CN112143694A

CN112143694A - Preparation method of three-dimensional cell sieving drug bracket of in-vitro osteoporosis drug

Info

Publication number: CN112143694A
Application number: CN202011024126.4A
Authority: CN
Inventors: 陈志钊; 何贤樱; 李志强; 张志坚; 王华倩; 杨静茹; 纪美琪; 范凯翔; 陈新鑫; 王晓东; 陈曌; 宫剑平
Original assignee: Guangzhou Baiyunshan Huacheng Pharmaceutical Co Ltd; Guangdong University of Technology
Current assignee: Guangzhou Baiyunshan Huacheng Pharmaceutical Co Ltd; Guangdong University of Technology
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-12-29
Anticipated expiration: 2040-09-25
Also published as: CN112143694B

Abstract

The invention discloses a preparation method of a three-dimensional cell sieving drug bracket of an in-vitro osteoporosis drug, which comprises the following steps: (1) uniformly mixing a sodium alginate solution and/or a gelatin solution, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution, and uniformly mixing and stirring to obtain a mixed solution A; (2) adding cholera toxin B subunit solution, stirring to obtain mixed solution B, dialyzing the mixed solution B, freeze-drying, and dispersing in physiological saline. The three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug obtained by the preparation method of the three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug is similar to the growth environment of in vivo cells, has good drug sieving capability and predictability, and solves the problem of poor drug sieving capability and predictability of the traditional two-dimensional osteoblast sieving drug model.

Description

Preparation method of three-dimensional cell sieving drug bracket of in-vitro osteoporosis drug

Technical Field

The invention relates to the technical field of three-dimensional cell sieving drug stents, in particular to a preparation method of a three-dimensional cell sieving drug stent of an in-vitro osteoporosis drug.

Background

Osteoporosis occurs frequently in the elderly and is usually the result of a balance between bone formation and bone resorption that is disrupted. Among them, osteoblasts are responsible for osteogenesis, and are transformed into osteocytes when they secrete osteoid and are embedded therein, and constitute bone tissues together with calcified osteoid. Therefore, when an in vitro osteoporosis drug screening model needs to be established, the osteoporosis drug screening can be carried out by constructing an osteoblast model. The conventional osteoblast model is usually based on a two-dimensional culture layer, namely osteoblasts are cultured on a plane, and then the effect of the medicament for treating osteoporosis in vivo is predicted by in vitro administration. However, although the conventional drug screening cell culture mode is convenient, the two-dimensional culture method cannot simulate the in vivo environment of osteoblasts, so that the drug screening capability and the predictability of the osteoblast model cultured in two dimensions are relatively poor.

In vivo, cells are not simply grown and tiled as in vitro monolayer cell culture, the cells are connected, and gap connection between osteoblasts can mediate signal transmission and mutually communicate, thereby playing an important role in the formation and development of bone tissues. Simple monolayer cell culture cannot construct extracellular matrix (ECM), and creates a microenvironment similar to that in vivo to form a three-dimensional cell structure. Since the extracellular matrix (ECM) is a supporting structure for osteoblasts to realize the processes of adhesion, differentiation, proliferation, migration and the like, the behavior of the osteoblasts can be changed, and growth factors provided by the ECM even influence the transformation of the osteoblasts into bone tissues and the regeneration of new bones. Therefore, the currently commonly used two-dimensional osteoblast model cannot simulate the growth condition which is similar to the growth condition of in vivo osteoblasts, the result obtained by using the two-dimensional osteoblast model as an osteoporosis drug screening model is greatly different from the in vivo drug action result, the drug screening model is not representative, and the drug screening capability and the predictability are poor.

Disclosure of Invention

Aiming at the problems brought forward by the background technology, the invention aims to provide a preparation method of a three-dimensional cell sieving drug bracket of an in-vitro osteoporosis drug, the obtained three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug is similar to the growth environment of in-vivo cells, the drug sieving capability and the predictability are good, and the problem that the drug sieving capability and the predictability of the traditional two-dimensional osteoblast sieving drug model are poor is solved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a three-dimensional cell sieving drug bracket of an in-vitro osteoporosis drug comprises the following steps:

(1) uniformly mixing a sodium alginate solution and/or a gelatin solution, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution, and uniformly mixing and stirring to obtain a mixed solution A;

(2) adding cholera toxin B subunit solution, stirring uniformly to obtain mixed solution B, dialyzing the mixed solution B, freeze-drying, and dispersing in physiological saline to obtain cholera toxin B subunit-sodium alginate solution or cholera toxin B subunit-sodium alginate-gelatin solution;

(3) dropwise adding the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution above the liquid level of the calcium chloride solution, stirring simultaneously, continuing stirring after the dropwise adding is finished, taking out the coagulated beads, and washing and freeze-drying to obtain a three-dimensional cell sieving drug support;

(4) sterilizing the three-dimensional cell sieving drug bracket, then air-drying, soaking the three-dimensional cell sieving drug bracket in osteoblast suspension, adding a culture medium culture dish, and then carrying out osteoblast culture to obtain the three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug.

Preferably, in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution 15-20 cm above the liquid level of the calcium chloride solution.

Preferably, in the step (3), the dropping speed of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution is 1-1.5 mL/min above the liquid level of the calcium chloride solution.

Preferably, in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution, and the stirring speed is 80-120 r/min;

after the dripping is finished, the stirring speed for continuously stirring is 180-220 r/min, and the stirring time is 8-12 min.

Preferably, the concentration of the sodium alginate solution is 15-30 mg/mL, and the concentration of the gelatin solution is 5-20 mg/mL.

Preferably, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 5-10 mg/mL, and the concentration of the N-hydroxysuccinimide in the mixed solution A is 2-5 mg/mL.

Preferably, the concentration of the cholera toxin B subunit solution is 1-4 mg/mL.

Preferably, the concentration of the calcium chloride solution is 15-30 mg/mL.

Preferably, in the step (1), the mixing volume ratio of the sodium alginate solution to the gelatin solution is 100: 0-25: 75;

in the step (2), the volume ratio of the cholera toxin B subunit solution to the sodium alginate solution or the sodium alginate-gelatin solution is 1: 4.

Preferably, the osteoblast suspension has a cell density of 1X 10⁶～1×10⁷one/mL.

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

1. utilizing carboxyl on a scaffold material (sodium alginate and/or gelatin) and amino on a cholera toxin B subunit (CTB), covalently bonding the sodium alginate and/or the gelatin with the CTB through an amide condensation reaction, enabling the CTB to be coupled to the sodium alginate and/or the gelatin, modifying the three-dimensional cell sieving drug scaffold of the in-vitro osteoporosis drug by means of a cholera toxin B subunit (CTB), increasing adhesion of osteoblasts on the three-dimensional cell sieving drug scaffold of the in-vitro osteoporosis drug through the combination between GM1 receptor of the osteoblasts and the CTB, simultaneously enabling the osteoblasts to grow in an environment simulating ECM, increasing connection among the osteoblasts, enabling the osteoblasts to form a three-dimensional structure in vitro, and enabling the obtained three-dimensional cell sieving drug scaffold of the in-vitro osteoporosis drug to be similar to the growth environment of cells in vivo, the drug screening ability and the predictability are good;

2. dropwise adding the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution into the calcium chloride solution from a high position, wherein calcium ions in the calcium chloride solution permeate from outside to inside to form gel with a larger outer-layer cross-linking density, and cells can survive in a network structure with a smaller inner cross-linking density and more pores to form a three-dimensional structure;

3. by controlling the dropping speed of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution into the calcium chloride solution, the stability of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution into the calcium chloride solution is ensured, and calcium ions permeate from outside to inside to form gel with a larger outer layer crosslinking density.

Detailed Description

The scheme adopts sodium alginate solution and/or gelatin as osteoblast support, has good biocompatibility due to the similarity of the structures of Sodium Alginate (SA) and gelatin (Gel) and the structure of an extracellular matrix (ECM) of an organism, and adopts calcium chloride as a cross-linking agent. Gelatin is a protein obtained by partial hydrolysis of collagen, has homology with collagen, and is important framework protein in ECM, so that the gelatin can simulate the ECM environment in vivo to a certain extent, promote adhesion between cells and better reflect the state of the cells in vivo. Furthermore, since osteoblasts secrete type I collagen, which affects their physiological functions, the addition of gelatin as a scaffold material for cell culture is advantageous for the inter-osteoblast communication, proliferation, differentiation, etc. Sodium alginate is a natural polysaccharide, can quickly form gel, and can perform ion exchange reaction with divalent cations to form a cross-linked network structure, thereby forming hydrogel. Cells grow in the hydrogel, and the surface of the sodium alginate or the sodium alginate-gelatin after gelation is not smooth, so that the adhesion of the cells is enhanced, the adherent growth of the cells is facilitated, a three-dimensional structure is formed, and the relation among the cells is enhanced. The calcium ion is also present in the organism and is necessary ion for each important physiological function in the organism, most of the calcium ion is present in the skeleton and plays a key role in the generation and development of bone tissues, the calcium chloride is used as a cross-linking agent, and the calcium chloride contains the calcium ion, so the growth of osteoblasts is facilitated. Because cholera toxin B subunit (CTB) is a nontoxic subunit of cholera toxin, monosialotetrahexosylganglioside (GM1) is the most important one of ganglioside substances, GM1 receptor is widely distributed in membrane structures of various cells of vertebrates and is a natural component of human cell membranes, CTB is a ligand of GM1 receptor and can be combined with GM1 receptor of osteoblasts in vitro, the treatment of blood cells by CTB can increase the total number of blood cells of micro-aggregates formed in vitro, and the chemical synthesis of calcium ions in cells stimulated by CTB is favorable for the adhesion of single blood cells on glass. Therefore, the material adopted for constructing the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug has good biocompatibility, has no toxic or harmful effect on osteoblasts, and is beneficial to realizing the processes of simulating signal transduction, adhesion migration, drug response and the like of cells in vivo;

1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is a water-soluble carbodiimide hydrochloride, is used as an activating reagent for carboxyl groups in an amide reaction, is used in combination with N-hydroxysuccinimide (NHS), and can promote an amidation reaction between carboxyl groups of sodium alginate and/or gelatin and amino groups of CTB (sodium alginate and/or gelatin has abundant carboxyl groups, and reacts with EDC first to form an unstable intermediate, and then forms a stable NHS-ester through a reaction between the unstable intermediate and NHS, during which the carboxyl groups of alginate are activated by EDC and NHS, and finally forms an amide bond through covalent bonding of NHS-ester and amino groups of CTB side chains, thereby synthesizing CTB-SA, and is not easily destroyed after formation due to covalent bonding, and gelatin molecule side chains are also rich in a large number of carboxyl groups, or after the carboxyl activation of the EDC/NHS, the carboxyl activation and the amino on the CTB side chain form amido bond to synthesize CTB-Gel), so that the sodium alginate and/or the gelatin are firmly combined with the CTB, and the coupling efficiency of the CTB on the sodium alginate and/or the gelatin is improved;

because the sodium alginate hydrogel system or the sodium alginate-gelatin hydrogel system is a semisolid substance with a reticular structure, the cell scaffold has good stability in vitro culture, most cells can be attached to the reticular structure of the hydrogel to grow, a three-dimensional structure different from planar growth is formed, and information transmission among the cells is facilitated. The blended gel system integrates the functions of ECM environment simulation and huge cell bearing force, and simultaneously the inherent elasticity of the gel also has a supplementary effect on the three-dimensional culture of osteoblast growth in a simulated body. Since cells are dynamic in vivo and are influenced not only by biochemical factors such as ECM and cytokines but also by physical factors such as tensile stress and fluid shear force, the elasticity of the hydrogel system also provides a simulation of the physical environment to some extent for the cells. The surface of the crosslinked sodium alginate hydrogel system or the sodium alginate-gelatin hydrogel system is sparse and porous, which is beneficial to material exchange, so that nutrient substances can permeate into the hydrogel and cell waste can be discharged. In addition, the CTB modifies the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug, so that the adhesion of osteoblasts on the bracket is greatly improved, the non-covalent combination between the receptor and the ligand can increase the movement and the adhesion of the osteoblasts to the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug by the combination between the CTB and the GM1 receptor expressed by the osteoblasts, and the CTB is a pentamer and can combine five GM1 receptors at one time, so that the situation that one CTB can combine GM1 receptors (no more than 5) on a plurality of osteoblasts or a plurality of GM1 receptors on 1 osteoblast can combine a plurality of CTBs can occur, namely the non-covalent combination between the bracket and the cells is enhanced by the modification of the CTB, the cell adhesion is promoted, the connection and the aggregation between the osteoblasts are increased, and the improvement between the osteoblasts and the osteoblasts is facilitated, the association and binding of osteoblasts to cell scaffolds.

The invention directly utilizes carboxyl on a bracket material (sodium alginate and/or gelatin) and amino on a cholera toxin B subunit (CTB) to covalently combine the sodium alginate and/or the gelatin with the CTB through an amide condensation reaction, the CTB can be coupled to the sodium alginate and/or the gelatin, the three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug is modified by means of a cholera toxin B subunit (CTB), the adhesion of osteoblasts on the three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug can be increased through the combination between GM1 receptor of the osteoblasts and the CTB, meanwhile, the osteoblasts grow in the environment simulating ECM, the connection between the osteoblasts is increased, the osteoblasts form a three-dimensional structure in vitro, and the obtained three-dimensional cell sieving drug bracket of the in vitro osteoporosis drug is similar to the growth environment of the cells in vivo, the drug screening ability and the predictability are good.

Further, in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution 15-20 cm above the liquid level of the calcium chloride solution.

And dropwise adding the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution into the calcium chloride solution from a high position, wherein calcium ions in the calcium chloride solution permeate from outside to inside to form gel with a larger outer-layer cross-linking density, and cells can survive in a network structure with a smaller inner cross-linking density and more pores to form a three-dimensional structure. If the dropping distance is smaller, the formation of coagulated bead particles with uniform size is not facilitated, because a liquid bridge with a specific shape and a certain volume is formed between two droplets when the dropping distance is smaller, the two separate droplets are connected and merged, larger particles can be formed in the free falling process, the shorter dropping distance causes that calcium ions are contacted when the droplets are not completely separated, and because the crosslinking speed is high, a series of water condensation beads are easily formed, and when the dropping distance is too large, the operation convenience is easily reduced, and the preparation efficiency is reduced.

Further, in the step (3), the dropping speed of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) above the liquid level of the calcium chloride solution into the calcium chloride solution is 1-1.5 mL/min.

Calcium ions permeate from outside to inside by controlling the dropping speed of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution into the calcium chloride solution, so that gel with a larger outer layer crosslinking density can be formed, if the dropping speed is too high, the stability of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution dropped into the calcium chloride solution is reduced, a liquid bridge with a specific shape and a certain volume is formed between two liquid drops, so that the two independent liquid drops are connected and merged, larger particles may be formed during the free fall, too fast drop acceleration causes the liquid to form a series of drops, and when the drop velocity is too slow, the operational convenience is easily reduced, and the preparation efficiency is reduced.

Further, in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution, and the stirring speed is 80-120 r/min;

Preferably, in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution, and the stirring speed is 100 r/min;

after the dripping is finished, the stirring speed for continuously stirring is 200r/min, and the stirring time is 10 min.

And (3) dropwise adding the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into a calcium chloride solution, and stirring at the same time to ensure that calcium ions in the calcium chloride fully react with the sodium alginate and/or the gelatin. And after the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution is dripped, stirring is continued, and the stirring speed is increased, so that calcium ions and sodium alginate and/or gelatin can continue to perform sufficient cross-linking reaction, and the hydrogel is cured.

Preferably, the concentration of the sodium alginate solution is 25mg/mL, and the concentration of the gelatin solution is 10 mg/mL.

By adding sodium alginate and/or gelatin, calcium chloride is used as a cross-linking agent. The sodium alginate and divalent cations can generate ion exchange reaction to form a cross-linked network structure, so that hydrogel is formed, gelatin is added as a culture scaffold material of cells to be beneficial to the communication, proliferation and differentiation among osteoblasts, the surface of the sodium alginate or sodium alginate-gelatin after gelation is not smooth, the adhesion of the cells is enhanced, the adherent growth of the cells is facilitated, a three-dimensional structure is formed, the communication among the cells is enhanced, and if the concentration of the sodium alginate solution or the gelatin solution is too low, the three-dimensional network structure of the prepared three-dimensional cell sieving medicament scaffold for the in-vitro osteoporosis medicament is loose, and the cell adhesion effect is poor.

Preferably, the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 8mg/mL, and the concentration of N-hydroxysuccinimide in the mixed solution A is 3 mg/mL.

The 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is water-soluble carbodiimide hydrochloride, is used as an activating reagent for carboxyl in an amide reaction, is used together with N-hydroxysuccinimide (NHS), can promote the amidation reaction between the carboxyl of sodium alginate and/or gelatin and the amino of CTB, has the concentration of 5-10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, has the concentration of 2-5 mg/mL of N-hydroxysuccinimide in the mixed solution A, ensures the carboxyl activating effect of the sodium alginate and/or gelatin, and improves the coupling efficiency of the CTB on the sodium alginate and/or gelatin.

Preferably, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide are dissolved by adopting a Tris buffer solution, and the pH values of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution are both 4.5-6.5.

The pH values of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution are both 4.5-6.5, so that the stability of amidation reaction between the carboxyl of sodium alginate and/or gelatin and the amino of CTB is ensured.

The cholera toxin B subunit in the cholera toxin B subunit solution is an active pentameric cholera toxin B subunit.

Preferably, the concentration of the cholera toxin B subunit solution is 2 mg/mL.

Preferably, the cholera toxin B subunit is dissolved by adopting a Tris buffer solution containing 0.2mol/L sodium chloride (NaCl), a supernatant is obtained after centrifugation, the protein concentration of CTB in the cholera toxin B subunit solution is measured by a BCA method, and the solution concentration of the cholera toxin B subunit is adjusted to be 1-4 mg/mL.

The CTB is adopted to modify the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug, the aim is to promote the adhesion of osteoblasts on the cell bracket by the mutual combination of GM1 receptors of the osteoblasts and the modified CTB, the modification of the CTB on the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug greatly improves the adhesion of the osteoblasts on the bracket, the non-covalent combination between the receptors and ligands can increase the movement and the adhesion of the osteoblasts to the bracket by the combination between the CTB and GM1 receptors expressed by the osteoblasts, and the CTB is a pentamer and can combine with five GM1 receptors at one time, so that the condition that 1 CTB combines with a plurality of osteoblasts or 1 osteoblasts combines with a plurality of CTBs can occur, namely the non-covalent combination between the modified enhanced cells of the CTB and the bracket can promote the cell adhesion, when the solution concentration of the cholera toxin B subunit is too low, too little CTB is bound with sodium alginate and/or gelatin, which reduces the adhesion effect of osteoblasts, and when the solution concentration of the cholera toxin B subunit is too high, it is easy to cause that too much CTB in the system is not bound with sodium alginate and/or gelatin, which causes waste.

Preferably, in the step (2), the mixed solution B is dialyzed at 4 ℃ for 3 times by using a dialysis bag with a molecular weight of 3500, wherein the dialysis is performed for 8 hours each time, and the dialysate needs to be replaced each time.

By carrying out the dialysis, small molecular substances in a mixed system can be removed, and the using effect of the prepared three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug is ensured.

Preferably, the concentration of the calcium chloride solution is 15-30 mg/mL.

Preferably, the concentration of the calcium chloride solution is 20 mg/mL.

Preferably, the calcium chloride solution is autoclaved.

Calcium chloride is used as a cross-linking agent, ion exchange reaction can occur between sodium alginate or gelatin and divalent cations to form a cross-linked network structure, hydrogel is further formed, the concentration of the calcium chloride solution is 15-30 mg/mL, and the concentration of calcium ions in the calcium chloride solution is guaranteed, so that the gelling effect of the sodium alginate or sodium alginate-gelatin is guaranteed, if the concentration of the calcium chloride solution is low, the gelling effect is poor, and the cell adhesion effect of the obtained in-vitro osteoporosis drug three-dimensional cell sieve drug scaffold is poor.

Further, in the step (1), the mixing volume ratio of the sodium alginate solution to the gelatin solution is 100: 0-25: 75;

Preferably, in the step (1), the mixing volume ratio of the sodium alginate solution to the gelatin solution is 50: 50.

Gelatin is a protein obtained by partial hydrolysis of collagen, has homology with collagen, and is important framework protein in ECM, so that the gelatin can simulate the ECM environment in vivo to a certain extent, promote adhesion between cells and better reflect the state of the cells in vivo. And furthermore, osteoblasts secrete type I collagen which influences the physiological function of osteoblasts, therefore, gelatin is added as a culture scaffold material of cells, which is beneficial to the connection, proliferation, differentiation and the like among osteoblasts, the cell adhesion effect of the three-dimensional cell sieving drug scaffold for the in vitro osteoporosis drug can be improved by adding the gelatin solution in the step (1), and the volume ratio of the cholera toxin B subunit solution to the sodium alginate solution or the sodium alginate-gelatin solution is 1:4, so that the combination effect of the sodium alginate and/or the gelatin and the CTB is ensured.

Further, the osteoblast suspension has a cell density of 1X 10⁶～1×10⁷Per mL。

Preferably, the osteoblast suspension has a cell density of 1X 10⁶one/mL.

The osteoblasts are inoculated on the three-dimensional cell sieving medicine bracket in the step (4), and because the obtained three-dimensional cell sieving medicine bracket of the in-vitro osteoporosis medicine is similar to the growth environment of in-vivo cells, the osteoblasts can be adhered to and grow on the three-dimensional cell sieving medicine bracket of the in-vitro osteoporosis medicine, and the medicine screening capability and the predictability are good.

In order to facilitate an understanding of the present invention, a more complete description of the present invention is provided below. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

A preparation method of a three-dimensional cell sieving drug bracket comprises the following steps:

(1) uniformly mixing a sodium alginate solution (prepared by sterile normal saline with the concentration of 15mg/mL) and a gelatin solution (prepared by sterile normal saline with the concentration of 5mg/mL), wherein the mixing volume ratio of the sodium alginate solution to the gelatin solution is 50:50, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 80mg/mL and a pH value of 6.5) and an N-hydroxysuccinimide solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 30mg/mL and a pH value of 6.5) after mixing, uniformly mixing and stirring for 30min to obtain a mixed solution A, wherein the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 5mg/mL, and the concentration of the N-hydroxysuccinimide in the mixed solution A is 2 mg/mL;

(2) adding 1mL of cholera toxin B subunit solution (1 mg/mL is prepared by adopting a trihydroxymethyl aminomethane (Tris) buffer solution containing 0.2mol/L sodium chloride (NaCl)), stirring at 4 ℃ for 12h, uniformly stirring to obtain a mixed solution B, dialyzing the mixed solution B for 3 times at 4 ℃ by using a dialysis bag with the molecular weight of 3500 for 8h each time, changing the dialyzate once per dialysis, and dispersing in 4mL of physiological saline after freeze-drying to obtain a cholera toxin B subunit-sodium alginate-gelatin solution for later use;

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution 15cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 15mg/mL) at the dripping speed of 1mL/min, stirring at the same time at the stirring speed of 100r/min, continuing stirring after finishing dripping, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 2

(1) uniformly mixing a sodium alginate solution (prepared by sterile normal saline to have a concentration of 25mg/mL) and a gelatin solution (prepared by sterile normal saline to have a concentration of 10mg/mL), wherein the mixing volume ratio of the sodium alginate solution to the gelatin solution is 50:50, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 80mg/mL and a pH value of 6.5) and an N-hydroxysuccinimide solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 30mg/mL and a pH value of 6.5) after mixing, uniformly mixing and stirring for 30min to obtain a mixed solution A, wherein the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 8mg/mL, and the concentration of the N-hydroxysuccinimide in the mixed solution A is 3 mg/mL;

(2) adding 1mL of cholera toxin B subunit solution (2 mg/mL is prepared by adopting a trihydroxymethyl aminomethane (Tris) buffer solution containing 0.2mol/L sodium chloride (NaCl)), stirring at 4 ℃ for 12h, uniformly stirring to obtain a mixed solution B, dialyzing the mixed solution B for 3 times at 4 ℃ by using a dialysis bag with the molecular weight of 3500 for 8h each time, changing the dialyzate once per dialysis, and dispersing in 4mL of physiological saline after freeze-drying to obtain a cholera toxin B subunit-sodium alginate-gelatin solution for later use;

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution 15cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 20mg/mL) at the dripping speed of 1mL/min, stirring at the same time at the stirring speed of 100r/min, continuing stirring after finishing dripping, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 3

(1) uniformly mixing a sodium alginate solution (prepared by sterile normal saline with the concentration of 30mg/mL) and a gelatin solution (prepared by sterile normal saline with the concentration of 20mg/mL), wherein the mixing volume ratio of the sodium alginate solution to the gelatin solution is 50:50, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 80mg/mL and a pH value of 6.5) and an N-hydroxysuccinimide solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 30mg/mL and a pH value of 6.5) after mixing, uniformly mixing and stirring for 30min to obtain a mixed solution A, wherein the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 10mg/mL, and the concentration of the N-hydroxysuccinimide in the mixed solution A is 5 mg/mL;

(2) adding 1mL of cholera toxin B subunit solution (4 mg/mL is prepared by adopting a trihydroxymethyl aminomethane (Tris) buffer solution containing 0.2mol/L sodium chloride (NaCl)), stirring at 4 ℃ for 12h, uniformly stirring to obtain a mixed solution B, dialyzing the mixed solution B for 3 times at 4 ℃ by using a dialysis bag with the molecular weight of 3500 for 8h each time, changing the dialyzate once per dialysis, and dispersing in 4mL of physiological saline after freeze-drying to obtain a cholera toxin B subunit-sodium alginate-gelatin solution for later use;

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution 15cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 30mg/mL) at the dripping speed of 1mL/min, stirring at the same time at the stirring speed of 100r/min, continuing stirring after finishing dripping, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 4

(1) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution (80 mg/mL concentration and 6.5 pH value prepared by adopting Tris buffer solution) and N-hydroxysuccinimide solution (30 mg/mL concentration and 6.5 pH value prepared by adopting Tris buffer solution) into sodium alginate solution (25 mg/mL concentration prepared by adopting sterile physiological saline) with volume of 4mL, mixing and stirring uniformly for 30min to obtain mixed solution A, so that the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 8mg/mL, the concentration of the N-hydroxysuccinimide in the mixed solution A is 3 mg/mL;

(2) adding 1mL of cholera toxin B subunit solution (2 mg/mL is prepared by adopting a trihydroxymethyl aminomethane (Tris) buffer solution containing 0.2mol/L sodium chloride (NaCl)), stirring at 4 ℃ for 12h, uniformly stirring to obtain a mixed solution B, dialyzing the mixed solution B for 3 times at 4 ℃ by using a dialysis bag with the molecular weight of 3500 for 8h each time, changing the dialyzate once per dialysis, and dispersing in 4mL of physiological saline after freeze-drying to obtain a cholera toxin B subunit-sodium alginate solution for later use;

(3) and (3) dripping the cholera toxin B subunit-sodium alginate solution prepared in the step (2) into the calcium chloride solution 15cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 20mg/mL), stirring at the same time at the speed of 1mL/min, continuing stirring after finishing dripping, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 5

(1) uniformly mixing a sodium alginate solution (prepared by sterile normal saline to have a concentration of 25mg/mL) and a gelatin solution (prepared by sterile normal saline to have a concentration of 10mg/mL), wherein the mixing volume ratio of the sodium alginate solution to the gelatin solution is 25:75, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 80mg/mL and a pH value of 6.5) and an N-hydroxysuccinimide solution (which is prepared by adopting a Tris buffer solution to obtain a solution with a concentration of 30mg/mL and a pH value of 6.5) after mixing, uniformly mixing and stirring for 30min to obtain a mixed solution A, wherein the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 8mg/mL, and the concentration of the N-hydroxysuccinimide in the mixed solution A is 3 mg/mL;

Example 6

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution 20cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 20mg/mL) at the dripping speed of 1.5mL/min, stirring at the stirring speed of 100r/min, continuing stirring after finishing dripping, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 7

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution at a dripping speed of 2mL/min at a position of 10cm above the liquid level of the calcium chloride solution (the concentration of the solution is 20mg/mL by using sterile physiological saline), stirring at the same time, continuing stirring after dripping is finished, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Example 8

(2) adding 1mL of cholera toxin B subunit solution (0.5 mg/mL is prepared by adopting a trihydroxymethyl aminomethane (Tris) buffer solution containing 0.2mol/L sodium chloride (NaCl)), stirring for 12h at 4 ℃, uniformly stirring to obtain a mixed solution B, dialyzing the mixed solution B for 3 times at 4 ℃ by using a dialysis bag with the molecular weight of 3500 for 8h each time, changing the dialyzate once for each dialysis, and dispersing the dialyzate in 4mL of physiological saline after freeze-drying to obtain a cholera toxin B subunit-sodium alginate-gelatin solution for later use;

Example 9

(3) and (3) dripping the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution 15cm above the liquid level of the calcium chloride solution (prepared by using sterile physiological saline with the concentration of 10mg/mL) at the dripping speed of 1mL/min, stirring at the same time, continuing stirring after dripping is finished, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell sieve medicine support.

Comparative example 1

(1) uniformly mixing a sodium alginate solution (prepared by sterile normal saline to have a concentration of 25mg/mL) and a gelatin solution (prepared by sterile normal saline to have a concentration of 10mg/mL), wherein the mixing volume ratio of the sodium alginate solution to the gelatin solution is 50:50, uniformly mixing and stirring to obtain a mixed solution A;

(2) and (2) dropwise adding the mixed solution A prepared in the step (1) into a calcium chloride solution (prepared by using sterile physiological saline with the concentration of 20mg/mL) 15cm above the liquid level of the calcium chloride solution at the dropping speed of 1mL/min, stirring at the same time, wherein the stirring speed is 100r/min, continuing stirring after the dropwise adding is finished, wherein the stirring speed is 200r/min, the stirring time is 10min, taking out the coagulated beads, washing with deionized water for 3 times, and freeze-drying to obtain the three-dimensional cell drug sieving bracket.

Performance testing

1. GM1 binding Activity assay

The three-dimensional cell sieve drug scaffolds prepared in examples 1-9 and comparative example 1 were placed in a 96-well plate, 200. mu.L of GM1 solution with a concentration of 50. mu.g/mL was added, and incubation was performed for 4 h. The scaffolds were rinsed 3 times with a known volume of deionized water and then incorporated into the above-mentioned incubated GM1 solution, and the GM1 content was determined colorimetrically, the amount of GM1 bound to the three-dimensional cell sieve drug scaffold-GM 1 before incubation-GM 1 after incubation, with 3 replicates per set of scaffolds, and the results are shown in the following table:

TABLE 1 results of binding Activity test of examples and comparative examples GM1

From the above test results, it can be seen that the three-dimensional cell sieve drug scaffold without CTB modification in comparative example 1 has no binding activity of GM1, while the three-dimensional cell sieve drug scaffold with CTB modification has good binding activity, while GM1 in examples 1-6 has good binding activity, GM1 has large gel particles due to too small dropping height and too fast dropping speed in example 7, and GM1 has poor binding activity due to low concentration of CTB solution and poor CTB modification effect of three-dimensional cell sieve drug scaffold in example 8, and GM1 has poor binding activity due to low concentration of calcium chloride solution in example 9.

2. Cell adhesion Performance assay

The three-dimensional cell sieving drug bracket prepared in the above examples 1 to 9 and comparative example 1 was washed with deionized water for 3 times, irradiated with ultraviolet light for 30min, washed with 70% ethanol solution for 3 times, air-dried, placed in a culture dish, and soaked in 1mL of osteoblast suspension (the cell density of the osteoblast suspension is 1X 10)⁶) Adding 1.5mL culture medium into a culture dish, culturing for 48h at 37 ℃, and culturing osteoblasts to obtain the three-dimensional cell sieving drug bracket of the in-vitro osteoporosis drug.

Washing with PBS buffer solution to obtain three-dimensional cell sieving medicine bracket of the in-vitro osteoporosis medicine for 3 times to remove non-adhered cells, then digesting the cells on the cell bracket with pancreatin, taking the liquid, placing the liquid in a 15mL sterile centrifuge tube, centrifuging at the rotating speed of 1000r/min for 5min, removing supernatant, adding PBS again to wash the cell bracket to collect residual adhered cell liquid, collecting the liquid, centrifuging, removing supernatant, re-suspending the cells with 5mL physiological saline, blowing uniformly, counting, and taking the average value after 3 times of counting. The test results are shown in the following table:

TABLE 2 results of cell adhesion Performance for examples and comparative examples

Test items	Number of adherent cells (number/mL)
		Example 1	8.5×10⁵
Example 2	9×10⁵
		Example 3	8.6×10⁵
Example 4	8.2×10⁵
		Example 5	7.5×10⁵
Example 6	8×10⁵
		Example 7	7×10⁵
Example 8	3.2×10⁵
		Example 9	6.3×10⁵
Comparative example 1	1×10⁵

From the above test results, the number of adhered cells of the three-dimensional cell sieve drug scaffold which is not subjected to CTB modification in comparative example 1 is the smallest, the number of adhered cells on the three-dimensional cell sieve drug scaffold which is subjected to CTB modification is larger, the three-dimensional cell sieve drug scaffolds in examples 1 to 6 can adhere a large number of cells, larger gel particles are formed due to too small dripping height and too fast dripping speed in example 7, and the binding activity of GM1 is affected, so that the number of adhered cells is reduced, the CTB modification effect of the three-dimensional cell sieve drug scaffold is poorer due to too low concentration of the CTB solution in example 8, so that the binding activity of GM1 is poor, the number of adhered cells is significantly reduced, the concentration of the calcium chloride solution in example 9 is lower, the gelling effect is poor, the binding activity of GM1 is affected, and the number of adhered cells is small.

3. Determination of cell proliferation

According to the results of the GM1 binding activity assay and the cell adhesion performance assay, example 2 was obtained as a preferred procedure, and the cell proliferation of the three-dimensional cell sieve drug scaffold cultured in two dimensions and not modified with CTB in comparative example 1 was compared under otherwise identical conditions.

According to the number of adherent cells in example 2, a cell suspension of the same number of cells was seeded in a 6-well plate, and cultured in the same medium at 37 ℃ for 48 hours as a two-dimensional culture control.

Thiazole blue (MTT) was added to a final concentration of 0.5mg/mL in example 2, comparative example 1 and two-position culture control, respectively, and after culturing in the dark at 37 ℃ for 4 hours, the culture solution was carefully poured out, 1.5mL of dimethyl sulfoxide (DMSO) was added, and after shaking at a low speed for 10 minutes, the DMSO was transferred to a 96-well plate at 150. mu.L per well to measure the absorbance at 570nm (the larger the absorbance, the stronger the cell activity, the higher the cell proliferation rate).

TABLE 3 determination of cell proliferation

Test items	Absorbance value
		Example 2	1.86
Comparative example 1	1.15
		Two-dimensional culture	0.85

From the above test results, it can be seen that, compared with the three-dimensional cell sieving drug scaffold which is two-dimensionally cultured and is not modified in comparative example 1, the three-dimensional cell sieving drug scaffold modified by CTB in example 2 has the highest proliferation rate of cells, the cells cultured by the three-dimensional cell sieving drug scaffold are superior to the proliferation condition of the cells cultured by two-dimensional, and the three-dimensional cell sieving drug scaffold modified by CTB is more beneficial to the proliferation of the cells.

4. Determination of TGF-beta 1, type I collagen expression

Washing the three-dimensional cell sieving drug bracket for 3 times by using PBS buffer solution to remove non-adhered cells, putting cell liquid on the three-dimensional cell sieving drug bracket for trypsinization of cells into a 15mL sterile centrifuge tube, centrifuging at the rotating speed of 1000r/min for 5min, removing supernatant, adding PBS again to wash the cell bracket to collect residual adhered cell liquid, collecting liquid, centrifuging and removing supernatant. The cells were lysed by adding cell lysis buffer and then freeze-thawed between-80 ℃ and 37 ℃ until the liquid was no longer viscous. Expression of TGF-beta 1 and type I collagen of osteoblasts cultured in two dimensions and three dimensions was measured by Western Blot, and osteoblast lysate cultured in two dimensions and three dimensions was centrifuged at 5000r/min for 15min, and the supernatant was taken out and put into a new EP tube, and protein concentration of each sample was measured by BCA method. Adding a sample buffer solution into a protein sample, carrying out water bath at 100 ℃ for 10min, taking the supernatant, carrying out SDS-PAGE, carrying out membrane transfer, primary antibody and secondary antibody incubation and protein detection after electrophoresis is finished, analyzing a target band, and calculating the relative expression quantity of TGF-beta 1 and type I collagen of each sample.

TABLE 4 TGF-. beta.1, type I collagen determination

Test items	TGF-β1	Type I collagen
			Example 2	1.86±0.19	1.75±0.32
Comparative example 1	1.56±0.22	1.45±0.26
			Two-dimensional culture	1.00	1.00

From the test results, compared with the three-dimensional cell sieving drug scaffold which is cultured in two dimensions and unmodified in the comparative example 1, the expression levels of TGF-beta 1 and type I collagen of the cells of the three-dimensional cell sieving drug scaffold which is modified by CTB in the example 2 are the highest, namely the expression condition of the cytoplasmic matrix of the three-dimensional cell sieving drug scaffold which is cultured in three dimensions is better than the expression condition of the cytoplasmic matrix which is cultured in two dimensions, and the three-dimensional cell sieving drug scaffold modified by CTB is more beneficial to creating a cell microenvironment similar to that in vivo.

5. Determination of cell proliferation condition of three-dimensional cell sieving medicine bracket of in-vitro osteoporosis medicine

Preparing a tortoise-deer kidney tonifying pill liquid medicine: weighing 15g of tortoise and deer kidney tonifying pills, grinding into powder, placing into a 250mL triangular flask, adding 150mL of 70% ethanol at room temperature, continuously stirring for 12h to extract active ingredients, centrifuging at the rotating speed of 5000r/min for 10min, collecting supernatant, drying by using a rotary evaporator, storing the obtained extract at-20 ℃, diluting to 10mg/mL by using PBS buffer solution when in use, and performing bacteria filtration treatment for later use.

Example 2 and comparative example 1 osteoblasts were cultured for 12h, and then 100. mu.L of Guilu kidney-tonifying pill liquid was added to the culture dish and cultured at 37 ℃ for 36 h. According to the number of adherent cells in example 2, a cell suspension of the same number of cells was inoculated into a 6-well plate, n was 3, and after 12 hours of culture, 100. mu.L of Guilu Bushen Wan liquid medicine was added, and the mixture was cultured in the same medium at 37 ℃ for 36 hours as a two-dimensional culture control.

Thiazole blue (MTT) was added to a final concentration of 0.5mg/mL in example 2, comparative example 1 and two-position culture control, respectively, and after culturing in the dark at 37 ℃ for 4 hours, the culture solution was carefully poured out, 1.5mL of dimethyl sulfoxide (DMSO) was added, and after shaking at a low speed for 10 minutes, the DMSO was transferred to a 96-well plate at 150. mu.L per well to measure the absorbance at 570nm (the larger the absorbance, the stronger the cell activity).

TABLE 5 three-dimensional cell sieving drug scaffold cell proliferation assay for in vitro osteoporosis drugs

Test items	Absorbance value
		Example 2	3.32
Comparative example 1	1.53
		Two-dimensional culture	1.05

From the test results, the tortoise-deer kidney-tonifying pill liquid medicine is beneficial to the proliferation and differentiation of osteoblasts through the cultivation of the tortoise-deer kidney-tonifying pill liquid medicine, but compared with the three-dimensional cell sieve medicine bracket which is subjected to two-dimensional cultivation and is not modified in the comparative example 1, the proliferation rate of the cells of the three-dimensional cell sieve medicine bracket which is subjected to CTB modification in the example 2 is obviously increased after the cultivation of the tortoise-deer kidney-tonifying pill liquid medicine, and the three-dimensional cell sieve medicine bracket which is subjected to CTB modification has good medicine sieving effect and good predictability.

6. Determination of expression condition of TGF-beta 1 and I type collagen of three-dimensional cell sieving medicine bracket of in-vitro osteoporosis medicine

TABLE 6 determination of three-dimensional cell-sieving drug scaffold TGF-beta 1, type I collagen for in vitro osteoporosis drugs

Test items	TGF-β1	Type I collagen
			Example 2	3.56±0.18	3.25±0.27
Comparative example 1	1.95±0.23	1.85±0.25
			Two-dimensional culture	1.00	1.00

From the test results, the cells of the three-dimensional cell sieve medicine stent subjected to CTB modification in the example 2 have obviously higher expression levels of TGF-beta 1 and type I collagen after being cultured in the Guilu kidney-tonifying pill medicine liquid, compared with the three-dimensional cell sieve medicine stent subjected to two-dimensional culture and unmodified in the comparative example 1, the Guilu kidney-tonifying pill medicine liquid is beneficial to the cytoplasmic matrix expression of osteoblasts, and the CTB-modified three-dimensional cell sieve medicine stent has good medicine sieving effect and good predictability.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A preparation method of a three-dimensional cell sieving drug bracket of an in-vitro osteoporosis drug is characterized by comprising the following steps:

2. The method for preparing the three-dimensional cell sieve medicine bracket for the in vitro osteoporosis medicine, according to claim 1, wherein in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution 15-20 cm above the liquid level of the calcium chloride solution.

3. The method for preparing the three-dimensional cell sieve drug scaffold for the in vitro osteoporosis drugs according to claim 1, wherein in the step (3), the dropping speed of the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) into the calcium chloride solution is 1-1.5 mL/min above the liquid level of the calcium chloride solution.

4. The method for preparing the three-dimensional cell sieve medicine bracket for the in vitro osteoporosis medicine, according to the claim 1, is characterized in that in the step (3), the cholera toxin B subunit-sodium alginate solution or the cholera toxin B subunit-sodium alginate-gelatin solution prepared in the step (2) is dripped into the calcium chloride solution, and the stirring speed is 80-120 r/min;

5. The method for preparing the three-dimensional cell sieving drug bracket for the in-vitro osteoporosis drug according to claim 1, wherein the concentration of the sodium alginate solution is 15-30 mg/mL, and the concentration of the gelatin solution is 5-20 mg/mL.

6. The method for preparing the three-dimensional cell sieve drug scaffold for the in vitro osteoporosis drug according to claim 1, wherein the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed solution A is 5-10 mg/mL, and the concentration of N-hydroxysuccinimide in the mixed solution A is 2-5 mg/mL.

7. The method for preparing the three-dimensional cell sieving drug bracket for the in-vitro osteoporosis drug according to claim 1, wherein the concentration of the cholera toxin B subunit solution is 1-4 mg/mL.

8. The method for preparing the three-dimensional cell sieving drug bracket for the in vitro osteoporosis drug according to claim 1, wherein the concentration of the calcium chloride solution is 15-30 mg/mL.

9. The preparation method of the three-dimensional cell sieving drug bracket for the in-vitro osteoporosis drug according to claim 1, wherein in the step (1), the mixing volume ratio of the sodium alginate solution to the gelatin solution is 100: 0-25: 75;

10. The method for preparing the three-dimensional cell sieving drug scaffold for in vitro osteoporosis drugs according to claim 1, wherein the cell density of the osteoblast suspension is 1 x 10⁶～1×10⁷one/mL.