CN112402701A - Diagnosis and treatment integrated gradient osteochondral bionic scaffold and preparation method thereof - Google Patents
Diagnosis and treatment integrated gradient osteochondral bionic scaffold and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a diagnosis and treatment integrated gradient osteochondral bionic scaffold and a preparation method thereof, which are realized by adopting a DLP photocuring 3D printing technology and comprise the following steps: respectively preparing sols of a cartilage layer, a cartilage calcified layer and a subchondral bone layer, and preparing the cartilage layer sol by using quercetin, methacrylated gelatin and a LAP solution; preparing cartilage calcification layer sol by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite and LAP solution; the subchondral bone layer sol is prepared by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite and LAP solution. The scaffold prepared by the method has structural gradient and material gradient, good mechanical property and biocompatibility, good bone induction capability and bone conduction capability, and can induce the regeneration of the bone tissue defect part. The gradient bracket has the MRI diagnosis function, and can realize accurate evaluation of the real-time condition of osteochondral defect repair in vivo.
Description
Technical Field
The invention belongs to the technical field of medical biomaterial preparation and tissue engineering, and particularly relates to a diagnosis and treatment integrated gradient osteochondral bionic scaffold and a preparation method thereof.
Background
According to statistics, 70% of people older than 65 years suffer from articular cartilage injuries of different degrees, which are common diseases caused by trauma, diseases, improper exercise, aging and the like, are often manifested as joint pain and dysfunction, and seriously affect the daily life of patients. Because the human articular cartilage belongs to avascular tissues, the required nutrition mainly comes from joint fluid, and the nutrition is insufficient to promote the repair and reconstruction of cartilage defects; and the relative renewal rate of chondrocytes is low, and when the defect of articular cartilage is >2mm, the defect can hardly be repaired by proliferation of chondrocytes, so that the damaged articular cartilage is difficult to regenerate by the autogenous repair ability.
The current clinical treatment methods mainly comprise: conservative treatment, joint cavity cleaning operation, micro fracture operation, cartilage cell transplantation, autologous bone or allogeneic bone cartilage transplantation, artificial joint replacement and the like. However, these methods have certain drawbacks, such as: insufficient donor source, rejection reaction, complications, poor long-term curative effect and the like.
In recent years, with the development of science and technology and the deep research of tissue engineering regeneration technology, a new solution is provided for the regeneration and repair of osteochondral injuries. The cartilage tissue is composed of articular cartilage and subchondral bone, is a complex tissue structure with progressive material gradient and physiological characteristics, and has very specific mechanical characteristics, metabolism and tissue transportation at different layers. Generally, a cartilage defect involves subchondral bone to cause a bone-cartilage defect, and when the subchondral bone is damaged, new cartilage is difficult to integrate with the subchondral bone, which in turn hinders repair of cartilage, so that repair of a single cartilage defect is difficult to achieve. Therefore, the invention constructs the osteochondral bionic scaffold with a multi-level gradient structure based on the cartilage structure and is simultaneously used for osteochondral defect repair.
In recent years, 3D printing technology is developed more and more mature in biological tissue engineering, the biological 3D printing technology can be used for accurately controlling the shape of osteochondral scaffold materials and the distribution of cells in the scaffold, artificial tissues highly similar to human tissue structures can be constructed, the two problems of the accuracy of the scaffold structures and the accuracy of cell implantation are solved, meanwhile, specific shape structures with different clinical requirements can be met, and personalized customization is achieved.
At present, the degradation of the bionic osteochondral scaffold is mostly evaluated by adopting a CT scanning mode, CT is a ray inspection method, functional changes of organ tissue structures are difficult to find, and an intuitive and noninvasive means for diagnosing the articular cartilage defect repair condition is urgently needed for more comprehensively evaluating the degradation of the bionic gradient osteochondral scaffold and tracking and diagnosing the repair condition of a bone defect part.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a diagnosis and treatment integrated gradient osteochondral bionic scaffold and a preparation method thereof. The osteochondral bionic scaffold prepared by the method has structural gradient and material gradient, good mechanical property and biocompatibility, good bone induction capability and bone conduction capability, and can induce the regeneration of bone tissue defect parts. In order to make up the defects of CT diagnosis, the gradient bracket has the MRI diagnosis function, and can realize accurate evaluation of the real-time condition of osteochondral defect repair in vivo.
In order to achieve the purpose, the invention provides a preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold, which is realized by adopting a DLP photocuring 3D printing technology and comprises the following steps: respectively preparing sol of cartilage layer, cartilage calcified layer and subchondral bone layer,
preparing a cartilage layer sol by using a solution of quercetin, methacrylic acid esterified gelatin (GelMA) and phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate (LAP);
preparing cartilage calcification layer sol by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite (Fe-HAP) and LAP solution;
the subchondral bone layer sol is prepared by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite and LAP solution.
Specifically, initiator LAP was dissolved with a Phosphate Buffered Saline (PBS) solution to obtain a LAP solution.
Specifically, 0.01-0.05 part of quercetin, 15-20 parts of GelMA and LAP solution are uniformly mixed to obtain the cartilage layer sol.
Specifically, 0.01-0.05 part of quercetin, 20-25 parts of GelMA, 2-3 parts of Fe-HAP and LAP solution are uniformly mixed to obtain the cartilage calcification layer sol.
Specifically, 0.01-0.05 part of quercetin, 25-30 parts of GelMA, 2-3 parts of Fe-HAP and LAP solution are uniformly mixed to obtain the subchondral bone layer sol.
Preferably, the step of preparing methacrylated gelatin (GelMA) comprises:
dissolving gelatin in a PBS solution, heating and dissolving to obtain a gelatin solution;
dripping methacrylic anhydride into the gelatin solution, controlling the pH value to be 8-9, and adding a PBS solution after a certain time to obtain a reaction solution;
putting the reaction solution into a dialysis bag, dialyzing with deionized water, and freeze-drying the dialysate in the bag to obtain the methacrylated gelatin.
Specifically, the preparation steps of methacrylated gelatin (GelMA) include:
(1) dissolving 1-10 parts of gelatin in 20-150 parts of PBS (phosphate buffer solution), completely dissolving the gelatin at 40-80 ℃ to obtain a gelatin solution, placing the gelatin solution on a constant-temperature magnetic stirrer for stirring, dropwise adding 1-5 parts of Methacrylic Anhydride (MA) by using a constant-pressure funnel, dropwise adding the Methacrylic Anhydride (MA) into the gelatin solution at the speed of 0.1-0.5 part per minute, dropwise adding NaOH solution in time to adjust the pH value of the solution, maintaining the pH value within the range of 8-9, and stopping the reaction by adding 1-5 times of the PBS after maintaining the stirring reaction for 4 hours.
(2) And (3) purification: transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, dialyzing with deionized water, changing water every 4-6 hours, dialyzing for 2 days, and freeze-drying the dialysate in the bag to obtain a foamy product, namely the methacrylated gelatin, and storing at-20 ℃ for later use.
Preferably, the pH of the PBS solution is 7.4.
Preferably, the preparation of iron-doped hydroxyapatite (Fe-HAP) comprises the steps of:
reacting Ca (OH)2Dissolving in water to obtain Ca (OH)2An aqueous solution;
FeCl is added2·4H2Dissolving O in water to obtain FeCl2·4H2An aqueous solution of O;
FeCl is added3·6H2Dissolving O in water to obtain FeCl3·6H2An aqueous solution of O;
dissolving phosphoric acid in water to prepare a phosphoric acid aqueous solution;
FeCl is added2·4H2Aqueous O solution and FeCl3·6H2O aqueous solution to Ca (OH)2And (3) obtaining a mixed solution from the aqueous solution, dripping the phosphoric acid aqueous solution into the mixed solution, heating and stirring for a period of time, aging, centrifuging, washing and drying the precipitate to obtain the iron-doped hydroxyapatite.
Specifically, the preparation method of the iron-doped hydroxyapatite (Fe-HAP) comprises the following steps:
(1) weighing 0.6-0.7 part of Ca (OH)2Dissolving in 100-500 parts of water, transferring into a reaction bottle, magnetically stirring and heating to 45-65 ℃ for later use; weighing 0.06-0.07 part of FeCl2·4H2Dissolving O in 75-100 parts of water, and weighing 0.06-0.07 part of FeCl3·6H2Dissolving O in 75-100 parts of water, and respectively considering the two prepared solutions as Fe3+And Fe2+(ii) a source of (a); after the above two solutions are completedAfter dissolution, both solutions were slowly added to Ca (OH) simultaneously2Suspension liquid; dissolving 0.4-0.5 part of phosphoric acid in 300-500 parts of water, slowly dripping the phosphoric acid solution into the mixed solution, and continuously heating and stirring for 2-4 hours.
(2) And (3) continuously heating the product obtained by the reaction (45-65 ℃), stirring for 2-4 h, finishing the reaction, aging at normal temperature for 24h, centrifuging at 1000rpm by using a centrifuge to obtain brownish black precipitate, washing the precipitate for 5 times by using pure water, and freeze-drying to obtain brownish black powder, namely iron-doped hydroxyapatite, and storing at-20 ℃ for later use.
Preferably, the molar ratio of the content of Fe to the content of Ca is 0.2.
Preferably, the molar ratio of the content of Ca and Fe to the content of P is 1.67.
Preferably, the mass concentration of the LAP solution is 0.5-1%.
Preferably, the steps further comprise:
establishing an osteochondral bionic scaffold model: designing a bone cartilage bionic scaffold model by means of three-dimensional modeling software according to a bionics principle and a size structure of a bone defect part, and exporting an STL format file;
the method comprises the steps of slicing a model, introducing the model into a DLP printer, adding sol of a subchondral bone layer into a material tank, printing the subchondral bone layer part of the model, replacing the material tank filled with the sol of the cartilage calcification layer after the last layer of the subchondral bone layer is printed, continuing to print the cartilage calcification layer, replacing the material tank filled with the sol of the cartilage layer after the last layer of the cartilage calcification layer is printed, and continuing to print the cartilage layer until the end of printing, thus obtaining a semi-finished support.
Preferably, when the bone defect part bracket model is established: the thickness of the cartilage layer is 2.3-3.5 mm, the aperture is 100-150 mu m, and the pore channels are mutually communicated; the thickness of the calcified layer of the cartilage is 0.01-0.5 mm, and the aperture is 0; the thickness of the subchondral bone layer is 3.5-5.5 mm, the aperture is 200-500 mu m, and the pore channels are mutually communicated.
Preferably, when the subchondral bone layer, the cartilage calcified layer and the cartilage layer are printed and jointed, the upper layer of jointed surface is cleaned by PBS solution, the water on the surface of the bracket is sucked dry by absorbent paper, and then the next layer of printing is continued.
Preferably, the wavelength of the light source during the gradient bracket printing is 405nm, and the light intensity is 2.0-4.5 mW/cm2The exposure time is 25-50 s, and the thickness of the printing layer is 5-100 μm.
Preferably, after the semi-finished stent is cleaned, secondary curing is carried out in an ultraviolet box.
Preferably, the secondary curing time of the post-treatment of the bracket is 10-20 min.
Preferably, the scaffold after the secondary curing is placed in PBS for full swelling, then is pre-frozen at the temperature of minus 20 ℃, and is frozen in a freeze dryer at the temperature of minus 55 to minus 40 ℃ for 24 to 72 hours.
Compared with the prior art, the invention has the beneficial effects that: the gradient support forming process adopts a Digital Light Processing (DLP) photocuring 3D printing technology, is more advanced than the traditional manufacturing process, can realize personalized customization, and is quicker and higher in precision than an extrusion biological 3D printing technology. The gradient scaffold comprises a cartilage layer, a cartilage calcification layer and a subchondral bone layer from top to bottom in sequence, has a bionic structure, is constructed by 3D printing to form an internal pore structure which is mutually communicated, and is suitable for repairing and reconstructing osteochondral bones and conveying nutrient substances. In the process of printing the scaffold, the bioactive phytohormone quercetin is adopted to induce osteogenic differentiation of cells on the 3D printing gradient repair scaffold, and the proliferation of osteoblasts is promoted. The gradient scaffold has good mechanical property and biocompatibility, can track the real-time condition of osteochondral defect repair through MRI, has good bone induction capability and bone conduction capability, and can induce the regeneration of bone tissue defect parts.
Drawings
FIG. 1 shows the results of the compressive strength test of the gradient scaffold of examples 1 to 6.
FIG. 2 results of cytotoxicity test of scaffolds with gradients of examples 1-6.
FIG. 3 results of the alkaline phosphatase activity tests of examples 1, 5 and 6.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold comprises the following steps:
(1) establishing an osteochondral scaffold model: according to the bionics principle and the size structure of the bone defect part, a bionic bone scaffold model is designed by using three-dimensional modeling software, and a 3D printer STL format file is finally exported by the model, wherein the model of the example is as follows: the cylindrical gradient scaffold with the diameter of 10mm and the height of 7mm is a cartilage layer, a cartilage calcification layer and a subchondral bone layer from top to bottom respectively, wherein the thickness of the cartilage layer is 3mm, the pore diameter is 150 mu m, pores are communicated with one another, the thickness of the cartilage calcification layer is 0.5mm, the pore diameter is 0, the thickness of the subchondral bone layer is 3.5mm, the pore diameter is 200-500 mu m, and the pores are communicated with one another.
(2) GelMA synthesis preparation: weighing 5g of gelatin by using an analytical balance, dissolving the gelatin in 50mL of PBS (pH is 7.4 and 0.01M), completely dissolving the gelatin at 60 ℃ to obtain 50mL of gelatin solution, placing the gelatin solution on a constant-temperature magnetic stirrer, stirring (50 ℃) by using a micro constant-pressure funnel, dropwise adding 1mL of Methacrylic Anhydride (MA) into the gelatin solution at the speed of 0.1mL/min, timely dropwise adding NaOH solution (1M) to adjust the pH of the solution, maintaining the pH to be about 8, keeping stirring for reaction for 3 hours, stopping the reaction by adding 3-5 times of the PBS solution, transferring the reaction solution into a dialysis bag with molecular weight cutoff of 8000Da, dialyzing for 3 days by using deionized water, changing water every 4 hours, and freeze-drying the dialysate in the dialysis bag to obtain a foamed product, namely GelMA, and storing at-20 ℃ for later use;
synthesis and preparation of Fe-HAP: 50g Ca (OH) are weighed2Dissolving in 400mL of water, transferring to a 1000mL reaction flask, magnetically stirring and heating to 65 ℃ for later use; 12.74g of FeCl were weighed2·4H2O was dissolved in 75mL of deionized water and 16.78g FeCl was weighed3·6H2Dissolving O in 75mL of deionized water; after the two solutions are completely dissolved, the two solutions are slowly added into Ca (OH) at the same time2Suspension liquid; another 44.4g of phosphoric acid was dissolved in 300mL of deionized water, and the phosphoric acid was dissolvedSlowly dripping the solution into the mixed solution, continuously heating and stirring, dripping for 2.5h, continuously heating the product obtained by reaction (65 ℃), stirring for 2h, finishing the reaction, aging at normal temperature for 24h, centrifuging at 1000rpm by using a centrifuge to obtain a brownish black precipitate, washing the precipitate for 5 times by using pure water, and freeze-drying to obtain brownish black powder to obtain Fe-HAP, and storing at-20 ℃.
(3) Sol preparation of cartilage layer, cartilage calcified layer and subchondral bone layer: dissolving initiator LAP with PBS (phosphate buffer solution) with pH value of 7.4 and 0.01M to obtain final concentration of 0.5% (w/w); weighing quercetin and GelMA, dissolving and mixing uniformly by using a LAP solution, wherein the final concentrations of the quercetin and the GelMA are respectively 0.03% (w/w) and 15% (w/w), and thus obtaining a cartilage layer sol; weighing quercetin, GelMA and Fe-HAP, dissolving and mixing uniformly by using a LAP solution, wherein the final concentrations of the quercetin, GelMA and Fe-HAP are respectively 0.03% (w/w), 20% (w/w) and 3% (w/w), and thus obtaining cartilage calcification layer sol; weighing quercetin, GelMA and Fe-HAP, dissolving and mixing uniformly by using LAP solution, wherein the final concentrations of the quercetin, GelMA and Fe-HAP are respectively 0.03% (w/w), 30% (w/w) and 3% (w/w), and thus obtaining the subchondral bone layer sol.
(4) Gradient support printing, slicing the designed model, guiding into a DLP printer, wherein the wavelength of a light source during printing is 405nm, and the light intensity is 2.65mW/cm2Exposure time 25s, print layer thickness 25 μm. Adding sol of a subchondral bone layer into a material tank, printing the part of the subchondral bone layer of the model, after printing the last layer of the subchondral bone layer, cleaning the section by PBS, replacing the material tank filled with the sol of the cartilage calcification layer, continuously printing the cartilage calcification layer, after printing the last layer of the cartilage calcification layer, cleaning the section by PBS, replacing the material tank filled with the sol of the cartilage layer, and continuously printing the cartilage layer until the end.
(5) Post-treating the stent, taking off the stent after printing, washing the uncured sol by pure water, and then using the stent with the wavelength of 405nm and the concentration of 3.5mW/cm2And (5) carrying out secondary curing for 10min by using a light intensity ultraviolet box.
(6) And fully swelling the treated scaffold in PBS, pre-freezing at-20 ℃, completely freezing, and placing in a freeze dryer with a cold trap temperature of-55 ℃ for 72 hours to obtain the diagnosis and treatment integrated gradient osteochondral bionic scaffold.
Example 2
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold is basically the same as that in embodiment 1, and the difference lies in that sol proportions of all layers are different:
chondral layer sol: weighing quercetin and GelMA, dissolving and mixing uniformly by using LAP solution, and obtaining the cartilage layer sol by respectively using the final concentrations of the quercetin and GelMA to be 0.03% (w/w) and 18% (w/w), thus obtaining the cartilage layer sol of example 2.
Cartilage calcification layer sol: weighing quercetin, GelMA and Fe-HAP, dissolving and mixing uniformly by using a LAP solution, wherein the final concentrations of the quercetin, GelMA and Fe-HAP are 0.03% (w/w), 23% (w/w) and 3% (w/w), respectively, and thus obtaining the cartilage calcification layer sol of example 2.
Sol of subchondral bone: quercetin, GelMA and Fe-HAP are weighed, and are dissolved and mixed uniformly by using a LAP solution, and the final concentrations of the quercetin, GelMA and Fe-HAP are respectively 0.03% (w/w), 28% (w/w) and 3% (w/w), so that the subchondral bone layer sol of example 2 is obtained.
Example 3
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold is basically the same as that in embodiment 1, and the difference lies in that sol proportions of all layers are different:
chondral layer sol: weighing quercetin and GelMA, dissolving and mixing uniformly by using LAP solution, and obtaining the cartilage layer sol by respectively using the final concentrations of the quercetin and GelMA to be 0.03% (w/w) and 20% (w/w), thus obtaining the cartilage layer sol of example 3.
Cartilage calcification layer sol: weighing quercetin, GelMA and Fe-HAP, dissolving and mixing uniformly by using a LAP solution, wherein the final concentrations of the quercetin, GelMA and Fe-HAP are 0.03% (w/w), 25% (w/w) and 3% (w/w), respectively, and thus obtaining the cartilage calcification layer sol of example 3.
Sol of subchondral bone: weighing quercetin, GelMA and Fe-HAP, dissolving and mixing uniformly by using a LAP solution, wherein the final concentrations of the quercetin, GelMA and Fe-HAP are 0.03% (w/w), 30% (w/w) and 3% (w/w), respectively, to obtain the subchondral bone layer sol of example 3.
Example 4
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold is basically the same as that in the embodiment 1, and the difference is that the Fe-HAP content of the gradient scaffold is different:
the final concentration of Fe-HAP in the chondral calcified layer sol and subchondral bone layer sol of this example was 2% (w/w).
Example 5
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold, which is basically the same as that in the embodiment 1, and is different in the content of quercetin in the gradient scaffold:
the final concentration of quercetin in the cartilage layer, the cartilage calcification layer sol and the subchondral bone layer sol in this example was 0.01% (w/w).
Example 6
A preparation method of a diagnosis and treatment integrated gradient osteochondral bionic scaffold, which is basically the same as that in the embodiment 1, and is different in the content of quercetin in the gradient scaffold:
the final concentration of quercetin in the cartilage layer, the cartilage calcification layer sol and the subchondral bone layer sol in this example was 0.05% (w/w).
The scaffolds of examples 1-6 were tested for performance:
the stents prepared in examples 1-6 were subjected to MRI scan, and the ROI technique was used to measure the T2 signal intensity of the gradient stent of each example (as shown in Table 1). As can be seen from Table 1, the T2 signal intensity detected in examples 1-6 indicates that the gradient stent has good MR development capability, and the T2 signal intensities of examples 1, 2, 3, 5 and 6 are similar, and compared with the former, the T2 signal intensity of example 4 is weaker and has an intensity difference of about 27%, indicating that the content of Fe-HAP affects the contrast performance of the stent.
TABLE 1 MRI scanning results
| Examples | 1 | 2 | 3 | 4 | 5 | 6 |
| T2 Signal Strength | 90.5 | 89.9 | 90.8 | 65.4 | 90.9 | 90.5 |
The mechanical compression performance test is performed on the stents of examples 1 to 6, as shown in fig. 1, the result shows that the compressive strength of example 3 is the maximum, and the compressive strength of example 2 is similar to that of examples 1, 4, 5 and 6 and lower than that of examples 2 and 3, which shows that the compressive strength of the stent is improved by increasing the GelMA concentration (wherein examples 1, 2, 3, 4, 5 and 6 in fig. 1 correspond to examples 1, 2, 3, 4, 5 and 6, respectively, and the same is applied below).
The scaffolds of examples 1-6 were subjected to cytotoxicity test according to MTT cytotoxicity assay, as shown in fig. 2, wherein control represents blank sample, and equal amount of PBS was added to blank group, and as a result, the scaffolds exhibited low cytotoxicity within 48h, indicating that the scaffolds had good biocompatibility, the cell activities of examples 2 and 3 at 24h and 48h were all lower than those of other groups, indicating that the cell activities of examples 2 and 3 at 24h and 48h showed a decreasing trend.
The scaffolds of examples 1, 5 and 6 were subjected to an alkaline phosphatase (ALP) test, as shown in fig. 3, where ALP is a marker for differentiation into osteoblasts, and mineralization of bone matrix was promoted, ALP activity was detected on day 7 in examples 1, 5 and 6, and ALP activity was more greatly differentiated on day 14 in examples 1, 5 and 6, indicating that ALP activity was the greatest in the scaffold of example 1 having 0.03% quercetin content, ALP activity was the next highest in the scaffold of example 6 having 0.05% quercetin content, and ALP activity was lower in the scaffold of example 5 having 0.01% quercetin content than in the former two.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The preparation method of the diagnosis and treatment integrated gradient osteochondral bionic scaffold is characterized by being realized by adopting a DLP photocuring 3D printing technology and comprising the following steps of: respectively preparing sol of cartilage layer, cartilage calcified layer and subchondral bone layer,
preparing cartilage layer sol by using quercetin, methacrylated gelatin and phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate solution;
preparing cartilage calcification layer sol by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite and phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate solution;
the subchondral bone layer sol is prepared by using quercetin, methacrylic acid esterified gelatin, iron-doped hydroxyapatite and phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate solution.
2. The method of claim 1, wherein the step of preparing the methacrylated gelatin comprises:
dissolving gelatin in a phosphate buffer solution, heating and dissolving to obtain a gelatin solution;
dripping methacrylic anhydride into the gelatin solution, and controlling the pH value to be 8-9 to obtain a reaction solution;
putting the reaction solution into a dialysis bag, dialyzing with deionized water, and freeze-drying the dialysate in the bag to obtain the methacrylated gelatin.
3. The method of claim 2, wherein the phosphate buffer solution has a pH of 7.4.
4. The method of claim 1, wherein the step of preparing iron-doped hydroxyapatite comprises:
reacting Ca (OH)2Dissolving in water to obtain Ca (OH)2An aqueous solution;
FeCl is added2·4H2Dissolving O in water to obtain FeCl2·4H2An aqueous solution of O;
FeCl is added3·6H2Dissolving O in water to obtain FeCl3·6H2An aqueous solution of O;
dissolving phosphoric acid in water to prepare a phosphoric acid aqueous solution;
FeCl is added2·4H2Aqueous O solution and FeCl3·6H2O aqueous solution to Ca (OH)2And (3) obtaining a mixed solution from the aqueous solution, dripping the phosphoric acid aqueous solution into the mixed solution, heating and stirring for a period of time, aging, centrifuging, washing and drying the precipitate to obtain the iron-doped hydroxyapatite.
5. The method according to claim 4, wherein the molar ratio of the Fe content to the Ca content is 0.2.
6. The method of claim 1, further comprising the steps of:
establishing an osteochondral bionic scaffold model: designing a bone cartilage bionic scaffold model by means of three-dimensional modeling software according to a bionics principle and a size structure of a bone defect part, and exporting an STL format file;
the method comprises the steps of slicing a model, introducing the model into a DLP printer, adding sol of a subchondral bone layer into a material tank, printing the subchondral bone layer part of the model, replacing the material tank filled with the sol of the cartilage calcification layer after the last layer of the subchondral bone layer is printed, continuing to print the cartilage calcification layer, replacing the material tank filled with the sol of the cartilage layer after the last layer of the cartilage calcification layer is printed, and continuing to print the cartilage layer until the end of printing, thus obtaining a semi-finished support.
7. The method of claim 6, further comprising the steps of:
when the subchondral bone layer, the cartilage calcified layer and the cartilage layer are printed and connected, a phosphate buffer solution is used for cleaning the upper layer of the connecting surface, water on the surface of the support is absorbed by absorbent paper, and then the next layer of the support is printed continuously.
8. The method of claim 6, further comprising the steps of:
and cleaning the semi-finished bracket, and then carrying out secondary curing in an ultraviolet box.
9. The method of claim 8, further comprising the steps of:
and placing the scaffold subjected to secondary curing in a phosphate buffer solution for full swelling, then pre-freezing at-20 ℃, and then freezing in a freeze dryer at the temperature of-55 to-40 ℃ for 24 to 72 hours.
10. A diagnosis and treatment integrated gradient osteochondral bionic scaffold, which is prepared by the preparation method of the diagnosis and treatment integrated gradient osteochondral bionic scaffold according to any one of claims 1 to 9.
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