TWI681784B - A cell scaffold and method of making same - Google Patents

Abstract

The present invention provides a cell scaffold, characterized in that the cell scaffold is made of hydroxypropylcellulose and the cell scaffold is chemically grafted with at least one drug. The invention further provides a method for modifying a hydroxypropyl cellulose cell scaffold, which comprises the following steps: degassing the scaffold, treating the scaffold with an oxidant, degassing the scaffold after the oxidant treatment, and drying the scaffold.

Description

Cell scaffold and preparation method thereof

The present invention provides a cell scaffold, characterized in that the cell scaffold is composed of hydroxypropyl cellulose, and the cell scaffold is grafted with at least one drug, and the grafted system is through chemical grafting. The present invention also provides a method for modifying a hydroxypropyl cellulose cell scaffold. The method includes the following steps: degassing the scaffold and treating the scaffold with an oxidant; degassing the scaffold after treating the oxidant; and The stent is dried.

In vitro cell research is mostly carried out in a two-dimensional environment. Cells cultured by two-dimensional cell technology will gradually lose their original state in an in vitro environment and are far from natural growth in the body. Although animal experiments are closer to the real situation of the human body, because there are many uncontrollable factors in the body and the environment will be complicated by the interaction between the body and the body, it is not conducive to exploring the specific mechanism.

In view of the limitations of two-dimensional cell culture technology and animal experiments, three-dimensional cell culture technology (TDCC) has been gradually developed in recent years. The three-dimensional cell culture technology refers to the co-cultivation of animal cells and materials with a three-dimensional structure, so that the cells can grow, proliferate and migrate in a three-dimensional space, thereby forming a three-dimensional model cell-carrier complex, so that the experimental environment can be better Of the simulated cell growth in the body.

Since the three-dimensional cell culture can not only retain the material structure foundation of the natural cell microenvironment, but also better simulate the cell growth microenvironment in vivo, the cells obtained by the three-dimensional cell culture can be formed The structure of three-dimensional tissues is significantly different from two-dimensional culture in terms of morphological structure, proliferation and differentiation, gene expression and cell function. Therefore, three-dimensional cell culture technology has been used in drug screening and evaluation, tumor treatment, virus monitoring, regeneration Many fields such as medicine have received much attention.

The three-dimensional cell culture technology can be divided into two types, with or without a cell scaffold. The non-scaffold part mainly uses physical methods to suspend the attached cells in the culture medium to achieve the purpose of three-dimensional culture; the three-dimensional cell culture technology with the cell scaffold makes the cell scaffold A sponge-like porous structure is simulated in three-dimensional space to provide cell attachment and growth, so that the cells attach to the scaffold and grow and migrate in three dimensions. The three-dimensional scaffold can provide an environment suitable for cells to attach and grow, and can even guide the cells to form tissues or support the shape of the tissue. The shape of the scaffold can be shaped according to the required shape to fit the defects of human tissues in the future. For most mammalian cell culture systems, if a three-dimensional structure cannot be provided for cell growth, the cells will not be able to differentiate and proliferate well. Therefore, three-dimensional scaffolds play a vital role in mammalian cell culture.

The scaffold is often a porous structure, which can increase the cell attachment space to accelerate the formation of tissue. The sources of scaffolds can be roughly divided into two types: natural and artificial. Natural materials are mainly obtained directly from organisms, such as collagen, chitin, algae, etc., and artificial materials include polylactate and polymer. Glycolic acid (polyglycolate) and so on. The material of the stent itself is preferably biodegradable (such as polylactic acid or chitosan, etc.), that is, it can be gradually decomposed in the body, and then replaced by the matrix of the body tissues. More importantly, the material itself or the products after decomposition cannot cause toxic damage to the body. It is best not to cause immune or inflammatory reactions after implantation, and can be closely and normally joined with the original tissue of the next part. Equivalent materials are quite biocompatible.

Hydroxypropyl Cellulose (HPC) is a new type of cellulose. After the hydroxyl groups in the cellulose are replaced by hydroxypropyl by etherification, the rigid hydroxypropyl cellulose is nonionic. Type of cellulose ether. Hydroxypropyl cellulose has good physical properties: 1. Solubility: Hydroxypropyl cellulose is soluble in water and organic solvents, such as methanol, ethanol, propylene glycol and other polymer solutions; 2. Water-soluble ): Hydroxypropyl cellulose is a white powder that is soluble in water. Heating the aqueous solution to about 45°C will cause phase separation. A white turbid state can be observed. If the aqueous solution is heated, a gel state will also be produced. In addition to low cost, hydroxypropyl cellulose has also been approved by the US Food and Drug Administration (FDA) in terms of drug delivery. Hydroxypropyl cellulose has been used in various fields of biomedical materials, and has great potential in terms of drug release and cytoskeleton.

Flavonoids have the properties of anti-inflammatory, anti-cancer, lipid-lowering, antioxidant, anti-thrombotic, anti-allergic, and reducing the occurrence of cardiovascular diseases such as arteriosclerosis. Among them, naringin and its hydrolysate (Naringenin, pomelo) Dermosin) has been pointed out to have many different biological and pharmacological properties, including increasing the gene expression of antioxidant enzymes in the body, such as superoxide dismutase (SOD) and catalase (catalas, CAT), reducing liver Mitochondrial hydrogen peroxide content to achieve anti-oxidation effect; on the other hand, naringin can block the activity of HMG-CoA reductase (HMG-CoA reductase), which in turn affects the synthesis of cholesterol in the blood, in order to reduce blood Cholesterol concentration. Recent studies have found that naringin is the same as statins and is an HMG-CoA inhibitor. It has the effect of treating osteoporosis and can effectively promote the growth of bone cells, but the naringin concentration is too high It will produce cytotoxicity, because the promotion of bone differentiation often occurs after a certain implantation time, in order to make naringin proliferate in the early bone cells and bone in the middle and late stages. All of them have a promoting effect. At present, the use of naringin is mostly carried out by multiple continuous injections, and long-term treatment cannot be achieved. Therefore, controlling the release rate and concentration of naringin to achieve long-lasting stimulation of the biological system will help more effective application of naringin and other flavonoids.

The purpose of the present invention is to prepare a cell scaffold with the ability to induce cell proliferation and differentiation, find out the best modification condition of the material by measuring the peroxide, and further avoid the drug in a short time by grafting the drug on the surface of the scaffold The internal release concentration is too high to produce cytotoxicity, or to enhance the cell affinity of the material itself, promote cell activity or differentiation ability.

The present invention provides a cell scaffold, characterized in that the cell scaffold is composed of hydroxypropyl cellulose, and the cell scaffold is grafted with at least one drug, and the grafted system is through chemical grafting.

The term "chemical grafting" as used in the present invention refers to the use of chemical reaction between the reactive groups on the surface of the material and the grafted monomers or macromolecular chains to achieve surface grafting, including: the functional groups and Through the bonding reaction between the functional groups on the graft; through chemical reagents reacting with the surface of the material or the polymer chain to generate an active center, which initiates the polymerization of the monomer; by placing the material in an oxidizing agent A peroxide is formed on the surface of the material, and the peroxide decomposes to generate free radicals to initiate the graft polymerization of the monomer on the surface of the material.

The "Flavonoid" referred to in the present invention refers to a series of compounds in which two benzene rings with phenolic hydroxyl groups are connected to each other through a central three-carbon atom, which includes but is not limited to anthoxanthins such as flavonoids , Flavonol (Flavanole) or 3-hydroxyflavone (3-hydroxyflavone); Flavanone (Flavanone) such as hesperidin (Hesperidin), naringin (Naringin), 3',4',5,7-tetrahydrodihydroflavones (Eriodictyol), high saccharin (Homoeriodictyol); dihydroflavonols (Flavanonols or 3-Hydroxyflavanone) or 2,3-dihydroflavones (2,3-dihydroflavonol); flavans (Flavams) such as flavan-3-ols (Flavan-3-ols), flavan-4-ols (Flavan-4-ols) and flavan-3,4-di Alcohol (Flavan-3,4-diols); Anthocyanidine (Anthocyanidine); and Isoflavones (Isoflavonoids).

In one embodiment, the cell scaffold is a three-dimensional cell scaffold.

In one embodiment, the drug is a flavonoid; in another embodiment, the flavonoid includes but is not limited to naringin.

In one embodiment, the chemical grafting method includes using an oxidizing agent for upgrading; in another embodiment, the oxidizing agent includes but is not limited to ozone.

In one embodiment, the cell growth scaffold is used as an intraosseous filler.

In one embodiment, the cell growth scaffold is used for cell culture; in another embodiment, the cell line is bone cells; in another embodiment, wherein the cell culture is in vitro; In another embodiment, the cell culture fluid of the cell culture is in a flowing state.

The present invention also provides a method for modifying a hydroxypropyl cellulose cell scaffold. The method includes the following steps: degassing the scaffold and treating the scaffold with an oxidant; degassing the scaffold after treating the oxidant; and The stent is dried.

In one embodiment, the oxidant is ozone; in another embodiment, the ozone is used to treat the stent at a flow rate of 2-12 liters per minute; in another embodiment, the ozone is The stent is processed at a flow rate of 4-10 liters per minute; in another embodiment, The treatment time of the ozone on the bracket is 20-130 minutes; in another embodiment, the treatment time of the ozone on the bracket is 30-120 minutes.

In one embodiment, the modified hydroxypropyl cellulose cell scaffold provided by the present invention further comprises the following steps: grafting a drug on the scaffold after drying; in another embodiment, wherein the drug is a flavonoid Compound; in another embodiment, wherein the flavonoid compound includes but is not limited to naringin.

Figure 1. The effect of ozone flow rate on the peroxide concentration on the surface of hydroxypropyl cellulose. The fixed ozone treatment time is 1 hour, changing the ozone flow rate from 0 to 12 liters per minute.

Figure 2. Effect of ozone treatment time on the peroxide concentration on the surface of hydroxypropyl cellulose. The ozone flow rate is fixed at 6 liters per minute, and the ozone treatment time is changed from 0 to 120 minutes.

Figure 3. Comparison of surface peroxide concentration of various polymers after ozone treatment.

Figure 4. FTIR spectrum of naringin.

Figure 5. FTIR spectrum of hydroxypropyl cellulose scaffolds modified with ozone and grafted with different naringin concentrations. pristine is a scaffold without ozone modification and naringin grafting, and 0.05%, 0.5% and 1% represent the weight percent concentration of naringin solution used in the modification procedure.

Figure 6. Surface profile of hydroxypropyl cellulose scaffold after ozone modification and naringin grafting. pristine is a scaffold without ozone modification and naringin grafting. 0%, 0.05%, 0.1% and 1% represent the weight percent concentration of naringin in the solution used in the grafting process after ozone modification .

Figure 7. The effect of different naringin concentration modification on the swelling rate of hydroxypropyl cellulose scaffold. pristine is a stent without ozone modification and naringin grafting, with 0.05%, 0.1% and 1% points Do not represent the weight percent concentration of naringin in the solution used in the modification procedure.

Figure 8. Naringin release concentration of hydroxypropyl cellulose scaffold in PBS. 0.05%, 0.1% and 1% respectively represent the weight percent concentration of naringin in the solution used in the modification procedure. O stands for ozone modification and then naringin grafting, A stands for direct adsorption and fixation of naringin. (a) Naringin release time: 0-120 hours (b) Naringin release time: 0-24 hours.

Figure 9. Calculation of naringin release concentration of hydroxypropyl cellulose scaffold in PBS using peroxide concentration as the maximum grafting amount. 0.05%, 0.1% and 1% respectively represent the weight percent concentration of naringin in the solution used in the modification procedure. (a) Naringin release time: 0-120 hours (b) Naringin release time: 0-24 hours.

Figure 10. Grafting naringin onto hydroxypropyl cellulose scaffold using ozone modification and direct adsorption method. After five days of cultivation, 7F2 activity was measured by mitochondrial activity test. ● (p<0.05) indicates that it is different from all other concentrations at the same incubation time. *(p<0.05) and **(p<0.01) indicate significant differences between the two groups (t-test, n=4). pristine is a scaffold that has not been surface-modified. O stands for hydroxypropyl cellulose scaffold which is modified by ozone before fixing naringin. A stands for directly adsorbing naringin to hydroxypropyl cellulose scaffold. 0.05%, 0.1% and 1% respectively represent the weight percent concentration of naringin in the solution used in the modification procedure.

Figure 11. Grafting naringin to hydroxypropyl cellulose scaffold using ozone modification method, and measuring 7F2 activity by mitochondrial activity test. *(p<0.05) and **(p<0.01) indicate significant differences between the two groups (t-test, n=4). pristine is a scaffold that has not undergone ozone modification and naringin grafting. 0.05%, 0.1% and 1% represent the weight percentage of naringin in the solution used in the modification procedure.

Figure 12. Observation of ozone modification of 7F2 by SEM after 1, 3, and 5 days of cell culture Fix the cell type on the naringin hydroxypropyl cellulose scaffold. pristine is a scaffold that has not undergone ozone modification and naringin grafting. 0.05%, 0.1% and 1% represent the weight percentage of naringin in the solution used in the modification procedure.

Figure 13. Observation of cell morphology of 7F2 on ozone-modified naringin hydroxypropyl cellulose scaffold by SEM after 5 days of cell culture. pristine is a scaffold that has not undergone ozone modification and naringin grafting. 0.05%, 0.1% and 1% represent the weight percentage of naringin in the solution used in the modification procedure. It can be found that the lamellipodia and filopodia of the modified group are widely distributed on the surface of the material, and 1% of them are widely distributed.

Figure 14. Observation of the actin filament tissue of 7F2 modified with ozone to fix naringin on hydroxypropyl cellulose scaffold by conjugated focus microscope after 5 days of cell culture. pristine is a scaffold that has not undergone surface modification. 0.05%, 0.1% and 1% represent the weight percent concentration of naringin in the solution used in the modification procedure.

Figure 15. Using ozone modification method to graft naringin to hydroxypropyl cellulose scaffold, 7F2 alkaline phosphatase (ALP) secretion performance. *(p<0.05) and **(p<0.01) indicate significant difference between the two groups (t-test, n=5). pristine is a scaffold that has not undergone ozone modification and naringin grafting. 0.05%, 0.1% and 1% represent the weight percentage of naringin in the solution used in the modification procedure.

Figure 16. Using X-ray EDS to analyze the calcium content of ozone modified naringin grafted on hydroxypropyl cellulose scaffold after 7F2 culture for 14 days. *(p<0.05) and **(p<0.01) indicate significant differences between the two groups (t-test, n=4). pristine is a scaffold that has not undergone surface modification. 0.05%, 0.1% and 1% represent the weight percent concentration of naringin in the solution used in the modification procedure.

Figure 17. Differences in cell activity between two-dimensional and three-dimensional environments. Observation of hydroxypropyl by SEM The surface morphology of the two-dimensional plane of cellulose and gelatin. (a) Front side of hydroxypropyl cellulose (b) Back side of hydroxypropyl cellulose

Figure 18. Differences in cell activity between two-dimensional and three-dimensional environments. The cell activity of 7F2 on the two-dimensional plane and three-dimensional scaffold of hydroxypropyl cellulose was measured by mitochondrial activity test. *(p<0.05) and **(p<0.01) indicate significant difference between the two groups (t-test, n=5).

Figure 19. The static and dynamic cell viability of 7F2 grown on a hydroxypropyl cellulose scaffold was determined by the mitochondrial activity test. *(p<0.05) and **(p<0.01) indicate significant differences between the two groups (t-test, n=4).

Example 1. Optimization of ozone upgrading.

For biomedical polymer materials, the surface properties of the material are the key factors for their affinity with cells and the adsorption surface of biomolecules. In industrial applications, many studies have used physical or chemical surface modification methods to modify polymer surfaces. In the chemical surface modification method, peroxides are generated on the surface of the substrate by the strong oxidizing properties of oxidants such as ozone, and free radicals are generated on the surface of the material by means of oxidation-reduction. The purpose of fixing the monomer on the surface of the substrate. The ozone modification method using ozone as an oxidant is easy to operate and suitable for samples of various shapes. It also greatly reduces the amount of organic solvents used. In addition to eliminating the biological toxicity caused by residual organic solvents, this procedure is also environmentally friendly (environmental-friendly). The variables that affect the peroxide concentration in the ozone modification method include ozone concentration and ozone modification time. Therefore, the ozone concentration and modification time are discussed.

Follow the steps below to upgrade the ozone: First place the holder in an Erlenmeyer flask, and degas by adding nitrogen for 15 minutes (min). Then at a normal temperature of 2-12 liters per hour (L/hr) ozone flow rate Ozone is introduced for 20-120 minutes. The conical flask modified by ozone was purged with nitrogen for 15 minutes to degas. Finally, store the modified bracket in a dry box.

For the discussion of ozone concentration, it can be found from the results in Figure 1 that the peroxide concentration on the surface of the material increases as the flow rate increases, which means that the ozone concentration in the reaction environment increases with the flow rate, and the number of activation points on the surface of the material also increases . However, when the flow rate continues to increase and the peroxide content reaches a peak, the content gradually becomes flat or even decreases, which means that the concentration of the activation point on the surface of the material may have reached a saturation state.

For the discussion of the modification time, it is found from the results in Fig. 2 that the peroxide content increases with time in the initial stage of modification, but it will gradually remain flat after more than one hour. The reason is presumed to be the crystal lattice of the material during modification The nature has changed.

Combined with the above test results, the material conditions fall within the flow rate of 2-12 liters per minute (L/min), and the modification time falls within 20-130 minutes. This condition is also used in subsequent experiments.

Example 2. Comparison of surface peroxide concentration.

Compare the optimized experiment results of the present invention with other ozone-treated polymers for the surface peroxide concentration. The peroxide content determination step is: the 1,1-diphenyl-2-trinitrophenylhydrazine (2,2-diphenyl-1-picrylhydrazyl, DPPH) mix with distilled water in dark place and prepare a 1 millivol molar concentration (mM) reaction solution. The modified stent is put into the solution of the previous step, and at the same time, a group of stents not ventilated with ozone is prepared and put into the same solution as a blank group (Blank). After 24 hours of reaction at 70°C in the dark, the absorption value was measured using a UV spectrometer at a wavelength of 520 nanometers (nm) and the DPPH loss was calculated.

The comparison results of surface peroxide concentration are shown in Figure 3. It can be found that the concentration of peroxide produced by hydroxypropyl cellulose (9.3×10 ^-7 gmol/cm ² (gmol/cm ² )) is at least 10 times higher than that of most polymers. The propylcellulose scaffold has a good modification efficiency, and this feature helps the subsequent drug grafting reaction.

Example 3. Ozone modification fixes flavonoids.

Select naringin as a flavonoid drug for grafting. Ozone modification and fixation of naringin follow the following steps: naringin is added to a round bottom flask and nitrogen is added, 10 minutes later, methanol is added, and the configuration is 0, 0.05, 0.1, 1 weight percent concentration (wt%) naringen Glycoside solution, then put the ozone-modified scaffolds into a round bottom flask and add the naringin solution of the previous step, during which nitrogen gas is continuously introduced, and then add 0.1% by weight of ferrous sulfate hexahydrate (Ammonium ferrous sulfate Hexahydrate, FAS), during which nitrogen gas was continuously introduced, and finally, placed in a 55°C oil bath for 24 hours after tightly sealing the bottle.

The scaffolds fixed with different naringin concentrations were measured by Fourier Infrared Spectroscopy (FTIR) with a wave number of 400 to 4000 cm ^-1 (cm ^-1 ), and then the functional groups were analyzed from the results for qualitative analysis.

It can be observed from the arrows in Figures 4 and 5 that the ozone-modified and naringin grafted groups have a C=O characteristic peak of naringin at the peak of 1665 cm ^-1 , which can prove that yuzu peel The glycoside was successfully grafted onto the hydroxypropyl cellulose scaffold.

Example 4. Surface modification of hydroxypropyl cellulose scaffold after ozone modification and naringin grafting.

The shape of the surface of the material is also one of the factors that affect cell growth. Therefore, after the ozone modification and naringin grafting, the surface shape of the hydroxypropyl cellulose scaffold was observed by SEM, and the surface shape of the material after modification was discussed. influences.

The results are shown in Figure 6. There is no obvious damage or corrosion on the surface of the bracket. The ozone and naringin modification conditions used in the present invention are not sufficient to affect the shape of the material surface.

Example 5. Hydrophilic and hydrophobic analysis of materials before and after naringin modification.

Hydrophobicity is an important property of biomedical substrates. In tissue engineering, the proliferation and differentiation of bone cells are not only affected by the surface shape of the material, but the hydrophobicity of the material itself is also the key to affecting cell adhesion and activity. Therefore, the swellability of the materials before and after modification is analyzed.

It can be seen from the results in FIG. 7 that there is no significant difference in the swelling property of the material after modification, so it can be ruled out that the material causes a difference in hydrophilicity and hydrophobicity after grafting.

Example 6. Drug release results of the material modified with ozone and grafted naringin.

Through the direct adsorption method of soaking the material directly into the naringin solution, and the way the material was modified by ozone and then grafted naringin, two different methods were tested to explore the effect of ozone modification on the release concentration. The steps of drug release are as follows: soaking the naringin-fixed scaffold in PBS and placing it in a 37°C incubator, taking points at 2, 4, 6, 8, 10, 24, 48, 72, 96 and 120 hours Measure the absorption value by UV spectrometer at the wavelength of 286 nm, and finally bring into the calibration line to convert the concentration of naringin.

From the results in FIG. 8, it can be found that the concentrations released by the materials are within the range of 1-100 ppm which can effectively stimulate cell activity and lower than the cytotoxicity (200 ppm). The release curve is divided into two stages: the first stage is the area where the release is faster (1 ^st stage), and the second stage is the area where the release is slower (2 ^nd stage). This release behavior has been quite in the past drug release research As common. The first stage of rapid release comes from the naringin, which is less stable, and it will be released into the liquid in a large amount and quickly in the beginning. After that, only the stable naringin remains on the surface of the material, and the release will enter slowly. The second stage. No matter what concentration of naringin is fixed on the stent, it has entered the second stage of stable release area after 24 hours of release. From the above results, it can be found that naringin can release higher concentrations of naringin by ozone modification grafting, and the release time is relatively long, which proves that the ozone modification method is superior in the long-acting release mode For direct adsorption.

Example 7. Grafting and releasing ratio of naringin.

In order to know the grafting and release ratio of naringin on the material, it is assumed that the optimal peroxide concentration is the maximum grafting amount of naringin and the release percentage is calculated from this.

From the results in Figure 9, it can be found that the release percentage still rises slowly at the fifth day, and the release percentage has not reached 100%. It is proved that the ozone modification grafting method used in the present invention is a long-acting release mode, and this release mode also helps to produce continuous stimulation when cultured with cells.

Example 8. Effect of different modification procedures on cell activity.

In order to know whether ozone modification affects the cell activity of fixing naringin on the surface of the scaffold, the present invention uses two different fixing procedures of ozone modification and direct adsorption to perform a cell activity test (MTT assay). The direct adsorption method is as described previously. The cell culture system is carried out in the following steps: using α-MEM as the cell culture medium, and culturing osteoblasts (7F2). The α-MEM culture medium contains 10% serum (FBS) and 1% antibiotics (Penicillin-streptomycin-amphotercin), and the cells are placed in a 37°C, 5% carbon dioxide cell incubator for culturing every three days Change the medium once.

From the results in Fig. 10, it can be found that naringin can improve the cell affinity of the material whether it is directly adsorbed or chemically bonded to the material. Among them, the chemical bonding method has a good stimulating activity effect on cells, which means that the material can release higher concentration of naringin after ozone modification, which is more effective for promoting the activity of bone cells; or the ozone-treated stent surface has More immobilized naringin can slowly stimulate bone cells by releasing it for a long time. Combining the above results, The way to improve the cell affinity of the material is to use ozone modification and activation better than physical adsorption.

Example 9. Effect of different grafting concentration on cell activity.

The purpose of this experiment is to explore the effect of different concentrations of naringin on cell viability during long-term culture during modification. The cell viability test (MTT assay) is carried out according to the following steps: First, the MTT stock solution is diluted with PBS at a concentration of 1:9 as a working solution for cell viability test. Next, after absorbing the culture liquid in the petri dish, PBS was added to rinse several times, and then the diluted working solution was added to the petri dish, and the petri dish was placed in a 37°C incubator to carry out the reaction. After about 4~6 hours of reaction, after sucking out the MTT working solution in the Petri dish, add DMSO for about 10~15 minutes to dissolve the formazan in the cells. Finally, aspirate each cell culture dish 4 times with an amount of approximately 200 μl/well, place it in a 96-well dish, and place it in an ELISA Reader, then use a wavelength of 570 nm to read the absorbance value.

The results are shown in Figure 11. It can be found that the scaffolds all have good biocompatibility, and no matter what the result is, the scaffold can be better promoted by bone modification after ozone modification and naringin grafting. The qualitative group had higher cell activity than the unmodified group. Among them, the high concentration of 1% has better performance. At the initial stage of culture, the higher concentration group had better effect of promoting cell activity. Combining the results of different days, it can be found that as the number of days increases, the difference in activity between the grafted naringin scaffold and the unmodified group increases, which proves that the material can improve cell affinity after grafting naringin. Fit and promote cell proliferation.

Example 10: Observation of cell type on scaffold.

Figures 12-13 show the results of cell type observation on the modified hydroxypropyl cellulose scaffold. Figure 12 shows that after one day of culture, all groups of 7F2 have been successfully attached to the surface of the material, and lamellipodia and filopodia can be seen around the cell body; After the third day of culture, more cells attached to the material can be observed, and cell-cell junctions begin to form; after the fifth day of culture, the modified groups have attached cells The area is significantly better than the unmodified group. Magnified analysis of the results on the fifth day (Figure 13), it can be found that the modified groups of lamellipodia and ilopodia are widely distributed on the surface of the material, and the distribution is 1%, which is also consistent with the results of cell activity . This result proves that the affinity of the material for cells after grafting naringin increases, making it easier for the cells to adhere to the surface of the material.

Example 11. Results of staining of actin skeleton and cell nucleus on a scaffold.

When the cell contacts the surface of the substrate, the chemical, physical, and structural signal molecules of the substrate are transferred to the nucleus through the linking protein and cytoskeleton, so the cytoskeleton plays an important role in transmitting signals inside the cell.

It can be found from Figure 14 that with DAPI staining the nucleus and Phalloidin staining the cytoskeleton, 7F2 of all groups can see obvious actin performance, showing that the cytoskeleton has formed a complete network, and it can be It was observed that the modified group showed higher stretched actin. This actin's performance should be caused by the extension of lamellipodia, indicating that there has been interaction between bone cells and the modified scaffold. , This result also shows that naringin can promote the extension of the cytoskeleton.

The conjugate microscope gradually analyzes the cell attachment staining from the inside of the material to the surface of the material, and the results can prove that osteoblasts not only grow on the surface of the scaffold, but also grow inside the material to form a three-dimensional growth pattern.

Example 12. The effect of different concentrations of naringin on the performance of alkaline phosphatase in 7F2 cells.

Alkaline phosphatase (Alkaline phosphatase, ALP) is regarded as one of the important indicators for inducing bone formation in the process of bone differentiation. According to the following ALP quantification steps, explore whether different concentrations of naringin fixed on the stent will affect the performance of 7F2 ALP: first configure it to contain 0.1 mol of glycine, 1 millimolar of zinc chloride and zinc chloride. 1 millimolar concentration of magnesium chloride solution, and using a 3 equivalent strength sodium hydroxide aqueous solution to adjust the pH of the solution to about 10.4, which is the base solution. Take 15 ml of the base solution and add a p-nitrophenyl phosphate (pNPP) tablet. After dissolving it in the dark and at room temperature, it is the reaction solution. Using 0.4 ml of cell lysis reagent, after suspending and lysing the cells in the Petri dish for about 15 minutes, and then uniformly mixing with 1.2 ml of the reaction solution, and reacting at room temperature in the dark for 30 minutes. After 30 minutes, 0.3 ml of an aqueous solution of sodium hydroxide (3 equivalents concentration) to stop the reaction was added and left at 4°C for 10 minutes. Use ELISA Reader to measure the absorbance under the wavelength range of 405 nm, and then calculate the ALP content from the calibration curve.

It can be seen from the results in Fig. 15 that on the third day of culture, there is no difference between the groups. This may be caused by the early stage of the bone cell culture just entering the differentiation stage. After the sixth day of culture, the ALP secretion performance of the modified group was higher than that of the unmodified group. This result showed that naringin began to promote bone cell differentiation. After cultivation to the tenth day, the ALP secretion performance of the modified group was still significantly higher than that of the unmodified group, and 0.05% of the modified group performed better. When the culture reaches the twelfth day, the ALP secretion of each group begins to decrease, which means that 7F2 will enter the next stage of differentiation. This result shows that grafted naringin can promote the secretion of ALP. After ozone modification and fixation of naringin on the scaffold, it has a significant promoting effect on the initial differentiation of 7F2. In the past literature, there are studies on cell proliferation and differentiation, some of which mentioned that when the cell proliferation is too fast, it may inhibit or delay the occurrence of differentiation, and this phenomenon is consistent with the cell activity and differentiation results of the present invention.

Example 13. Effect of different concentrations of naringin on the scaffold on mineralization of bone cells in the later stage.

When osteoblasts at the end of differentiation are specialized into osteoprogenitor cells, the cells will secrete osteocalcin. At this time, calcium ions and phosphate ions are gradually deposited, allowing osteoblasts to undergo bone mineralization. In this experiment, EDS elements are used to analyze late bone cells The content of calcium was used to observe the effect of different concentrations of naringin on the scaffold on mineralization of bone cells in the later period.

It can be seen from Figure 16 that the modified group had higher calcium secretion than the unmodified group, showing that after the ozone modification step, the naringin fixed on the hydroxypropyl cellulose scaffold can effectively promote bone Cells are mineralized to achieve bone regeneration. This result can also be speculated that increasing the modified concentration of naringin may inhibit the occurrence of bone mineralization. This situation is also consistent with the previous ALP secretion results.

Example 14. Differences in cell activity between two-dimensional and three-dimensional environments.

In order to explore the effect of three-dimensional scaffold materials on cells, the same material as the scaffold was used to create a two-dimensional planar environment and cultured with osteoblasts. From the cell activity results of different environments in Figures 17-18, it can be found that there is no difference between the two materials and the three materials on the first day of culture. This may be because the cells were just attached to the material at the beginning of the culture, but not yet There is no obvious difference in the next stage of hyperplasia. After the third day of cultivation, the three-dimensional culture began to be superior to the two-dimensional environment. The reason is presumed that the cells began to grow in the three-dimensional direction on the scaffold, resulting in more cell growth space. This result can prove that the ability of the scaffold to provide cells in three-dimensional growth has been fully demonstrated, and once again proves that the scaffold in this study is a three-dimensional growth model. Based on the comprehensive results, the three-dimensional scaffold culture is superior to the two-dimensional planar environment.

Example 15. The difference between static and dynamic cell culture.

Use the peristaltic pump as the power to deliver the culture medium Culture in the bioreactor to explore the activity of cells in different culture systems.

From the results in Figure 19, it can be found that when using a dynamic system to culture cells on the scaffold, as the number of days increases, the activity gap between static and dynamic also increases, which may be due to the addition of physical such as flow Stimulation and increased nutrient and mass transfer effects through continuous delivery of culture medium. This result also shows that the mechanical properties of the hydroxypropyl cellulose scaffold itself are sufficient to withstand the simulation of dynamic systems, indicating that it can have better cell activity in a more similar in vivo environment Performance, from the cultivation of different systems, we can see that dynamic systems are better than static cultivation.

pristine‧‧‧Stent without surface modification

O‧‧‧Hydroxypropyl cellulose scaffold is modified by ozone before fixing naringin

A‧‧‧Direct adsorption of naringin on hydroxypropyl cellulose scaffold

0.05%, 0.1% and 1% ‧‧‧ The weight percent concentration of naringin in the solution used in the modification procedure

Claims (9)

A three-dimensional cell scaffold, characterized in that the three-dimensional cell scaffold is composed of hydroxypropyl cellulose, and the three-dimensional cell scaffold is grafted with at least one drug. The grafting system is through a chemical grafting method, wherein the chemical grafting method is a system For the use of oxidants for upgrading. For example, the three-dimensional cell scaffold of patent application item 1, wherein the drug is a flavonoid compound. For example, the three-dimensional cell scaffold of the first patent application scope, wherein the three-dimensional cell growth scaffold is used as a bone filling. A method for modifying a hydroxypropyl cellulose cell scaffold. The method includes the following steps: degassing the scaffold and treating the scaffold with an oxidizing agent; degassing the scaffold after the oxidizing agent treatment; and drying the scaffold. For example, the method for modifying the hydroxypropyl cellulose cell scaffold in the patent application scope item 4, wherein the oxidant treats the scaffold at a flow rate of 2-12 liters per minute. For example, the method for modifying the hydroxypropyl cellulose cell scaffold in the patent application scope item 4, wherein the treatment time of the scaffold by the oxidant is 20-130 minutes. For example, the method for modifying the hydroxypropyl cellulose cell scaffold in the patent application item 4 further includes the following steps: grafting the drug on the scaffold after the drying treatment. For example, the method for modifying the hydroxypropyl cellulose cell scaffold in the 7th range of the patent application, wherein the drug is a flavonoid compound. A modified hydroxypropyl cellulose cell scaffold is modified by a method such as item 4 of the patent application.

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