CN111588908B - Biological 3d printed active biofilm for improving AMIC technology cartilage repair and preparation method thereof - Google Patents
Biological 3d printed active biofilm for improving AMIC technology cartilage repair and preparation method thereof Download PDFInfo
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- CN111588908B CN111588908B CN202010167197.3A CN202010167197A CN111588908B CN 111588908 B CN111588908 B CN 111588908B CN 202010167197 A CN202010167197 A CN 202010167197A CN 111588908 B CN111588908 B CN 111588908B
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Abstract
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.
Description
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 biolayer matrix (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.
Example 1
1) Printing material preparation
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.
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.
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.
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.
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.
1.6) the lyophilized fibronectin powder was diluted with medium, vortexed to dissolve it sufficiently, and prepared into a solution of 100. mu.g/ml, and stored at-20 ℃ for further use.
2) Configuration of the printed hydrogel:
mixing and uniformly 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 to obtain 10ml of sodium alginate/gelatin/hyaluronic acid biological ink with the final concentration of 2.5% (w/v), the concentration of hyaluronic acid of 0.1% (w/v), the concentration of gelatin of 5% (w/v), the cell concentration of 106cells/ml and the concentration of fibrinectin of 20 mu g/ml.
And pouring the sodium alginate/gelatin/hyaluronic acid solution which is fully and uniformly dissolved into a printing needle cylinder for centrifugal defoaming.
3) Printing and post-treatment of hydrogel active biofilms
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 an extrusion biological 3d printer (EFL-BP6601), setting the temperature of a nozzle needle cylinder of the printer to be 35 ℃, and setting the temperature of a platform to be 5 ℃. The air pressure is started, the extrusion pressure is 8kpa, the distance between the nozzle and the floor surface is 0.2mm, the nozzle specification is 20G, and the moving speed is 360 mm/min. Relevant parameters of the biological membrane are adjusted, 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. And starting printing after the nozzle stably extrudes the hydrogel microfilaments. And (3) after printing is finished, fully curing the hydrogel film scaffold by using a calcium chloride solution for 4% (w/v) for 40s to finally obtain the cell-loaded hydrogel film scaffold.
Example 2
1) Printing material preparation
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.
1.2) preparation of gelatin solution: heating 4g of gelatin in 20ml of deionized water in 48 ℃ water bath, stirring by using a constant-temperature magnetic stirrer, and adjusting the rotating speed to 300r/min to fully dissolve the gelatin to prepare a gelatin solution.
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.
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 8% (w/v). For storage at room temperature, ready for use.
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.
1.6) the lyophilized fibronectin powder was diluted with medium, vortexed to dissolve it sufficiently, and prepared into a solution of 100. mu.g/ml, and stored at-20 ℃ for further use.
2) Configuration of the printed hydrogel:
mixing and uniformly 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 liquid, 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 8% (w/v), the cell concentration is 106cells/ml and the concentration of fibrinectin is 10ml of sodium alginate/gelatin/hyaluronic acid biological ink with the concentration of 20 mu g/ml.
And pouring the sodium alginate/gelatin/hyaluronic acid solution which is fully and uniformly dissolved into a printing needle cylinder for centrifugal defoaming.
3) Printing and post-treatment of hydrogel active biofilms
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 an extrusion biological 3d printer (EFL-BP6601), setting the temperature of a nozzle needle cylinder of the printer to be 35 ℃, and setting the temperature of a platform to be 5 ℃. The air pressure is started, the extrusion pressure is 8kpa, the distance between the nozzle and the floor surface is 0.2mm, the nozzle specification is 20G, and the moving speed is 360 mm/min. Relevant parameters of the biological membrane are adjusted, 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. And starting printing after the nozzle stably extrudes the hydrogel microfilaments. And (3) after printing is finished, fully curing the hydrogel film scaffold by using a calcium chloride solution for 4% (w/v) for 40s to finally obtain the cell-loaded hydrogel film scaffold.
Example 3
1) Printing material preparation
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.
1.2) preparation of gelatin solution: heating 5g of gelatin in 20ml of deionized water in 48 ℃ water bath, stirring by using a constant-temperature magnetic stirrer, and adjusting the rotating speed to 300r/min to fully dissolve the gelatin to prepare a gelatin solution.
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.
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 10% (w/v). For storage at room temperature, ready for use.
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.
1.6) the lyophilized fibronectin powder was diluted with medium, vortexed to dissolve it sufficiently, and prepared into a solution of 100. mu.g/ml, and stored at-20 ℃ for further use.
2) Configuration of the printed hydrogel:
mixing and uniformly 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 liquid, 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 10% (w/v), the cell concentration is 106cells/ml and the concentration of fibrinectin is 10ml of sodium alginate/gelatin/hyaluronic acid biological ink with the concentration of 20 mu g/ml.
And pouring the sodium alginate/gelatin/hyaluronic acid solution which is fully and uniformly dissolved into a printing needle cylinder for centrifugal defoaming.
3) Printing and post-treatment of hydrogel active biofilms
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 an extrusion biological 3d printer (EFL-BP6601), setting the temperature of a nozzle needle cylinder of the printer to be 35 ℃, and setting the temperature of a platform to be 5 ℃. The air pressure is started, the extrusion pressure is 8kpa, the distance between the nozzle and the floor surface is 0.2mm, the nozzle specification is 20G, and the moving speed is 360 mm/min. Relevant parameters of the biological membrane are adjusted, 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. And starting printing after the nozzle stably extrudes the hydrogel microfilaments. And (3) after printing is finished, fully curing the hydrogel film scaffold by using a calcium chloride solution for 4% (w/v) for 40s to finally obtain the cell-loaded hydrogel film scaffold.
Example 4 (comparative example)
1) Printing material preparation
1.1) preparation of tyramine root graft modified gelatin: 500ml of 50mM morpholine ethanesulfonic acid buffer was prepared, 10g of gelatin powder was added, and the mixture was stirred and dissolved sufficiently at 50 ℃. Adding 5g of tyramine hydrochloride, stirring and fully dissolving; after the solution is cooled to room temperature, sequentially adding 0.37g/0.11g of carboxyl activating agent N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to activate carboxyl on a gelatin molecular chain, and reacting for 12 hours at room temperature; and (3) filling the reaction product into a dialysis bag with the molecular weight cutoff of 10000-12000, dialyzing for 4 days in a deionized water environment, finally removing water by using a freeze dryer to obtain a white spongy modified product, and storing the white spongy modified product in a moisture-proof cabinet for later use.
1.2) preparation of silk fibroin solution: adding 4g of degummed silk into a beaker, and then adding 20ml of 9.3mol/l lithium bromide solution; placing the beaker in a water bath kettle at 60 ℃, and heating to dissolve degummed silk for 4 hours; after full dissolution, transferring the solution into a dialysis bag with molecular weight cutoff of 3500, dialyzing for 2 days in an ionic water environment, and changing water for 2-3 times every day; after the dialysis is finished, centrifuging the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; and after the centrifugation is finished, obtaining a clear silk fibroin solution, wherein the concentration of the silk fibroin solution is obtained by a drying specific gravity method, and the silk fibroin solution is diluted to 2.5 w/v% by deionized water.
2) Preparation of printing paste
Adding modified gelatin into silk fibroin solution with concentration of 2.5 w/v% to obtain modified gelatin with concentration of 15 w/v%, dissolving at 50 deg.C for about 2 hr, and mixing; adding horseradish peroxidase after the gelatin is dissolved to make the concentration of the gelatin be 60 Units/ml; after mixing, the mixture was transferred to a charging barrel.
3) Printing and post-processing of hydrogel scaffolds
Transferring the charging barrel filled with the printing paste in the step 2) into a 3D printer, setting the temperature of the charging barrel to be 29 ℃, setting the temperature of a printing deposition platform to be 4 ℃, and keeping the temperature of the charging barrel for 2 hours; setting the external appearance of the bracket as a cuboid with 10 × 4mm, and setting the internal structure as follows: the space between the fiber yarns is 0.6mm, and the included angle between every two layers of the fiber yarns is 90 degrees; printing is started after the extrusion pressure is set to be 2bar and the drafting speed is set to be 15 mm/s; and after printing is finished, soaking the printed structure in hydrogen peroxide solution for 30min to initiate enzyme-catalyzed crosslinking between the silk fibroin and the modified gelatin, and washing the silk fibroin and the modified gelatin for three times by using deionized water to obtain the SF2.5GT15 hydrogel scaffold printed in 3D.
The above examples of the present invention are merely examples for illustrating the present invention and are not intended to limit the embodiments of the present invention; any modification, equivalent replacement, etc. made within the spirit and principle of the present invention should be within the protection scope of the claims of the present invention.
Example 5
The invention also provides a cartilage repair biological 3D printer, and referring to fig. 14-17, the cartilage repair biological 3D printer comprises a machine body frame 5, a back plate 2 is fixedly connected to the position, close to the rear side, of the top end of the machine body frame 5, a fixing support 10 is fixedly connected to the back plate 2, a sliding groove 11 is formed in the surface of the fixing support 10, a forming platform 1 is arranged on the surface of the fixing support 10, the forming platform 1 is in sliding connection with the sliding groove 11 in the surface of the fixing support 10, a stripping device 4 is fixedly connected to the center position of the top end of the machine body frame 5, a material containing device 3 is arranged at the top end of the stripping device 4, the material containing device 3 abuts against the stripping device 4, a material supplementing device 6 is fixedly connected to the top end of the stripping device 4, and a DLP projection optical machine 7 is fixedly connected to the interior of the machine body frame 5.
The forming platform 1 comprises a forming panel 101 and a printing nozzle 104, the top end of the forming panel 101 is fixedly connected with an upper piece and a lower piece in sequence, the top end of the upper piece is embedded with an adjusting device 102, a nozzle moving rod 103 is arranged on the side surface of the printing nozzle 104, and the nozzle moving rod 103 is fixedly connected with the side surface of the forming platform 1.
Stripping off device 4 is including backup pad 404, guide bar 403, motor 401, connector 402, peel off apron 405, motor 401 top and connector 402 fixed connection, connector 402 offsets with guide bar 403 top and peel off apron 405, and peels off apron 405 avris fixedly connected with fixture block.
The material containing device 3 comprises a trough bottom plate 304, a jumping film ring 303, a trough ring 302 and a glass platform 301, wherein the trough bottom plate 304 is fixed through a clamping groove formed between a dragging plate on a stripping cover plate 405 and a clamping block, the jumping film ring 303 is fixedly connected with the trough bottom plate 304, the top end of the jumping film ring 303 is fixedly connected with the trough ring 302, and the glass platform 301 is abutted to the trough ring 302.
The feeding device 6 comprises a biological ink container 602, a sliding table 601 is fixedly connected to the side surface of the biological ink container 602, a push rod 603 is arranged on the back surface of the biological ink container 602, and the push rod 603 is fixedly connected with the sliding table 601.
DLP projection ray apparatus 7 bottom is equipped with adjusts slide rail 8, and DLP projection ray apparatus 7 is fixed with adjusting slide rail 8, it is equipped with reflector 9 to adjust the slide rail 8 inboard, and reflector 9 and the inboard fixed connection of slide rail.
A stripping device 4 is fixedly connected to the center of the top end of a body frame 5 of a cartilage repair biological 3D printer, a material containing device 3 is arranged at the top end of the stripping device 4, the material containing device 3 is abutted against the stripping device 4, a material supplementing device 6 is fixedly connected to the top end of the stripping device 4, a DLP projection optical machine 7 is fixedly connected to the interior of the body frame 5, a forming platform 1 comprises a forming panel 101 and a printing nozzle 104, an upper piece and a lower piece are fixedly connected to the top end of the forming panel 101 in sequence, an adjusting device 102 is embedded at the top end of the upper piece, a nozzle moving rod 103 is arranged on the side surface of the printing nozzle 104, the nozzle moving rod 103 is fixedly connected with the side surface of the forming platform 1, the three precise adjusting devices 102 are used by the forming platform 1 to realize the adjustment of the initial height and the levelness of a forming plane by utilizing the three-point surface fixing principle, so as to improve the printing precision, and the high-precision projection of the DLP projection optical machine 7 solidifies the biological ink, the forming precision can reach dozens of micrometers, the device can be used for printing extremely complex shapes, the printing nozzle 104 with high degree of freedom can print in a defective cartilage focus cavity, repair of defective cartilage can be realized only by minimally invasive focus surfaces, preparation and culture of cartilage particles are not needed, the treatment period can be greatly shortened, treatment cost can be reduced, pain, abrasion and economic pressure of a patient can be relieved, the supplementing device 6 comprises a biological ink container 602, a sliding table 601 is fixedly connected to the side surface of the biological ink container 602, a push rod 603 is arranged on the back surface of the biological ink container 602, the push rod 603 is fixedly connected with the sliding table 601, an adjusting slide rail 8 is arranged at the bottom end of a DLP projection optical machine 7, the DLP projection optical machine 7 is fixed with the adjusting slide rail 8, a reflector 9 is arranged on the inner side of the adjusting slide rail 8, and the reflector 9 is fixedly connected with the inner side of the slide rail.
The working principle of the printer of the invention is as follows: aiming at the printing of biological materials, a distributed temperature control module and a material supplementing device 6 are additionally arranged on a main body, the distributed temperature control module heats and preserves the biological materials at three positions of a material containing device 3, the material supplementing device 6 and a forming platform 1 in the whole printing process so as to prevent the condensation of the materials, and the smooth printing is ensured. The feeding module feeds regularly to reduce concentration variations due to evaporation of moisture from air-exposed biological material in the trough and to save expensive printing material.
In the implementation of the invention, the adjusting device and the DLP projector are adopted to realize the printing of high-precision complex modeling, the three precise adjusting devices are used by the forming platform to realize the adjustment of the initial height and the levelness of the forming plane by utilizing the three-point surface fixing principle, thereby improving the printing precision, the high-precision projection of the DLP projector solidifies the biological ink, the forming precision can reach tens of microns, and the DLP projector can be used for printing extremely complex modeling.
The invention provides a biological 3D printer for cartilage repair, which relates to the technical field of cartilage repair and comprises a machine body frame, wherein a back plate is fixedly connected to the position, close to the rear side, of the top end of the machine body frame, a fixing support is fixedly connected with a back plate, a sliding groove is formed in the surface of the fixing support, a forming platform is arranged on the surface of the fixing support and is in sliding connection with the sliding groove in the surface of the fixing support, a stripping device is fixedly connected to the central position of the top end of the machine body frame, a material containing device is arranged on the top end of the stripping device and is abutted against the stripping device, a material supplementing device is fixedly connected to the top end of the stripping device, a DLP projection optical machine is fixedly connected inside the machine body frame, the initial height and the levelness of a forming plane are adjusted by using three precise adjusting devices through a forming platform and utilizing a three-point surface fixing principle, so that the printing precision is improved, the biological ink is solidified through the high-precision projection of the DLP projection optical machine, the forming precision can reach tens of microns, and the printing machine can be used for printing extremely complicated models.
In the implementation of the invention, the printing nozzle is adopted to realize the repair of the cartilage defect, the printing nozzle with high degree of freedom can be used for printing in the lesion cavity of the defect cartilage, the repair of the defect cartilage can be realized only by minimally invasive lesion surfaces, the preparation and culture of cartilage particles are not needed, the treatment period can be greatly shortened, the treatment cost can be reduced, and the pain and the abrasion of patients and the economic pressure can be favorably reduced.
Claims (6)
1. 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 prepare a jelly-shaped gel state, 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; starting air pressure, adjusting relevant parameters of the biological membrane, and starting printing after the nozzle stably extrudes hydrogel microfilaments; fully curing the hydrogel film scaffold by using a calcium chloride solution after printing is finished, and finally obtaining 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 a hydrogel pre-prepared solution; all other operations are operated in a super clean bench;
step 1.2) preparation of gelatin solution: heating 20ml of deionized water and 2.5g of gelatin in a 48 ℃ water bath, stirring by using a constant-temperature magnetic stirrer, and adjusting the rotating speed to 300r/min to fully dissolve the gelatin to prepare a 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 a 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 solution, so that the sodium alginate, the gelatin or the hyaluronic acid solution is fully dissolved uniformly, and finally, the concentration of the sodium alginate is 2.5% w/v, the concentration of the hyaluronic acid is 0.2% w/v, and the concentration of the gelatin is 5% w/v; storing at room temperature for later use;
step 1.5) digesting chondrocytes, adding the digested chondrocytes into a cartilage culture medium which normally contains 10% serum and 1% double antibody, adding a 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 are normally cultured cartilage precursor cells after 3 generations;
step 1.6) diluting the fibronectin freeze-dried powder of fibronectin with a culture medium, fully dissolving the diluted solution by vortex, preparing a solution of 100 mu g/ml, and storing the solution at-20 ℃ for later use; the step 2) configuration of printing the hydrogel comprises the following steps:
step 2.1) 2ml of a solution of fibrinectin at a concentration of 100. mu.g/ml, 2ml of a solution of cells at a concentration of 5X106Mixing and stirring the cell suspension/ml and 6ml of sodium alginate/gelatin/hyaluronic acid hydrogel pre-prepared solution uniformly, wherein the final sodium alginate concentration is 1.5% w/v, the hyaluronic acid concentration is 0.12% w/v, the gelatin concentration is 3% w/v, and the cell concentration is 1 × 10610ml of sodium alginate/gelatin/hyaluronic acid biological ink with each cell/ml and the concentration of fibronectin being 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.
2. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 1, wherein: in the step 3), the concentration of the calcium chloride solution is 4% w/v, and the curing and crosslinking time is 40 s.
3. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 1, wherein: in the step 3), the temperature of the syringe is set to 35 ℃, and the temperature of the platform is set to 5 ℃.
4. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 1, 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.
5. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 1, 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.
6. The method of preparing an active biofilm of biological 3d printed modified AMIC for cartilage repair as claimed in claim 1, wherein: the extrusion type biological 3D printer in the step 3) is EFL-BP 6601.
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