Three dimensional tracheal substitute replacing respiratory organs and method of producing the same

Abstract

The present invention relates to a bellows framework having a concave-convex structure on at least one of outer and inner sides using three-dimensional printing technology and a method of producing thereof, and an artificial tracheal replacement comprising an epithelium part formed on the inner side of the bellows framework and an annular cartilage part formed along the circumference of concave-convex grooves on the outer side and a method of producing thereof.

Claims

1. A method of producing an artificial tracheal replacement, comprising preparing a porous bellows framework having a concave-convex structure using three-dimensional printing technology, comprising (a) preparing a thermoplastic polymer solution melt by inputting a thermoplastic polymer into a syringe and heating; and (b) dispensing the thermoplastic polymer through a dispensing head nozzle of a three-dimensional printer by applying a physical force, wherein the dispensing the thermoplastic polymer is performed by printing the thermoplastic polymer solution melt in a dispensing section and a non-dispensing section to produce porous bellows having pores, and wherein the bellows framework is a bellows framework having a surface concave-convex structure which has concave-convex convolutions and grooves in a longitudinal direction on at least one side of outer and inner sides, preparing an annular cartilage part by printing a bio-ink for forming a cartilage part along the circumference of grooves on the outer side of the bellows framework, and preparing an epithelium part by printing a bio-ink for forming an epithelium part on the inner side of the bellows framework.

2. The method of producing according to claim 1, further comprising performing at least a process selected from the group consisting of heating treatment and oxygen plasma treatment, on the produced porous bellows framework.

3. The method of producing according to claim 2, wherein the heating treatment is performed by heating treatment process on the porous bellows framework at 40 to 200° C. for 10 minutes to 60 minutes, and the oxygen plasma treatment process is characterized by treating at 50 to 150 W for 30 minutes to 3 hours.

4. The method of producing according to claim 1, wherein the printing in a dispensing section and a non-dispensing section is controlled by adjusting movement speed of the dispensing head in each section, and the wall thickness and pore formation of the porous bellows framework is controlled by adjusting the distance of the dispensing section and the distance of the non-dispensing section.

5. The method of producing according to claim 1, wherein the dispensing is conducted at an air pressure of 400 to 600 kPa in the dispensing section, or the movement speed of the dispensing head is 60 to 150 nm/min.

6. The method of producing according to claim 1, wherein the length of the non-dispensing section is 300 to 800 μm, and the movement speed of the dispensing head in the non-dispensing section is 200 to 600 mm/min, or the distance between non-dispensing sections is 100 to 1000 μm.

7. The preparation method according to claim 1, wherein the preparation of the cartilage part and preparation of the epithelium part are carried out simultaneously or sequentially.

8. The preparation method according to claim 1, wherein the annular cartilage part and the epithelium part is prepared by printing a bio-ink under the condition of the air pressure of 20 kPa to 200 kPa and the rotation speed of the bellows framework of 3 to 180 DPS (degree per second).

9. The preparation method according to claim 1, wherein the bio-ink comprises a biodegradable hydrogel polymer, and the hydrogel polymer is one or more kinds selected from the group consisting of alginate, gelatin, fibrin, hyaluronic acid, and decellularized tissue derived hydrogel.

10. The preparation method according to claim 9, wherein the bio-ink further comprises one or more kinds selected from the group consisting of a cell, a growth factor, and a non-biodegradable polymer.

11. The preparation method according to claim 1, wherein the preparation method further comprises preparing an absorption prevention part of cartilage part on the outer side of the cartilage part with three-dimensional printing, wherein the absorption prevention part of cartilage part is prepared by dispensing and printing the melt obtained by heating a biodegradable thermoplastic polymer to 60° C. to 200° C., at the air pressure of 400 to 600 kPa, and laminating at least a printing layer with a thickness of 80 to 200 μm.

12. The preparation method according to claim 1, wherein the preparation of the cartilage part and preparation of the epithelium part are carried out sequentially.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

(2) FIG. 1 is a drawing which shows a tubular frame structure of the trachea of a human body.

(3) FIG. 2 is a drawing which shows a tracheal replacement for reconstruction of extensive circular defects of the trachea according to one example of the present invention.

(4) FIG. 3 is a schematic diagram which illustrates a process of preparing a tracheal replacement using a sacrificial layer according to one example of the present invention.

(5) FIG. 4 is a schematic diagram which illustrates a 3D printing process for preparation of a hollow type of porous bellows structure without using a sacrificial layer according to one example of the present invention.

(6) FIG. 5 shows the effect on pore formation according to changes of the temperature of PCL, the transfer speed of the dispensing head, and the length of B section according to one example of the present invention.

(7) FIG. 6 shows the effect on pore formation according to the change of the distance between B sections according to one example of the present invention.

(8) FIG. 7 shows a PCL bellows framework of two dimensions considering preclinical evaluation according to one example of the present invention.

(9) FIG. 8 is a drawing which shows the flexural behavior of the bellows framework according to the change of time of heating treatment at 60° C. according to one example of the present invention.

(10) FIG. 9 is a drawing which shows the effect of oxygen plasma treatment for surface hydrophilicity of the hollow type of rectangular PCL structure in a size of 8×8×13 mm prepared as a rabbit model according to one example of the present invention.

(11) FIG. 10 is a drawing which shows the effect of oxygen plasma treatment for surface hydrophilicity of the hollow type of rectangular structure in a size of 18×18×33 mm prepared as a dog model according to one example of the present invention.

(12) FIG. 11 is an approximate schematic diagram of the method for preparing an artificial tracheal replacement using the three-dimensional printing technology of the present invention. The step of preparation of the bellows support and the step of preparation of the cartilage and epithelium parts are separated from each other, and heating treatment or oxygen plasma treatment for enhancing physical properties of the bellows structure may be performed therebetween.

(13) FIG. 12 shows photographs of cartilage part samples undergoing rotary printing in outer grooves of the PCL bellows framework prepared according to one example of the present invention.

(14) FIG. 13 is a drawing which shows the contraction of the cartilage part at the first day after printing according to one example of the present invention.

(15) FIG. 14 is a drawing which shows the epithelium part undergoing rotary printing (A) and the epithelium part at the third day after printing (B) according to one example of the present invention.

(16) FIG. 15 is the result of the animal experiment for verifying the membrane type of absorption prevention part of cartilage part prepared according to one example of the present invention and its effect.

(17) FIG. 16 is a drawing which shows the inclination angel of bellows framework convolutions, and the length of grooves, and the part of the length of convolutions.

(18) FIG. 17 shows the numerical values as a graph, by comparing relative expression levels of factors related to cartilage differentiation (COL2, ACAN, SOX9) in each group through PCR, after preparing a bellows framework in which each cartilage part is printed by dispensing bio-ink having 4 kinds of cell density (1×10.sup.6/ml (Group I), 2×10.sup.6/ml (Group II), 5×10.sup.6/ml (Group III), 1×10.sup.7/ml (Group IV)) to outer grooves of the PCL bellows framework.

(19) FIG. 18 shows the numerical values as a graph, by comparing relative expression levels of factors related to mucosal tissue differentiation (MUSIN 5AC, KARATIN 14, BETA-TUBULIN) in each group through PCR, after preparing a bellows framework in which each epithelium part is printed by dispensing bio-ink having 4 kinds of cell density (1×10.sup.6/ml (Group I), 2×10.sup.6/ml (Group II), 5×10.sup.6/ml (Group III), 1×10.sup.7/ml (Group IV)) to internal surfaces of the PCL bellows framework.

DETAILED DESCRIPTION OF THE INVENTION

(20) The present invention will be described in more detail by the following examples, but the scope of the present invention is not intended to be limited by the following examples.

Example 1: Test of Conditions of Preparation of a Porous Bellows Framework

(21) In the present invention, for preparation of a porous bellows framework, PCL was used. A certain amount (15˜1,000 mg) of PCL was loaded in a 10 cc stainless syringe in the inside of the dispensing head, and the syringe was heated to 65˜80° C., to melt PCL. The melted PCL was made to be dispensed through a metal nozzle (N20, inner diameter: 200 μm, Musashi Engineering) at the end of the syringe at an air pressure of 500 kPa. After fixing the height of PCL laminating to 100 μm (three layers in total), and the speed of dispensing PCL in sections A and C to 85 mm/min, the formation of pores was observed according to changes of the temperature of PCL, the distance of B section, and the transfer speed in this section (305, 405 and 505 mm/min) (FIG. 5).

(22) As the result of the experiment, it was confirmed that the pores were formed best, when the temperature of melting PCL to 80° C., and the distance of B section to 600 μm, and the transfer speed of the dispensing head in the B section to 305 mm/min. In case of the transfer speed of the dispensing head, there was no big difference in three conditions (305, 405 and 505 mm/min), and to minimize vibrations of the head occurring when the dispensing head transfers rapidly, it is better to select the smallest transfer speed (305 mm/min).

(23) Then, the experiment about how much densely the pores were formed when PCL was loaded as three layers at a height of 100 μm under the established conditions was carried out. The distance of B section of transferring at a speed of 305 mm/min without dispensing PCL was fixed to 600 μm, and the distance between B sections was changed to 100˜600 μm, and the formation of pores was observed (FIG. 6).

(24) As the result of the experiment, it was confirmed that the pores were formed well until the distance between B sections was 100 μm at a minimum. Accordingly, pores can be formed at an interval of at least 400 μm in the hollow wall type of structure. This means that the porosity of the bellows framework can be controlled if needed.

(25) In the present experiment, bellows frameworks of two dimensions were prepared in consideration of preclinical evaluation of the tracheal replacement (animal experiment using rabbit and beagle dog) (FIG. 7). As a result, in the present invention, a bellows framework having various dimensions and porosity as necessary can be prepared by three-dimensional printing technology of the dispensing method.

Example 2: Evaluation of Structural Integrity of the Bellows Framework (Heating Treatment)

(26) In the present experiment, a bellows framework was prepared using PCL, and the melting point of PCL was 60° C. Therefore, for heating treatment of the prepared bellows framework, the internal temperature of a vacuum oven (OV-11, JEIOTECH, Korea) was set to 60° C., and for heating treatment, the bellows framework was stored in the oven for 10˜30 minutes. After that, to evaluate the structural integrity of the bellows framework undergoing heating treatment under each condition, the three-point bending test was performed. In the three-point bending test, to apply bending to the bellows framework, it was conducted by supporting the convolution part at both ends of the bellows framework and pressing the central part between convolutions at both ends (the part in the middle of the bellows framework in a longitudinal direction). In addition, the size of load occurring by pressing the central part of the bellows framework and applying bending was observed.

(27) As shown in FIG. 8, as the time of heating treatment increases, the displacement point at which the bellows framework causes rupture under bending increases. In addition, it can be seen that the condition of heating treatment at 60° C. for 25 minutes is the most effective. Furthermore, it can be confirmed that the difference of the distance-load diagram of each sample under the condition of heating treatment for 25 minutes is the smallest.

Example 3: Evaluation of Hydrophilicity of the Bellows Framework (Hydrophilicity Treatment)

(28) When the bellows framework is hydrophobic, it is a great possibility that the printed epithelium part and cartilage part are separated from the bellows framework in the process of in vitro culture, and therefore, it is needed to increase the hydrophobicity of the bellows framework prepared with material 1 if needed. In the above Example 2, PCL was used in preparation of the bellows framework, and this material has the very high hydrophobicity. Thus, the hydrophilicity of the surface of the bellows framework was increased through a process of oxygen plasma treatment.

(29) At first, considering the preclinical evaluation of the tracheal replacement (animal experiment using rabbit and dog), rectangular cylinder structures of two kinds of dimensions were prepared. In addition, the rectangular cylinder structures prepared using Plasma Cleaner (PDC-001, Harrick Plasma) were subjected to oxygen plasma treatment under each condition (Group I: 100 W+30 minutes treatment, Group II: 100 W+60 minutes treatment, Group III: 100 W+30 minutes treatment+overturning the top and bottom of the structure and then 30 minutes treatment, Group IV: 100 W+60 minutes treatment+overturning the top and bottom of the structure and then 60 minutes treatment). Then, the contact angle at each position (A, B) in the internal and outer surfaces was measured to verify the effect of oxygen plasma treatment (FIG. 9).

(30) In the rectangular cylinder structure of 8×8×13 mm considering the dimension for the rabbit animal model, the longer the oxygen plasma treatment is carried out, the better the hydrophilicity in the internal and outer surfaces becomes (FIG. 9, Groups I and II). In addition, in the same treatment time, by comprising the process of overturning the positions of the top (A) and the bottom (B) of the structure between treatment, the uniformity of the surface hydrophilicity of the total structure is raised (FIG. 9, Groups II and III). However, there is no big difference between oxygen plasma treatment for one hour and two hours (FIG. 9, Groups III and IV), it can be concluded that the oxygen plasma treatment for 1 hour including the process of replacing the positions of the top 9A) and the bottom (B) of the structure (100 W+30 minutes treatment+overturning the top and the bottom of the structure and then 30 minutes treatment) is the most effective (FIG. 9, Group III).

(31) In case of the rectangular cylinder structure in a size of 18×18×33 mm considering the dimension for the dog animal model, as the result of observing the internal and outer surface hydrophilicity in the top, middle and bottom parts of the structure, it could be confirmed that the condition of plasma treatment for 2 hours in Group 11 (100 W+60 minutes treatment+overturning the top and the bottom of the structure and then 60 minutes treatment) had an excellent effect of improving the cylinder surface hydrophilicity than the condition of plasma treatment for 1 hour in Group I (100 W+30 minutes treatment+overturning the top and the bottom of the structure and then 30 minutes treatment) (FIG. 10, Groups I and II).

(32) In this way, by adding the heating treatment and oxygen plasma treatment processes of the bellows framework between the printing process of the bellows framework and the printing process of the epithelium par and the cartilage part, the structural integrity and surface hydrophilicity of the bellows framework prepared earlier were increased. Such an additional treatment process is possible by separating the printing process of the bellows framework and the printing process of the epithelium part and the cartilage part, and therefore it can be added or skipped depending on physical properties of the bellows framework material.

Example 4: Printing the Cartilage Part of the Artificial Trachea

(33) Then, the printing process of the cartilage part and the epithelium part is followed. This printing process uses a motorized rotation stage and a curved nozzle (FIG. 11). At first, the prepared bellows framework is on the rotation stage for printing of the cartilage part, and the dispensing head equipped with the cartilage part material moves, and the end of the curved nozzle is positioned at the outer groove part of the bellows framework. In addition, the bellows on the stage is rotated together as the rotation stage is rotated at a certain speed at the same time when the cartilage part material starts to be dispensed. When the bellows framework rotates 360°, the rotation stage stops to rotate, and at the same time, dispensing the cartilage part material is terminated together. Then, the dispensing head moves and the end of the curved nozzle is positioned at other groove part of the bellows framework, and the material is dispensed and the bellows framework rotates 360°. Such a process is repeated until the cartilage part material is filled in all the outer groove parts of the bellows framework.

(34) As the cartilage material, liquid collagen including human septal chondrocytes (hNSCs) (3% atelocollagen, Therafill, Sewon Cellontech., Seoul, Korea) was used. To secure a sufficient cell number (1×10.sup.7×1×10.sup.8/ml), hNSCs were cultured in vitro in a DMEM culture solution containing 10% fetal bovine serum (FBS) for a certain period, and the culture solution (cell suspension) including hNSCs was mixed with the liquid collagen at a volume ratio of 1:10 so as to have a cell density of 1×10.sup.7/ml, to prepare bio-ink. After loading the prepared bio-ink in a syringe in the dispensing head, it was dispensed through a curved nozzle (CPN-25G-A45, inner diameter: 250 μm, Musashi Engineering). Then, the appropriate cartilage part printing condition was established in the range of the air pressure of 50˜120 kPa and the rotation speed of 4.5˜33 DPS (degree Per Second).

(35) As shown in FIG. 12, the cartilage part material was successively printed in the outer groove of the bellows framework in the various air pressure and rotation speed ranges. It was confirmed that the cartilage part material was flowed down in the air pressure of 70 kPa and the rotation speed of 9 DPS, but it was confirmed that in other conditions, the printed cartilage part material was not flowed down even when dispensing a larger amount of cartilage part materials than the volume of the original outer groove, and it was remained well in the outer groove of the bellows framework.

(36) The bellows framework in which the cartilage part was printed was incubated at 37° C. for about 30 minutes and then was cultured in a 37° C., 5% CO.sub.2 incubator overnight, for cross-linking of collagen. In this process, it was confirmed that the contraction of the printed cartilage part material occurred (FIG. 13) and the cartilage part material printed under the condition of the air pressure of 60 kPa and 10.5 DPS and 9 DPS, and the air pressure of 70 kPa and 27 DPS and 25.5 DPS was contracted to the volume of the outer groove of the bellows framework. Accordingly, in consideration of the level of contraction of the printed cartilage part and the printing time, it can be concluded that the printing condition using the air pressure of 70 kPa and the rotation speed of 25.5 DPS is the most suitable.

(37) In addition, bio-ink was prepared by the same method as above using another collagen used in the clinic (3% atelocollagen, UBIOSIS, Korea), and the cartilage part printing experiment was progressed. The cartilage part printing when using another collagen was performed under the condition of the air pressure of 120 kPa and 12 DPS. (Even if the same collage, if using a different manufacturer's product, the printing condition will be different.)

Example 5: Printing of the Epithelium Part of the Artificial Trachea

(38) Then, the printing process of the epithelium part of the internal surface of the bellows. The end of the curved nozzle of the dispensing head equipped with the epithelium part material is positioned at the groove part in the inside of the bellows framework, and as same as the cartilage part material is printed, the epithelium part material is dispensed and at the same time, the stage rotates 360°. In this way, after the epithelium part material is filled in all the internal groove parts of the bellows framework, the epithelium part material is filled on the surface between the internal groove and groove in the same way.

(39) In the present invention, as the epithelium part material, collagen including human nasal inferior turbinate derived mesenchymal stem cells (hTMSCs) was used. To secure a sufficient cell number (1×10.sup.6˜1×10.sup.7/ml), hTMSCs which were cultured in vitro in a DMEM culture solution containing 10% fetal bovine serum (FBS) for a certain period were mixed with 3% liquid collagen and were loaded in the syringe in the dispensing head, and then were dispensed through the curved nozzle. To print the epithelium part material in the internal groove of the bellows framework, the condition as same as printing of the cartilage part was used (FIG. 14), and to print the epithelium part material on the other internal surface except for the internal groove, the rotation speed two times faster than the printing condition of the cartilage part was used.

Example 6: Printing of the Absorption Prevention Part of Cartilage Part of the Artificial Trachea

(40) 6-1. Printing of the Membrane Type of Absorption Prevention Part of Cartilage Part

(41) Following Example 5, to prevent the internal absorption of the printed cartilage part, printing of the absorption prevention part of cartilage part can be followed. In the present invention, to prepare a membrane type of absorption prevention part of cartilage part, PCL was used. PCL in a certain amount (15˜1,000 mg) was loaded in the 10 cc stainless syringe in the inside of the dispensing head, and the syringe was heated to 65˜80° C., to melt PCL. The melted PCL was made to be dispensed through a metal nozzle (N20, inner diameter: 200 μm, Musashi Engineering) at the end of the syringe at the air pressure of 500 kPa. The membrane is dispensed as drawing a certain pattern so as to have the same pattern with stent when surrounding the tracheal replacement, and this is printed in two layers, or three layers. Then, the second pattern is drawn while dispensing PCL on the first pattern, and this is also printed in two layers or three layers. The height of loading PCL is 100 μm (FIG. 15(A)).

(42) The membrane type of the absorption prevention part of cartilage part was prepared, and the level of cartilage part absorption of the tracheal replacement according to its application was verified through an experiment using a nude mouse. The animal experiment using the nude mouse was performed for two months in total, and the level of absorption of the cartilage part was observed by extracting the tracheal replacement at the first month and second month after grafting the tracheal replacement subcutaneously in the nude mouse. It was confirmed that in case of the tracheal replacement applying the absorption prevention part of cartilage part (Group II), the volume of the cartilage part printed initially was maintained well. However, it could be seen that the cartilage part was significantly absorbed and the volume of the cartilage part was significantly reduced, when grafting only the tracheal replacement without the absorption prevention part of cartilage part to the nude mouse subcutaneously (Group I) (FIG. 15(A)).

(43) 6-2. Printing of a Tube Type of Absorption Prevention Part of Cartilage Part

(44) To prepare a tube type of absorption prevention part of cartilage part, PCL was used. PCL in a certain amount (15˜1,000 mg) was loaded in the 10 cc stainless syringe of the inside of the dispensing head, and the syringe was heated to 65˜80° C., to melt PCL. The melted PCL was dispensed through a metal nozzle (N20, inner diameter: 200 μm, Musashi Engineering) at the end of the syringe at the air pressure of 500 kPa. The syringe was positioned on the rotation axis according to the initial position set in advance, and for preparation of the tube type, PCL was dispensed in a longitudinal direction of the rotation axis according to the code. At the same time, for forming a stent type of pattern, the rotation axis was stopped and rotated according to the code. Then, the second pattern was made by dispensing PCL on the first pattern, and it was printed in two layers or three layers. Each height at which PCL is laminated is 100 μm.

Example 7: Cell Content of the Cartilage Part and the Epithelium Part

(45) In printing of the cartilage part and the epithelium part, chondrocytes, stem cells and the like which facilitates cartilage formation as the cartilage part material can be comprised, and mucosal cells, stem cells and the like as the epithelium part material can be comprised. In this case, at first, to carry out rotary printing of the cartilage part and the epithelium part of the tracheal replacement, securing a sufficient amount of cell number is necessary, and since the effect of tissue regeneration of the cartilage part and the epithelium part can be affected depending on the concentration of cells comprised in the printed cartilage part or epithelium part, it is needed to determine the optimal content of cells showing the most excellent tissue regeneration effect while considering the rotary printing efficiency.

(46) Accordingly, to determine the optimal content of stem cells comprised in the cartilage part and epithelium part, the following experiment was carried out.

(47) 7-1. Determination of the Optimal Cell Content of the Cartilage Part of Tracheal Replacement

(48) To secure a sufficient cell number (1×10.sup.6˜1×10.sup.7/ml), hNSCs (Human neural stem cells) which were cultured in vitro in a DMEM culture solution containing 10% fetal bovine serum (FBS) for a certain period were mixed with 3% liquid collagen, to prepare bio-ink having 4 kinds of cell density in total (1×10.sup.6/ml (Group I), 2×10.sup.6/ml (Group II), 5×10.sup.6/ml (Group III), 1×10.sup.7/ml (Group IV)). After loading the prepared bio-ink in the syringe in the inside of the dispensing head, it was dispensed to the outer groove part of the PCL bellows framework through the curved nozzle. In this experiment, the PCL bellows framework having only one outer groove was used.

(49) The bellows framework of which cartilage part was printed was cultured in a 37° C., 5% CO.sub.2 incubator for one week, after keeping it at 37° C. for about 30 minutes for cross-linking of collagen. Then, the DMEM culture solution containing 10% fetal bovine serum (FBS) was newly replaced per 2˜3 days. After that, to confirm the effect of cartilage regeneration by each cell content, the expression levels of each factor related to cartilage differentiation (COL2, ACAN, SOX9) were compared by polymerase chain reaction (PCR), and then, the expression levels of each factor related to cartilage differentiation were represented by relative values to the expression of GAPDH which was used as a control gene (See FIG. 17).

(50) As shown in FIG. 17, as the result of comparing the relative values of factors related to cartilage differentiation by each group (Group I to Group IV), it was shown that the expression was highest in the group printed with the bio-ink having the cell content of 2×10.sup.6/ml (Group II). Therefore, it could be seen that it was most suitable to comprise the cell content of 2×10.sup.6/ml, when using bio-ink comprising hNSCs, in preparation of the tracheal replacement support by printing the cartilage part on the porous bellows surface of the present invention.

(51) 7-2. Determination of the Optimal Cell Content of the Epithelium Part of the Tracheal Replacement

(52) To secure a sufficient cell number (1×10.sup.6˜1×10.sup.7/ml), hTMSCs (Human turbinate mesenchymal stromal cells) which were cultured in vitro in a DMEM culture solution containing 10% fetal bovine serum (FBS) for a certain period were mixed with 3% liquid collagen, to prepare bio-ink having 4 kinds of cell density in total (1×10.sup.6/ml (Group I), 2×10.sup.6/ml (Group II), 5×10.sup.6/ml (Group III), 1×10.sup.7/ml (Group IV)). After loading the prepared bio-ink in the syringe in the inside of the dispensing head, it was dispensed to the outer groove part of the PCL bellows framework through the curved nozzle. In this experiment, the PCL bellows framework having only one outer groove was used.

(53) The bellows framework of which epithelium part was printed was cultured in a 37° C., 5% CO.sub.2 incubator for one week, after keeping it at 37° C. for about 30 minutes for cross-linking of collagen. Then, the DMEM culture solution containing 10% fetal bovine serum (FBS) was newly replaced per 2˜3 days. After that, to confirm the effect of mucosal tissue regeneration by each cell content, the expression levels of each factor related to mucosal tissue differentiation (MUSIN 5AC, KARATIN 14, BETA-TUBULIN) were compared by polymerase chain reaction (PCR). Then, the expression levels of each marker were represented by relative values to the expression of GAPDH which was used as a control gene (See FIG. 18).

(54) As shown in FIG. 18, the relative expression of MUSIN 5AC was shown as the highest in the group printed with the bio-ink having the cell content of 1×10.sup.6/ml (Group I) among factors related to mucosal tissue differentiation by each group, but in case of KARATIN 14 and BETA-TUBULIN, the group printed with the bio-ink having the cell content of 2×10.sup.6/ml (Group II) was the highest. Therefore, it could be seen that it was most suitable to comprise the cell content of 2×10.sup.6/ml, when using bio-ink comprising hTMSCs, in preparation of the tracheal replacement support by printing the epithelium part on the porous bellows surface of the present invention.