TWI622411B - Porous Biomedical Scaffold, Manufacturing Method thereof, and Biomedical Composite Material Comprising the Same - Google Patents

Abstract

The invention relates to a porous biomedical stent, a preparation method thereof, and a biomedical composite material comprising the same, wherein the porous biomedical stent is composed of a cross-linked keratin and a chitosan chitosan. The biomedical composite material is composed of the porous biomedical stent and a cell.

Description

Porous biomedical stent, preparation method thereof, and biomedical composite material containing same

The present invention relates to a porous biomedical stent, a preparation method thereof, and a biomedical composite material comprising the same, and more particularly to a biomedical stent for tissue engineering and a biomedical composite material comprising the same.

The advancement of modern medicine has prolonged human life and provided a better quality of life. Regenerative medicine and tissue engineering are emerging medical methods. Among them, the concept of tissue engineering is to culture or construct a functional tissue in vitro, and then replace and repair the originally damaged tissue or organ. Tissue engineering has three main elements, namely cells, scaffolds, and growth factors. Cell lines are attached to the scaffold for growth, while growth factors induce proliferation or differentiation of cells in the scaffold.

The cells commonly used in tissue engineering include autologous cells or stem cells. The autologous cells must be taken out from the patient's own tissues, and the cells are separated and cultured in a scaffold. Autologous cells can avoid immune system rejection caused by tissue transplantation and are an ideal source of cells. However, some tissues in the human body are not easy to obtain, or the available tissues are limited. Therefore, the mesenchymal stem cells are often used as a source of cells, and growth factors or cell culture environments are used to induce mesenchymal stem cells to differentiate into specific cells. Mesenchymal stem cells are multi-functional stem cells, which have a wide range of sources and can be isolated from bone marrow, fat, placenta, pulp and other parts. The quantity source is relatively stable, which is the most promising stem cell species in tissue engineering.

Furthermore, tissue engineered scaffolds are typically prepared using materials that are biocompatible and degradable, in addition to promoting cell attachment to them, as well as having a mechanical strength to maintain the stent. The tissue engineering scaffold may be composed of a synthetic polymer or a natural polymer, or may be composed of a composite material of the two. The preferred stent needs to have excellent biocompatibility and is a degradable material, and its degradation rate must also match the growth rate of the new tissue.

Finally, growth factors play a role in inducing stem cell differentiation to induce stem cells to differentiate toward a particular cell, allowing stem cells to differentiate into specific cells in the scaffold and gradually form specific tissues.

The scaffolds used in tissue engineering must meet non-toxic, biocompatible, biodegradable, mechanical strength, and cell attachment requirements. Therefore, the preparation of a stent that meets the above conditions is one of the important development goals in tissue engineering.

In order to achieve the above object, the present invention provides a porous biomedical stent, a preparation method thereof, and a biomedical composite material comprising the same.

The preparation method of the porous biomedical stent provided by the invention mainly comprises the following steps: (1) mixing a keratin and a chitosan chitosan in a solvent to prepare a mixture; (2) irradiating a light to The mixture is such that the keratin in the mixture is crosslinked with the azide chitosan to obtain a crosslinked mixture; (3) the crosslinked mixture is prepared into a porous biomedical stent by freeze drying.

In step (1), the keratin may be extracted from animal hair or nails. In a preferred embodiment, the keratin is extracted from human hair. Further, the azidated chitosan is as shown in the following chemical formula: Among them, 300 < n < 1200.

In the step (1), the content ratio of the keratin to the azide chitosan is 0.01 to 1, and preferably 0.02 to 0.5.

In the step (1), the solvent is preferably water, more preferably deionized water.

In the step (2), the light is preferably UV light having an irradiation intensity of 65 to 100 mW/cm ² and a wavelength of 280 to 380 nm, wherein preferably 100 mW/cm ² and a wavelength of 365 nm.

Another object of the present invention is to provide a porous biomedical stent comprising a cross-linked keratin and a chitosan chitosan having a pore size of 100 to 500 μm, preferably 200 to 350 μm.

In the above porous biomedical stent, the ratio of the keratin to the azidated chitosan content may be 0.01 to 1, and further preferably 0.02 to 0.5.

The Young's coefficient of the above biomedical stent may be 0.01 to 0.05 MPa, and preferably 0.03 to 0.04 MPa. The biomedical stent may have a porosity of 30% to 50%, and more preferably 35% to 50%. In this way, the porous biomedical stent of the present invention can provide sufficient mechanical strength and maintain the structural integrity of the pores for the cells to grow and attach thereto.

The porous biomedical stent provided by the invention has good biocompatibility and is favorable for cell attachment, and the appropriate porosity can supply the cell with an excellent growth environment. The above advantages make the porous biomedical stent of the present invention conform to the conditions of the stent as a tissue engineering.

Accordingly, the present invention further provides a biomedical composite for tissue engineering, comprising a porous biomedical stent and a cell, wherein the porous biomedical stent comprises a cross-linked keratin and an azide Butanose having a pore size of 100 to 500 μm; and the cell line is cultured on the porous biomedical stent.

The biomedical composite material further includes a culture solution filled in the pores of the porous biomedical stent to supply cell nutrition.

In the above biomedical composite material, the cell density may be 1.0 × 10 ⁵ to 5 × 10 ⁵ cells/scaffold, and further preferably 1.5 × 10 ⁵ to 2.5 × 10 ⁵ cells/scaffold.

In the above biomedical composite material, the cell may be a tissue cell isolated from a biological tissue, for example, a hard bone cell, a chondrocyte, an adipocyte, an epithelial cell, or the like, or a stem cell having a differentiation function, such as an embryonic stem cell. Mesenchymal stem cells, etc. However, in a preferred embodiment, the cells are stem cells, and among them, mesenchymal stem cells are preferred because they are easier to obtain. The type of the mesenchymal stem cells is not particularly limited, and may be a mesenchymal stem cell having a differentiation function in any tissue of the living body, and may be, for example, adipose stem cells, bone marrow stem cells, dental pulp stem cells, placental stem cells, or the like.

Furthermore, when the cells contained in the biomedical composite material described above are stem cells, the culture solution may further comprise at least one growth factor to induce stem cells to differentiate toward a specific cell. The growth factor to be used should be determined depending on the type of tissue to be repaired or replaced by the tissue engineering, and any molecule known in the art for promoting cell differentiation is not particularly limited. For example, when the biomedical composite is used to repair a hard bone, the growth factor may include Dexamethasone, β-glycerol phosphate, Ascorbic acid, and 1α, 25-dihydroxyvitamin D3 to promote differentiation of stem cells into hard bone cells. Other types of growth factors can be, for example, epidermal growth factor, transforming growth factor-α, transforming growth factor-β, bone morphogenetic protein, nerve growth factor, fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage Colonic stimulating factor, platelet-derived growth factor, erythropoietin, thrombopoietin, or hepatocyte growth factor, etc., but are not limited thereto.

[Kenatin extraction]

The keratin protein in the present invention is extracted from human hair, and the hair is washed with secondary water, air-dried, and then immersed in a solvent of chloroform/methanol (2:1, v/v) for 12 hours to evaporate the solvent to remove residual hair. Fat. The hair (5 g) was then immersed in a mixed solution containing 25 mM Tris, 2.6 M thiourea, 5 M urea, and 5% 2-mercaptoethanol, and maintained at 50 ° C for three days. . Next, the extraction solution was taken out, dialyzed against 1 liter of water using a MWCO 1 kDa dialysis cassette for 36 hours, and water was changed every 12 hours to obtain keratin.

[Synthesis of azidated chitosan]

Mix 80 mg of 4-azidobenzoic acid (ABA), 1 mL of EDC/NHS (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (47.75 mg) / N-Hydroxysuccinimide (23 mg) in water, and 30 mL Chitosan (400 mg) solution (water/DMSP (1:1, v/v)) and adjusted to pH between 4.5 and 5.5 with 1 M HCl, protected from light and stirred overnight, centrifuged for mixing The solution was taken from the supernatant and dialyzed against 1000 mL of secondary water for ten times, then lyophilized and reconfigured to a solution of 20 mg/mL of azide chitosan.

[Preparation of porous biomedical stent]

A mixed solution of keratin (KE) and azide chitosan (CHI) in different proportions is placed in a 48-well plate, and the mixing ratio of keratin and azide in the mixed solution is as follows. Table 1 shows. 800 μL of the mixed solution was injected into each well of a 48-well plate, and irradiated with UV light (UniVex, T-500UV, 100 mW/cm ² at 365 nm) for photocrosslinking for 30 minutes, followed by freeze-drying to prepare for implementation. Porous biomedical stents of Examples 1-3 and Comparative Examples 1-2. The mixing ratio of keratin to azide chitosan is shown in Table 1.

Table 1  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Keratin (mg/mL) </td><td> Azide chitosan (mg/mL) </td></tr><tr><td> Example 1 </td><td> KECHI/0.5:10 </td><td> 0.5 < /td><td> 10 </td></tr><tr><td> Example 2 </td><td> KECHI/2.5:10 </td><td> 2.5 </td><td > 10 </td></tr><tr><td> Example 3 </td><td> KECHI/5:10 </td><td> 5 </td><td> 10 </td ></tr><tr><td> Comparative Example 1 </td><td> CHI </td><td> - </td><td> 10 </td></tr><tr>< Td> Comparative Example 2 </td><td> KE </td><td> 10 </td><td> - </td></tr></TBODY></TABLE>

[Mechanical strength test]

The porous biomedical stents of the above Examples 1-3 and Comparative Example 1 were placed on a pressure test stage, and a pressure gauge was measured using a liquid crystal push force gauge (FGP Digital Force Gauge, SHIMPO). The Young's coefficient obtained after testing is shown in Table 2.

Table 2  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Example 1 </td><td> Example 2 < /td><td> Example 3 </td><td> Comparative Example 1 </td></tr><tr><td> Young's Coefficient (MPa) </td><td> 0.03892 </td ><td> 0.01942 </td><td> 0.01842 </td><td> 0.03240 </td></tr></TBODY></TABLE>

It can be known from the test results that as the proportion of chitosan azide increases, the mechanical strength of the stent can be increased.

[Porosity test]

The calculation of the porosity of the support is as follows: Porosity = V _p / V _s × 100 (%); where V _p = (W _h - W _o ) / ρ; W _h is the porous biomedical stent after being immersed in water Weight; W _o is the weight of the porous biomedical stent; V _s is the volume of the porous biomedical stent; V _p is the volume of the pores in the porous biomedical stent; ρ is the density of water.

The porosity of the porous biomedical stents of Examples 1-3 and Comparative Example 1 was calculated by the above calculation method, and the results are shown in Table 3.

table 3  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Example 1 </td><td> Example 2 < /td><td> Example 3 </td><td> Comparative Example 1 </td></tr><tr><td> Porosity</td><td> 38.87 (%) </td> <td> 41.77 (%) </td><td> 45.82 (%) </td><td> 38.56 (%) </td></tr></TBODY></TABLE>

The test results shown in Table 3 show that the porosity of the porous biomedical stent increases as the keratin content increases.

[Cell separation and culture]

Human adipose tissue was washed with buffered saline, and human adipose tissue was minced, and finely divided adipose tissue was immersed in a three-in-one containing 1 mg/mL type I collagenase (Gibco, Carlbad, CA) and 1%. The digestion solution of antibiotic (penicillin-streptomycin-amphotericin B solution, Biological industries, Kibbutz Beit Haemek, Israel) was digested at 37 ° C for 60 minutes. The digestion solution was then filtered and centrifuged to obtain primary cells, and suspended in a cell culture medium at a density of 8000 cells/cm ² . The cell culture medium contained DMEM/F12 medium (Hyclone®, Thermo Scientific, Waltham, MA), 10 % fetal bovine serum (FBS, Biological industries), 1 ng/mL basic fibroblast growth factor (bFGF, R&D systems, Minneapolis, MN). Cell passage was performed when the primary cells were cultured to 9 minutes.

[Steps of cell culture in porous biomedical stent]

The porous biomedical stent prepared above was cut to a thickness of 2 mm, and human adipose stem cells were injected into a 20 μL culture medium containing 2×10 ⁵ cells from a different angle of the porous biomedical stent (density of 2×10). ⁵ cells/scaffold), after standing for 3 hours, add 1 mL of cell culture medium to cover the scaffold, culture at 37 ° C, 5% CO ₂ incubator, change the cell culture medium once a day, and carry out 1 period. , 4, 7 days of incubation time test.

[Cell survival test]

The WST-1 reagent (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulonate) was diluted with the culture medium (1:10, v/ v), added to the culture wells of Examples 1-3 and Comparative Example 1 containing culture for 1, 4, and 7 days, 300 μL per well, and allowed to stand at 37 ° C, 5% CO ₂ incubator for 40 minutes. The absorbance (450 nm, reference length 630 nm) was then tested using ELISA and converted to Cell viability. The results of the cell survival test are shown in Fig. 1. It can be seen from the test results that the porous biomedical stent containing keratin in Examples 1-3 is more suitable for cell growth than the chitosan scaffold, and the keratin content is more, such as implementation. Example 3, the more suitable for cell growth.

[Biocompatibility test]

The cells in the scaffolds of Examples 1-3 and Comparative Example 1 were cultured for 1 and 4 days using LIVE/DEAD kit (LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells, ThermoFisher Scientific), and observed using a fluorescence microscope. The staining results are shown in Figure 2. Among them, the green fluorescence is a living cell, and the red fluorescence is a dead cell. It can be observed from FIG. 2 that the cell green fluorescence ratio of the examples 1-3 is higher, indicating that the biocompatibility is better, and the comparative example is better. 1 has more red fluorescence and a higher proportion of cell death, so it shows that Examples 1-3 to which keratin is added have superior biocompatibility compared to Example 1.

[Cell type observation]

In Examples 1-3 and Comparative Example 1, the scaffolds were cultured for 1 day and 4 days, and the cells in the scaffold were fixed with 3.7% formaldehyde for 15 minutes, then 0.1% Triton-X100 was added, and after soaking for 10 minutes, the ghost pen rings were respectively added. Peptides (Phalloidin, 1:300, Thermo) were soaked for 1 hour and DAPI (1:500, Termo) soaked for 5 minutes. Subsequently, it was washed with a buffered physiological saline solution, and observed by a fluorescence microscope, and the result of the staining was as shown in FIG. 3, in which blue fluorescence was a nucleus and red fluorescence was a cytoskeleton. As can be observed from Fig. 3, in the examples 1-3, the cell attachment was better than that of the comparative example 1, and it was shown that the examples 1 to 3 were more suitable for cell attachment growth than the comparative example 1.

Based on the above experimental results, it is shown that the porous biomedical stent provided by the invention has excellent biocompatibility and is favorable for cell attachment, and the internal porosity of the stent is high, providing a large amount of space for cells to grow therein, suitable for tissue engineering or Regenerative medicine.

no.

Figure 1 is a graphical representation of the results of a cell survival assay in accordance with an embodiment of the present invention. 2 is a schematic diagram showing the results of biocompatible cell staining of an embodiment of the present invention. Fig. 3 is a schematic diagram showing the results of immunofluorescence staining of an embodiment of the present invention.

Claims (17)

A method for preparing a porous biomedical stent comprises the steps of: (1) mixing a keratin and a stack of chitosan chitosan in a solvent to prepare a mixture, wherein the azidated chitosan is The following chemical formula shows: Wherein, 300 < n <1200; (2) irradiating a light to the mixture such that the keratin in the mixture is crosslinked with the azide chitosan to obtain a crosslinked mixture; (3) using freeze drying The crosslinked mixture was prepared as a porous biomedical stent. The preparation method according to claim 1, wherein in the step (1), the keratin is extracted from animal hair or nails. The preparation method according to claim 1, wherein in the step (1), the content ratio of the keratin to the azide chitosan is 0.01 to 1. The preparation method according to claim 3, wherein in the step (1), the content ratio of the keratin to the azide chitosan is 0.02 to 0.5. The preparation method according to claim 1, wherein in the step (1), the solvent is water. The preparation method according to claim 1, wherein in the step (2), the light is UV light. A porous biomedical stent comprising a cross-linked keratin and a chitosan chitosan having a pore size of 100 to 500 μm. The biomedical stent according to claim 7, wherein the ratio of the keratin to the azide chitosan is 0.01 to 1. The porous biomedical stent according to claim 8, wherein the ratio of the keratin to the azide chitosan is 0.02 to 0.5. The porous biomedical stent according to claim 7, wherein the biomedical stent has a Young's modulus of 0.01 to 0.05 MPa. The porous biomedical stent according to claim 7, wherein the biomedical stent has a porosity of 30% to 50%. A biomedical composite material comprising a porous biomedical stent; and a cell, wherein the porous biomedical stent is composed of a cross-linked keratin and a chitosan chitosan, and the pore size thereof The cells are 100 to 500 μm; the cell line is cultured on the porous biomedical stent. The biomedical composite material according to claim 12, further comprising a culture solution filled in the porous biomedical stent. The biomedical composite material according to claim 12, wherein the cell has a density of 1.0 × 10 ⁵ to 5 × 10 ⁵ cells/scaffold. The biomedical composite material according to claim 12, wherein the cell line is a stem cell. The biomedical composite material according to claim 15, wherein the stem cell line is a leaf stem cell. The biomedical composite material according to claim 13, wherein the culture solution further comprises at least one growth factor.