CN112891620A - Artificial bone material carrying anti-tumor medicine and method for preparing artificial bone - Google Patents
Artificial bone material carrying anti-tumor medicine and method for preparing artificial bone Download PDFInfo
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Abstract
The invention discloses an artificial bone material carrying anti-tumor drugs and a method for preparing the artificial bone, wherein the method comprises the following steps: dissolving hydroxyapatite particles, collagen powder, a dispersing agent, an anti-tumor drug and trace elements in deionized water, adjusting the pH value, and uniformly mixing to obtain slurry; ball-milling and ultrasonically oscillating the slurry to prepare an ink suspension; and (2) loading the ink suspension into a spraying barrel of 3D direct-writing forming equipment, connecting a computer with the direct-writing forming equipment, setting a program of the 3D direct-writing equipment and an imitated bone structure model, superposing the ink suspension layer by layer in the air by controlling the rheological property of the ink suspension, printing, and drying to obtain the artificial bone material carrying the anti-tumor drug. The artificial bone material can play the dual roles of repairing bone defect and drug carrier, has good biocompatibility and bone regeneration inducing capability, improves the local drug concentration of the anti-tumor drug, lightens the systemic drug reaction, and inhibits the recurrence and metastasis of tumor.
Description
Technical Field
The invention belongs to the field of biomedical material preparation and molding, and particularly relates to an artificial bone material carrying an anti-tumor drug and a method for preparing an artificial bone.
Background
Bone tissue malignancies are common in the clinic, primary bone malignancies with the first four high-incidence being plasmacytomas, osteosarcomas, chondrosarcomas, and ewing's sarcoma. In addition, bone tissue is also the third most metastatic site of a systemic malignancy, common to bone metastases in lung, prostate and breast cancers. For the treatment of bone tumor, before the development of chemotherapy scheme, mainly amputation or local wide excision and radiotherapy cause great damage to patients, seriously affect the postoperative life quality, have higher fatality rate and have 5-year survival rate less than 20%. With the research and development of chemotherapeutic drugs, the application of multi-drug combination chemotherapy and the application of new adjuvant chemotherapy proposed in recent years, the survival rate of patients is obviously improved. The principle of the combined chemotherapy is that several medicaments with the treatment effect on tumors are combined to generate additive or synergistic effect, so that the toxic effect on cells is not increased while the drug resistance generated by cells due to single-medicament chemotherapy is eliminated. Neoadjuvant chemotherapy was proposed in the last 70 th century, emphasizing that chemotherapy was performed 6-10 weeks before surgery, followed by tumor resection. The post-operative chemotherapy regimen is then specified in terms of the degree of necrosis of the tumor tissue. The implementation of neoadjuvant chemotherapy can limit tumor tissues, shrink, reduce the chance of tumor metastasis and dissemination, and kill potential metastatic foci early. The survival rate of the patients after the operation after the new adjuvant chemotherapy treatment reported in China reaches 60 percent. Therefore, the application of the chemotherapeutic drugs and the multi-drug combined chemotherapy play a key role in improving the survival rate of bone tumor patients and improving prognosis.
For patients who have undergone tumor osteotomy after neoadjuvant chemotherapy, postoperative chemotherapy is required to avoid tumor metastasis, and bone defects also need to be repaired to maintain stability of the original bone structure at the focus. Therefore, it is necessary to implant bone tissue into a bone defect site to repair the bone defect. The bone filling materials mainly adopted clinically at present comprise autogenous bone, allogeneic bone, xenogeneic bone, bone cement and biological ceramic. The autogenous bone avoids immunological rejection reaction, has higher survival rate after implantation, and the autogenous bone tissue is mainly taken from the proximal end of the ilium or the tibia. However, the tissue for harvesting the autogenous bone is limited, the bone tissue cannot completely meet the morphological requirements of the defect part, the trauma of the patient is aggravated, and the infection and pain of the bone harvesting part can occur after the operation. The allogeneic bone has certain antigenicity, although the allogeneic bone can be treated by the antigen to avoid immunological rejection reaction, the allogeneic bone does not contain any living cells and only can play a role in bone conduction, and the osteoinductivity is poor. The heterogeneous bone has sufficient source, low price, certain osteoinductivity and good adhesion with cells, and is an ideal bone filling material. However, there are antigenic differences between species, and how to reduce immune rejection is still under investigation. The bone cement mainly comprises acrylic ester bone cement and calcium phosphate bone cement. The acrylic ester bone cement has good plasticity and mechanical property, but does not have osteoconductivity and has poor biocompatibility. Compared with the calcium phosphate bone cement, the biocompatibility of the calcium phosphate bone cement is greatly improved, but the defects of low compressive strength, easy influence on the curing process and the like still exist. The bioceramic is a filling material with good bone conductivity and has good biocompatibility. Mainly comprises beta-TCP, hydroxyapatite, bioactive glass and the like. The scaffold can not only carry a framework which can simulate natural bones macroscopically and microscopically, but also promote the increase of the number and activity of osteoclasts, and promote the dissolution of implanted bones and the deposition of new bones. The artificial bone compound is constructed by adding bone marrow mesenchymal stem cells into coral hydroxyapatite in the Pimenta racemosa and the like, and the bone forming speed and quality on the artificial bone are found to be close to those of the self-transplanted bone. However, the existing biological ceramic preparation materials imitate inorganic components of natural bones and can meet the requirements of the natural bones in the aspects of porosity, pore diameter and compressive strength, but the toughness of the natural bones given by organic components is the defect of the biological ceramic materials. Therefore, the preparation of the bone tissue engineering scaffold which can meet the requirements of bone defect repair on both biological performance and mechanical performance becomes a current research hotspot.
In order to enable the prepared bone scaffold to meet the requirements of patients in an individualized manner and simultaneously have a good bone-imitating structure in a macroscopic view and a microscopic view, the 3D printing technology is widely applied to the field of bone tissue engineering. The direct writing forming technology is a novel 3D printing technology. When the ink jet printer works, a three-dimensional model is established by a computer and is input into direct-writing forming equipment, and after curable printing ink is configured, the curable printing ink is ejected from a nozzle with a certain diameter by controlling the rheological property of the ink and is cured and formed in the air. In order to achieve rapid curing of ink after ejection and to maintain the shape, it is essential to provide an appropriate ink material. Compared with the traditional 3D printing technology, the forming process of the direct-writing forming technology is simple and convenient, and the efficiency is high. Meanwhile, the ink material can be various, and the metal, nonmetal, organic material and inorganic material can be used for preparing the ink. Even biologically active substances such as cells, proteins, growth factors, etc. can be printed directly. Based on the above advantages, the direct writing molding technology has been applied to the research of bone tissue engineering scaffolds.
In conclusion, the direct-writing forming technology is applied to the preparation of the artificial bone, so that the macroscopic and microstructure bone imitation is met, the individualized customization of the bone scaffold is realized, the composite scaffold simultaneously containing inorganic materials and organic materials can be prepared, the bone is highly imitated in components, the mechanical strength and the biocompatibility of the bone scaffold are greatly improved, and the bone defect of a patient is better repaired. Meanwhile, for bone tumor patients, the chemotherapeutic drug is added into the bone scaffold, so that the local drug concentration of the focus can be improved, the systemic reactivity of the drug can be reduced, and the recurrence and metastasis of the tumor can be inhibited while the regeneration of the induced bone is promoted. Therefore, for those skilled in the art, the development of artificial bone materials carrying anti-tumor drugs is of great significance for the postoperative repair of bone defects.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an artificial bone material carrying an anti-tumor drug and a method for preparing the artificial bone material carrying the drug by using a 3D direct writing forming technology. Aims to provide a material for promoting and inducing bone regeneration, simultaneously can inhibit the recurrence and metastasis of tumors of tumor patients and improve the prognosis of the patients.
In order to achieve the purpose, the artificial bone material loaded with the anti-tumor drug provided by the invention comprises hydroxyapatite particles, collagen powder, a dispersing agent, an anti-tumor drug, trace elements and deionized water.
In order to achieve the aim, the method for preparing the artificial bone material carrying the anti-tumor drug by using the 3D direct writing forming technology comprises the following steps:
(1) dissolving hydroxyapatite particles, collagen powder, a dispersing agent, an anti-tumor drug and trace elements in deionized water, adjusting the pH value, and uniformly mixing to obtain slurry, wherein the particle size range of the hydroxyapatite particles is 0.1-10um, and the anti-tumor drug comprises one or more of methotrexate, adriamycin, cisplatin and 5-FU;
(2) ball-milling and ultrasonically oscillating the slurry to prepare an ink suspension;
(3) the method comprises the following steps of (1) filling an ink suspension into a temperature-controllable charging barrel of 3D direct-writing forming equipment, connecting a computer with the direct-writing forming equipment, setting a program of the 3D direct-writing equipment and a bone-like structure model, spraying the ink suspension out of a spray head by controlling rheological property, superposing the ink suspension layer by layer in the air, and carrying out self-curing forming to obtain the artificial bone material carrying the anti-tumor drug; the bone-like structure model is established by acquiring CT information of a bone defect part of a patient and establishing a three-dimensional model through computer modeling software.
Preferably, the ratio of the added hydroxyapatite particles to the collagen powder is 3: 2.
preferably, the dispersant is one or more of citrate, polyacrylic acid, ammonium polyacrylate and polyacetyl imine.
Preferably, the amount of the dispersant added is 0.1-4% of the mass of the hydroxyapatite particles.
Preferably, the content of the anti-tumor drug in the slurry is 1-10 mg/mL.
Preferably, the pH is adjusted to a range of 4 to 10.
Preferably, the pH adjusting agent comprises one or more of carbonic acid, phosphoric acid, citric acid, magnesium hydroxide and calcium hydroxide.
Preferably, the trace elements comprise one or more of magnesium, zinc and chlorine.
Preferably, the ball milling time in the step (2) is 24 hours, and the rotating speed is 50-250 rmp; the ultrasonic oscillation time is 12 h.
Compared with the prior art, the technical scheme adopted by the invention at least has the following advantages:
1) the printing ink suspension prepared by the invention has a solid phase volume fraction of 50-66%. The high solid phase volume fraction can ensure that the ink material smoothly passes through the nozzle and can be quickly cured and molded after being sprayed.
2) The artificial bone material preparation method provided by the invention can realize personalized customization according to the bone defect condition of a patient, and has the advantages of simple preparation process, high production efficiency and low material cost.
3) The artificial bone material carrying the anti-tumor drug realizes bone imitation on a macroscopic structure and microscopic pores, adopts hydroxyapatite/beta-TCP and collagen in composition, imitates organic and inorganic components of natural bone, and has good biocompatibility and osteoinductivity and greatly improved mechanical property compared with the traditional bone scaffold.
4) The drug-loaded artificial bone can play double roles of repairing bone defect and drug carrier, has good biocompatibility and bone regeneration inducing capability, and simultaneously improves the local drug concentration of the anti-tumor drug, lightens the systemic drug reaction and inhibits the recurrence and metastasis of tumor.
Detailed Description
In order that the invention may be better understood, the invention will now be further described by way of specific embodiments, which are not intended to limit the scope of the invention.
Example 1
beta-TCP (one kind of hydroxyapatite) with the grain diameter of 0.8um, medical collagen powder, deionized water, polyacetyl imine, methotrexate and ammonium chloride are mixed to form ink slurry; wherein, the content of the polyacetylimine is 0.6 percent of the content of the beta-TCP dry powder, the dosage of the ammonium chloride is 0.06 percent of the mass of the ink, the concentration of the methotrexate is 6mg/mL, and the PH value is adjusted to be 4 by adopting phosphoric acid. The solid content of the obtained ink slurry was 44%.
And (3) placing the mixed slurry on a ball mill, ball-milling for 24h at the rotating speed of 90rmp, and ultrasonically oscillating for 12h to obtain the required ink suspension. The obtained ink suspension has good shear thinning property within 10s-1The viscosity at shear rate is 2 pas; has good viscoelasticity and an elastic modulus of up to 10 at 1pa3Pa, good shape retention, and can smoothly pass through the needle nozzle to block and maintain the three-dimensional shape.
The ink suspension was loaded into a barrel of a 3D direct write molding apparatus with a nozzle diameter of 160 um. Connecting a computer with direct-writing forming equipment, setting a program of the 3D direct-writing equipment and a bone-imitating structure model, starting printing in the air in a layer-by-layer superposition mode to obtain a three-dimensional structure, drying the obtained three-dimensional structure at room temperature for 6h, then drying at 60 ℃ for 12h, and then drying at 80 ℃ for 12h to obtain a green body of the three-dimensional structure, namely the methotrexate-loaded artificial bone. The bone-like structure model is established by acquiring CT information of a bone defect part of a patient and establishing a three-dimensional model through computer modeling software.
In vitro drug release experiments: the methotrexate-loaded artificial bone prepared in the example is used as a sample for detection. The release medium was 3ml PBS, temperature 37.0 ℃. The assay was performed every 48 hours, with an equivalent amount of PBS changed. PBS containing the released drug was stored at-45 ℃ prior to assay. The results show that: the medicine is released continuously, and the medicine concentration can be maintained at 0.1-1.0 mu g/ml for 12 days.
Example 2:
the particle size of the hydroxyapatite is 1um, medical collagen powder, deionized water, polyacrylic acid, adriamycin and zinc acetate are mixed to form ink slurry; wherein, the content of polyacrylic acid is 0.8 percent of the dry powder content of hydroxyapatite, the dosage of zinc acetate is 0.05 percent of the mass of the ink, the concentration of adriamycin is 10mg/mL, and the PH value is adjusted to be 9 by adopting magnesium hydroxide. The solid content of the prepared ink slurry is 56%.
And (3) placing the mixed slurry on a ball mill, ball-milling for 24h at the rotating speed of 100rmp, and ultrasonically oscillating for 12h to obtain the required ink suspension. The obtained ink suspension has good shear thinning property within 10s-1Viscosity at shear rate of 3Pa · s); has good viscoelasticity, and the elastic modulus of the elastic gel is up to 10 at 10pa3Pa, good shape retention, and can smoothly pass through the needle nozzle to block and maintain the three-dimensional shape.
The ink suspension was loaded into the barrel of a 3D direct write apparatus with a nozzle diameter of 100 um. Connecting a computer with direct-writing forming equipment, setting a program of the 3D direct-writing equipment and a bone-imitating structure model, starting and printing in the air in a layer-by-layer superposition mode to obtain a three-dimensional structure, drying the obtained three-dimensional structure at room temperature for 6 hours, then drying at 60 ℃ for 12 hours, and then drying at 80 ℃ for 12 hours to obtain a green body of the three-dimensional structure, namely the adriamycin-loaded artificial bone. The bone-like structure model is established by acquiring CT information of a bone defect part of a patient and establishing a three-dimensional model through computer modeling software.
In vitro drug release experiments: the doxorubicin-loaded artificial bone prepared in this example was used as a sample for detection. The release medium was 3ml PBS, temperature 37.0 ℃. The assay was performed every 48 hours, with an equivalent amount of PBS changed. PBS containing the released drug was stored at-45 ℃ prior to assay. The results show that: the drug release reaches the peak in the fourth day, the drug release rate is faster in the first 14 days, and the drug concentration is still greater than the half-inhibitory concentration of the adriamycin in the 40 th day.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An artificial bone material carrying an anti-tumor drug is characterized by comprising hydroxyapatite particles, collagen powder, a dispersing agent, an anti-tumor drug, trace elements and deionized water.
2. A method for preparing an artificial bone material carrying an anti-tumor drug by using a 3D direct writing molding technology is characterized by comprising the following steps:
(1) dissolving hydroxyapatite particles, collagen powder, a dispersing agent, an anti-tumor drug and trace elements in deionized water, adjusting the pH value, and uniformly mixing to obtain slurry, wherein the particle size range of the hydroxyapatite particles is 0.1-10um, and the anti-tumor drug comprises one or more of methotrexate, adriamycin, cisplatin and 5-FU;
(2) ball-milling and ultrasonically oscillating the slurry to prepare an ink suspension;
(3) the method comprises the following steps of (1) filling an ink suspension into a temperature-controllable charging barrel of 3D direct-writing forming equipment, connecting a computer with the direct-writing forming equipment, setting a program of the 3D direct-writing equipment and a bone-like structure model, spraying the ink suspension out of a spray head by controlling rheological property, superposing the ink suspension layer by layer in the air, and carrying out self-curing forming to obtain the artificial bone material carrying the anti-tumor drug; the bone-like structure model is established by acquiring CT information of a bone defect part of a patient and establishing a three-dimensional model through computer modeling software.
3. The method for preparing an artificial bone material carrying an anti-tumor drug by using a 3D direct writing molding technology according to claim 2, wherein the ratio of the added hydroxyapatite particles to the added collagen powder is 3: 2.
4. the method for preparing the artificial bone material loaded with the anti-tumor drug by using the 3D direct writing molding technology as claimed in claim 2, wherein the dispersant is one or more of citrate, polyacrylic acid, ammonium polyacrylate and polyacetyl imine.
5. The method for preparing an artificial bone material carrying an anti-tumor drug by using a 3D direct-write molding technique according to claim 4, wherein the amount of the dispersant added is 0.1-4% of the mass of the hydroxyapatite particles.
6. The method for preparing an artificial bone material carrying an anti-tumor drug by using a 3D direct writing molding technology as claimed in claim 2, wherein the content of the anti-tumor drug in the slurry is 1-10 mg/mL.
7. The method for preparing an artificial bone material loaded with an anti-tumor drug using a 3D direct write molding technique according to claim 2, wherein the pH is adjusted to a range of 4 to 10.
8. The method for preparing an artificial bone material loaded with an anti-tumor drug according to claim 7, wherein the pH adjusting agent comprises one or more of carbonic acid, phosphoric acid, citric acid, magnesium hydroxide, and calcium hydroxide.
9. The method for preparing the artificial bone material loaded with the anti-tumor drug by using the 3D direct-writing molding technology as claimed in claim 2, wherein the trace elements comprise one or more of magnesium, zinc and chlorine.
10. The method for preparing an artificial bone material carrying an anti-tumor drug by using a 3D direct-write molding technology as claimed in claim 2, wherein the ball milling time in the step (2) is 24 hours, and the rotating speed is 50-250 rmp; the ultrasonic oscillation time is 12 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115227868A (en) * | 2022-07-20 | 2022-10-25 | 中南大学湘雅医院 | Bone defect repairing material and magnesium pretreatment acellular tissue engineering bone scaffold |
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