosslinking to generate gel when meeting calcium ions, so the sodium alginate hydrogel is widely used for 3d printing of cartilage tissues. Gelatin as a protein has the performance of dissolving at high temperature and forming gel at low temperature, and can obviously improve the viscosity of hydrogel and improve the printing performance. Gelatin is a protein obtained by partial hydrolysis of collagen, and has homology with collagen, and collagen is a main component of articular hyaline cartilage, and is widely used for 3d printing of cartilage due to its good biocompatibility and cartilage-promoting ability. Hyaluronic Acid (HA) is an acidic mucopolysaccharide macromolecular substance widely existing in connective tissues of human and animals, HAs obvious cartilage-promoting capability on various physiological functions of cells and cell aggregation in the process of regulating tissue formation, and shows the application value of the HA in cartilage tissue engineering.

The discovery of Cartilage Precursor Cells (CPC) provides a new clue for cartilage repair. In normal cartilage, a cartilage precursor cell with stem cell characteristics is present and has the ability to clone and potentially differentiate. Chondrocyte precursor cells can be isolated by fibronectin adhesion, possess properties similar to those of stem cells for self-clonal proliferation, are primitive cells located in cartilage tissue, have self-proliferation ability, and have the potential to differentiate toward cartilage. Fibronectin (fibronectin) is a high molecular weight (450kDa) glycoprotein. fibronectin promotes cell-cell and cell-substrate adhesion and cell migration, all of which are necessary for maintaining cell structure and function. The dose-dependent increase in chondrocyte migration and cell metabolic rate, and thus increase in protein, RNA and DNA synthesis, of fibrinectin, whereas CPC has a stronger ability to adhere to fibrinectin than chondrocytes, indicating that fibrinectin is more beneficial for CPC activation and cartilage formation.

The method which is commonly adopted at present for large-scale talus cartilage injury is that the focus is removed and then the same variant bone is transplanted, when the variant bone is taken, the three-dimensional solid geometry of the receptor talus focus which is manually cut by visual inspection is cut out from the variant talus to repair and rebuild, the method has strong subjectivity and inaccuracy, the talus is irregular in shape, the size shape radian of each talus is different, the obtained variant bone can not be accurately matched with the receptor talus to be rebuilt, the condition that the size shape radian of the cut variant bone and the size shape radian of the receptor talus are different and the joint surface is not smooth easily occurs, the postoperative joint internal stress is abnormal, the osteoarthritis and the like, the curative effect is poor or the operation fails, therefore, a cartilage 3D printer capable of accurately repairing is needed.

Disclosure of Invention

The invention aims to overcome the defects of the conventional AMIC technology, and an active biological membrane of the improved AMIC is prepared by using a biological 3d printing technology to realize a better cartilage injury repair effect.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: firstly, sodium alginate, gelatin and hyaluronic acid are used as raw materials to prepare mixed hydrogel, cartilage precursor cells and fibronectin (fibronectin) are mixed in the hydrogel, a deposition type biological 3D printing technology is utilized to construct a porous hydrogel biomembrane, and chemical crosslinking is realized through calcium chloride soaking to enhance mechanical properties.

An active biofilm for biological 3d printing of improved AMIC for cartilage repair, comprising: the biological membrane is prepared by taking sodium alginate/gelatin/hyaluronic acid as raw materials to prepare mixed hydrogel, cartilage precursor cells and fibronectin (fibronectin) are mixed in the hydrogel, a deposition type biological 3D printing technology is utilized to construct the porous hydrogel biological membrane, and chemical crosslinking is realized through calcium chloride soaking to enhance mechanical properties.

A method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair comprising the steps of:

step 1), preparing a printing material;

step 2), preparing printing hydrogel;

step 3) printing and post-processing of the hydrogel active biological membrane:

pretreating the biological ink in the step 2) for 10min to form a gel state suitable for jelly, transferring the printing needle cylinder filled with the biological ink into a 3d printer, and setting the temperature of a printer nozzle and the temperature of a printer platform. And starting air pressure, adjusting relevant parameters of the biological membrane, and starting printing after the nozzle stably extrudes the hydrogel microfilaments. And after printing, fully curing the hydrogel film scaffold by using a calcium chloride solution to finally obtain the cell-loaded hydrogel film scaffold.

The step 1) of preparing the printing material comprises the following steps:

step 1.1) sterile treatment: performing ultraviolet disinfection on gelatin, sodium alginate, hyaluronic acid and calcium chloride powder for 24 hours, and placing a magnetic heating stirrer on an ultra-clean bench to prepare the hydrogel pre-prepared liquid. All other operations are operated in a clean bench.

Step 1.2) preparation of gelatin solution: heating 2.5g gelatin in 20ml deionized water in 48 deg.C water bath, stirring with constant temperature magnetic stirrer at 300r/min, and dissolving completely to obtain gelatin solution.

Step 1.3) preparation of sodium alginate/gelatin solution: adding 20ml of deionized water and 1.25g of sodium alginate into the gelatin solution, heating in a water bath at 48 ℃ at the rotating speed of 300r/min, and fully dissolving the mixture into the sodium alginate/gelatin solution.

Step 1.4) preparation of sodium alginate/gelatin/hyaluronic acid mixed solution: adding 10ml of deionized water and 0.1g of hyaluronic acid into the sodium alginate/gelatin solution, heating in a water bath at 48 ℃ at the rotating speed of 300r/min to fully dissolve the sodium alginate/gelatin/hyaluronic acid solution, wherein the final sodium alginate concentration is 10% (w/v), the hyaluronic acid concentration is 0.1% (w/v) and the gelatin concentration is 5% (w/v); for storage at room temperature, ready for use.

Step 1.5) digesting the chondrocytes, adding the digested chondrocytes into a cartilage culture medium which normally contains 10% serum and 1% double antibody, adding the chondrocyte suspension into a 10cm culture dish coated with prepared fibronectin in advance, adsorbing for 20min, removing the culture medium, obtaining cartilage precursor cells adsorbed at the bottom of the culture dish, and normally placing the cartilage precursor cells in a cell culture box with the carbon dioxide concentration of 5% and the temperature of 37 ℃ for later use. The cartilage precursor cells described in the present invention are cartilage precursor cells obtained after 3 normal culture generations.

Step 1.6) the lyophilized fibronectin powder is diluted with a culture medium, sufficiently dissolved by vortex, prepared into a solution of 100 mu g/ml, and stored at-20 ℃ for later use.

The step 2) configuration of printing the hydrogel comprises the following steps:

step 2.1) mixing and stirring 2ml of fibrinectin solution with the concentration of 100 mu g/ml, 2ml of cell suspension with the cell concentration of 5x106cells/ml and 6ml of sodium alginate/gelatin/hyaluronic acid hydrogel pre-prepared solution uniformly, wherein the final concentration of sodium alginate is 2.5% (w/v), the concentration of hyaluronic acid is 0.1% (w/v), the concentration of gelatin is 5% (w/v), the cell concentration is 106cells/ml and the concentration of fibrinectin is 10ml of sodium alginate/gelatin/hyaluronic acid bio-ink with the concentration of 20 mu g/ml;

and 2.2) pouring the fully and uniformly dissolved sodium alginate/gelatin/hyaluronic acid solution into a printing needle cylinder for centrifugal defoaming.

In the step 1.2), the step 1.3) and the step 1.4), the dosage of the gelatin (sigma) is 0.5g, the dosage of the sodium alginate (alatin) is 0.25g, the dosage of the hyaluronic acid (Mecline) is 0.02g, and the ratio of the gelatin (sigma) to the sodium alginate to the hyaluronic acid (Mecline) is 100:25: 2.

In step 3), the concentration of the calcium chloride (Solarbio) solution is 4% (w/v), and the solid-phase crosslinking time is 40 s.

In the step 3), the temperature of the syringe is set to 35 ℃, and the temperature of the platform is set to 5 ℃.

In step 3), the shape and size of the active biofilm can be designed according to actual needs, and the internal structure of the stent is that the height of the printing stent is 0.11mm, the length and the width are 2.2mm and 2.2mm, and the height of each layer is 0.18 mm.

The extrusion pressure in step 3) was 8kpa, the nozzle was 0.2mm from the floor surface, the nozzle specification was 20G, and the moving speed was 360 mm/min.

The extrusion type biological 3D printer in the step 3) is EFL-BP 6601.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the materials used in the invention are all natural materials, have low immunogenicity, good biocompatibility and wide sources, and simultaneously have certain mechanical properties and can provide good support for cartilage regeneration.

2. In terms of the preparation method, the 3d printed biological membrane is used for replacing an I/III collagen membrane prepared by the traditional method, on one hand, the 3d printed biological membrane can accurately control the pores, the void ratio, the structure and the like of the membrane, on the other hand, the 300-micron and 500-micron gaps can promote the regeneration of cartilage, and meanwhile, the excellent void ratio structure and the like can also promote the exchange and communication of substances among cells, thus being beneficial to the adhesion of cell factors or cells and the growth and proliferation of the cells in the biological membrane. And the model can be made according to the actual condition of the damaged cartilage of the patient, so that the personalized treatment is really realized.

3. As for the preparation material, the biological membrane prepared by mixing sodium alginate, gelatin and hyaluronic acid is used for replacing the collagen membrane I/III. Sodium alginate as a natural polysaccharide has good histocompatibility and biodegradability, and sodium alginate can rapidly generate ion exchange crosslinking to generate gel when meeting calcium ions, so the sodium alginate hydrogel is widely used for 3d printing of cartilage tissues. Gelatin as a protein has the performance of dissolving at high temperature and forming gel at low temperature, and can obviously improve the viscosity of hydrogel and improve the printing performance. The gelatin is a protein obtained by partial hydrolysis of collagen, has homology with the collagen, is used as the main component of the articular hyaline cartilage, has good biocompatibility and obvious cartilage promoting capacity. Hyaluronic Acid (HA) is an acidic mucopolysaccharide macromolecular substance widely present in human and animal connective tissues, and HAs a remarkable cartilage-promoting ability to perform various physiological functions of cells and regulate cell aggregation during tissue formation. Has obvious chondrogenesis promoting effect relative to a single collagen membrane.

4. The invention mixes cartilage precursor cells and fibrinectin into mixed hydrogel to prepare active biomembrane improved AMIC technology, (1) the fibrinectin has obvious effects of promoting proliferation and chondrogenic differentiation on the cartilage precursor cells and bone marrow mesenchymal stem cells. (2) The co-growth of the cartilage precursor cells present in the membrane with the released mesenchymal stem cells promotes chondrogenic differentiation of the mesenchymal stem cells. (3) The membrane cartilage precursor cells can be effectively differentiated into cartilage cells, and the cartilage repair effect is promoted. In general, the improved AMIC of the present invention produces biofilms with significantly improved cartilage repair compared to collagen membranes of the inactive single material.

5. The invention provides an active biological membrane based on sodium alginate/gelatin/hyaluronic acid mixed hydrogel combined cartilage precursor cells and fibronectin (fibronectin) for biological 3D printing of cartilage repair and a preparation method thereof, which are used for improving an autologous matrix induced cartilage regeneration (AMIC) technology used clinically. The biological membrane takes sodium alginate, gelatin and hyaluronic acid as basic hydrogel materials, mature third generation cartilage precursor cells and fibronectin are mixed after the preparation of the hydrogel is completed, a porous biological hydrogel biological membrane is constructed by a sedimentary biological 3d printing technology, and the mechanical property is increased by further crosslinking through calcium chloride soaking. The biofilm is used for replacing a collagen membrane collagen I/III bilayerrmatrix (Chondro-Gide; Geistlich Pharma AG) which is used clinically, namely, the biofilm is used for combining the microfracture technology of subchondral bone. The active biological membrane combines the excellent performances of gelatin, sodium alginate and hyaluronic acid, has good biocompatibility, mechanical property and proper gap and porosity, is added with cartilage precursor cells and fibronectin, improves the regeneration capacity of cartilage, and can be used for improving the AMIC technology and repairing cartilage defects.

Drawings

Fig. 1 is a schematic diagram of the printing process of the active biofilm 3 d.

FIG. 2 is a diagram illustrating the general effect of printing according to the embodiment.

Fig. 3 is a schematic view of a printing scheme using gelatin 5%.

Fig. 4 is a schematic view of a printing scheme using 8% gelatin.

Fig. 5 is a schematic view of a printing scheme using 10% gelatin.

Fig. 6 is a mechanical test stress-strain curve of the example.

FIG. 7 shows the mechanical test modulus of elasticity of the examples.

FIG. 8 is a schematic view of an entire SEM of an embodiment.

FIG. 9 is a schematic view of a local area of a scanning electron microscope according to an embodiment.

FIG. 10 is a schematic view of a local area of a scanning electron microscope according to an embodiment.

FIG. 11 is a schematic of shear-thinning for the rheological property test of the examples.

FIG. 12 is a frequency sweep diagram of rheological property testing of the examples.

FIG. 13 is a schematic temperature profile of the rheological property test of the examples.

FIG. 14 is a graph showing the results of live-dead staining of the cells of example after in vitro culture for 7 days after printing.

FIG. 15 is a flow chart of a method of making the present invention.

Fig. 16 is a schematic structural diagram of a biological 3D printer for cartilage repair proposed by the present invention;

fig. 17 is a front view of a biological 3D printer for cartilage repair proposed by the present invention;

fig. 18 is a right side view of a biological 3D printer diagram of cartilage repair as proposed by the present invention;

fig. 19 is a front view of a molding platform of a cartilage repair bio-3D printer according to the present invention.

Illustration of the drawings: 1. a forming platform; 101. forming a panel; 102. an adjustment device; 103. a nozzle moving rod; 104. printing a spray head; 2. a back plate; 3. a material containing device; 301. a glass platform; 302. a trough ring; 303. a film jumping ring; 304. a trough bottom plate; 4. a peeling device; 401. a motor; 402. a connector; 403. a guide bar; 404. a support plate; 405. stripping the cover plate; 5. a fuselage frame; 6. a material supplementing device; 601. a sliding table; 602. a bio-ink container; 603. a push rod; 7. DLP projection light machine; 8. adjusting the slide rail; 9. a reflective mirror; 10. fixing a bracket; 11. a chute.

Detailed Description

The invention will be further elucidated with reference to the drawings and specific examples, without being limited thereto.

As shown in fig. 1-18.