KR102214090B1 - Development of 3d polymeric hybrid structures for substitution of acellular dermal matrix on breast reconstruction - Google Patents

Abstract

The present invention relates to a polymer composite structure for replacing the cell-free allogeneic dermis for breast reconstruction.
In the present invention, a polymer composite structure is manufactured using a 3d printing technique and an electrospinning technique, and it is possible to replace the conventional cell-free allogeneic dermal formulation, which is expensive and difficult to maintain a limited supply quantity and quality. In addition, the polymer composite structure according to the present invention is used as an essential medical device for breast reconstruction surgery combined with implant implantation and autologous fat graft because it is easy to fix the human body by using sutures, does not cause inflammation in the body, and has excellent mechanical properties. It is possible.

Description

DEVELOPMENT OF 3D POLYMERIC HYBRID STRUCTURES FOR SUBSTITUTION OF ACELLULAR DERMAL MATRIX ON BREAST RECONSTRUCTION}

The present invention relates to the development of a three-dimensional polymer composite structure for the replacement of the cell-free allogeneic dermis for breast reconstruction.

Breast reconstruction is a plastic surgery to restore the original breast shape in female patients who have undergone mastectomy for breast cancer treatment. According to the Korean Breast Cancer Association, breast cancer is reported to occur in 13.5% of women in their 30s and 38.1% of women in their 40s. This phenomenon seems to be caused by the increase in early detection of breast cancer due to the development of breast cancer screening cycle and diagnostic technology. However, it is reported that most women suffer from severe mental distress after breast cancer treatment due to poor quality of life due to breast cancer incidence.

Breast reconstruction performed to improve the quality of life of these women has been performed with autologous flap and silicone artificial breast implants after determining oncological safety. In the case of autologous flap surgery, there are disadvantages of additional scarring on the donor, asymmetry of the left and right breasts, and a long recovery period after surgery. Therefore, in recent years, there is a trend that many simple silicone artificial breast implants are performed with few side effects such as muscle damage. In addition, in the process of silicone breast implantation, a cell-free allodermis is used to wrap the implant or fix and suture the human body after the silicone breast is inserted to increase the side effects and plasticity satisfaction that accompany the surgery.

The cell-free alloderm is a medical device for dermal regeneration manufactured by decellularizing a dermal tissue obtained from the skin of a corpse donated abroad, and is a medical device that can be applied to various skin damage treatments. The cell-free allodermis has disadvantages that are considerably expensive compared to other medical devices, limited supply, and difficult to maintain a certain quality. Therefore, there is a need for a new technology that can replace the cell-free allodermis used in breast reconstruction.

On the other hand, the polymer nanofiber nonwoven fabric manufactured by the electrospinning method has a nonwoven fabric form in which nanofibers having a diameter of 1 um or less are entangled. The nanofiber nonwoven fabric may adjust cell affinity and mechanical properties according to the polymer type of the nanofiber. In particular, in the case of fabricating a nanofiber nonwoven fabric using a highly biocompatible polymer, it can be applied as a support for cell culture, so many studies using this have been conducted. Synthetic polymers are used as such biocompatible polymers, and unlike natural polymers, the synthetic polymers have excellent mechanical properties and have a uniform molecular weight, so that it is easy to maintain quality when developing medical devices using them. In particular, among synthetic polymers, polycaprolactone is known as a biocompatible polymer that is completely absorbed by sugar metabolism in the human body, and it is widely used as an implant for human insertion. However, when polycaprolactone is manufactured in the form of a nanofiber nonwoven fabric using an electrospinning technique, it is difficult to manufacture a nonwoven fabric having a thickness of 500 um or more, and there is a limitation in that it is impossible to fix in the body using a suture.

1. Korean Patent Registration No. 10-1127112 2. Korean Patent Registration No. 10-1747387

An object of the present invention is to solve the above-described problem, and an object of the present invention is to provide a medical device in the form of a biocompatible polymer composite structure capable of replacing the cell-free allodermis used in conventional breast reconstruction.

The present invention is a three-dimensional polymer composite; And

Including a nanofiber layer formed on one surface of the three-dimensional polymer composite,

The three-dimensional polymer composite and the nanofiber layer each contain a biodegradable polymer,

The three-dimensional polymer composite has a circular grating structure including two or more circular lines, and at least one straight line is arranged in a radial shape based on the center of the circular grid. It provides a three-dimensional polymer composite structure.

The present invention also includes the steps of (S1) 3d printing a first polymer solution containing a biodegradable polymer to prepare a three-dimensional polymer composite; And

(S2) comprising the step of electrospinning a second polymer solution containing a biodegradable polymer on one surface of the three-dimensional polymer composite to prepare a nanofiber layer,

The three-dimensional polymer composite has a circular grating structure including two or more circular lines, and at least one straight line is arranged in a radial shape based on the center of the circular grid. It provides a method of manufacturing a three-dimensional polymer composite structure.

The three-dimensional polymer composite structure according to the present invention can be manufactured using a 3D printing technique and an electrospinning technique, and can replace the conventional cell-free allogeneic dermal preparation, which is expensive and difficult to maintain a limited supply quantity and quality.

In addition, the three-dimensional polymer composite structure according to the present invention is easy to fix in the body using sutures, can wrap an artificial breast implant, and has excellent mechanical properties without causing inflammation in the body, making it an essential medical device for breast reconstruction surgery. Can be used.

In addition, in the present invention, autologous adipose tissue is supported inside the polymer composite structure and inserted like an artificial breast implant, so that side effects caused by surgery can be minimized through fusion with surrounding adipose tissue, and cosmetic/plastic effects can be maximized. I can.

1 is a schematic diagram showing the manufacturing process of the three-dimensional polymer composite structure of the present invention and a photograph of the three-dimensional polymer composite structure prepared in the embodiment of the present invention.
FIG. 2 shows photographs of a 3D polymer composite structure prepared in an Example of the present invention and a 3D polymer composite prepared in Comparative Example, silicone and cell-free allodermis (ADM, ALLODERM, Allergan, USA).
3 shows a measurement method and a calculation formula for analyzing the transmittance strength of the three-dimensional polymer composite structure prepared in the embodiment of the present invention.
4 shows the results of measuring the permeation strength of the three-dimensional polymer composite structure prepared in Example of the present invention and the three-dimensional polymer composite prepared in Comparative Example, silicone and cell-free allodermis.
5 shows the surface of the three-dimensional polymer composite structure prepared in the embodiment of the present invention and an image at a 45-degree angle.
6 and 7 show cell affinity evaluation results obtained after culturing fat-derived stem cells on the surface of the three-dimensional polymer composite structure prepared in the example of the present invention.
8 and 9 show the results of evaluating lipodifferentiation of adipose-derived stem cells cultured in the three-dimensional polymer composite structure prepared in the example of the present invention.
10 shows the results of analyzing the degree of fat differentiation of adipose-derived stem cells cultured in the three-dimensional polymer composite structure prepared in the example of the present invention through qPCR.
11 and 12 show the results of observing and quantifying the degree of fat differentiation of adipose-derived stem cells cultured in the three-dimensional polymer composite structure prepared in the embodiment of the present invention through a confocal fluorescence microscope.
13 and 14 are to analyze the biocompatibility of the three-dimensional polymer composite structure prepared in the embodiment of the present invention, the three-dimensional polymer composite structure under the dorsal skin of the SD rat over 1 to 3 weeks with a silicone implant. The results of animal experiments obtained by transplanting together are shown.
Figure 15 is to analyze the possibility of simultaneous application to breast implant implantation and autologous fat grafting of the three-dimensional polymer composite structure prepared in the embodiment of the present invention, collected from the groin of SD rats subcutaneously over 1 to 4 weeks. This is the animal test result obtained by transplanting one autologous fat, a three-dimensional polymer composite structure, and a silicone implant together (the result of analyzing the activity of fat).

The present invention is a three-dimensional polymer composite; And

It relates to a three-dimensional polymer composite structure including a nanofiber layer formed on one surface of the three-dimensional polymer composite.

In an embodiment of the present invention, the surface shape and physical properties of the 3D polymer composite structure are analyzed, and after culturing fat-derived stem cells in the 3D polymer composite structure, affinity with the cells and the ability to induce fat differentiation are analyzed. I did. Through this, it was confirmed that the three-dimensional polymer composite structure has improved physical properties compared to the case of using the conventional nanofiber nonwoven fabric, and has excellent cell affinity and ability to induce fat differentiation. In addition, the possibility of application to breast reconstruction combined with implant implantation and autologous fat grafting of the 3D polymer composite structure was confirmed.

Hereinafter, the three-dimensional polymer composite structure of the present invention will be described in more detail.

In the present invention, the average diameter of the three-dimensional polymer composite structure may be 10 to 15 cm or 12 to 15 cm. In the above diameter range, when used as a graft material for human breast reconstruction, the breast implant can be wrapped, and mechanical properties can be maintained when the peripheral muscle is sutured. In the examples of the present invention, the average diameter was made to be 1.5 cm for cell and animal experiments.

In addition, the thickness of the three-dimensional polymer composite structure may be 1.0 to 5.0 mm or 1.5 to 4.0 mm. In the case of the conventional nanofiber nonwoven fabric, since it is impossible to manufacture a nonwoven fabric having a thickness of 500 um or more, it was impossible to fix it in the body using a suture. In the present invention, by providing a three-dimensional polymer composite structure having the above thickness range, it is possible to overcome the limitations of the conventional nanofiber nonwoven fabric, and fix it in the body using a suture.

In the present invention, the 3D polymer composite may support a nanofiber layer to be described later, and may impart strength to the 3D polymer composite structure.

The three-dimensional polymer composite includes a biodegradable polymer, and specifically may include a biodegradable synthetic polymer. Compared with natural polymers, the biodegradable synthetic polymer has excellent mechanical properties and has a uniform molecular weight, so that it is easy to maintain quality when applied as an in vivo implant, that is, a medical device. In addition, cell affinity and mechanical properties can be adjusted according to the type of polymer.

In one embodiment, the biodegradable polymer is polycaprolactone (Polycaprolactone), polylactic acid (Poly (l-lactic acid)), poly glucolic acid (Poly (glycolic acid)), polylactic-glucolic Seed copolymer (Poly (lactic-co-glycolic acid)), polylactic-caprolactone copolymer (Poly (L-Lactide-co-ε-Caprolactone)) and a mixture thereof, including at least one selected from the group consisting of It can be, and specifically, it may be polycaprolactone (Polycaprolactone). Since the polycaprolactone is completely absorbed by sugar metabolism in the human body, it can be easily applied to the three-dimensional polymer composite structure of the present invention.

In addition, in one embodiment, the molecular weight (Mw) of the biodegradable polymer may be 10,000 to 100,000 to 10,000 to 800,000. In the case of having a low molecular weight, since there is a risk of toxicity and the decomposition rate becomes too fast, it is preferable to use a polymer having a molecular weight in the above range.

In the present invention, the surface of the 3D polymer composite, that is, the surface on which the nanofiber layer is formed, has a circular grating structure including two or more circular lines. In the present invention, "grating structure" means a pattern having a periodic or quasi-periodic structure, and the grid structure includes a liner grating, a circular grating, a radial grating, and an EC A grating (elongated circular grating) or a matched radial parallel grating (MRP). In addition, a circular grating structure means a pattern composed of two or more circular lines based on a center point. In this case, the circle is not limited to a perfect circle, and may mean any shape without an angle, including an ellipse. In the present invention, when a three-dimensional polymer composite having a circular grid structure is used as an implant material for breast reconstruction, the breast implant can be stably wrapped, and physical properties suitable for use can be provided.

In one embodiment, the circular grid structure may include 20 to 30 or 23 to 27 circular lines. The number of circular lines may be appropriately adjusted according to the required purpose of the object to which the 3D polymer composite structure is applied. In the embodiment of the present invention, five circular lines were prepared for cell and animal experiments.

In one embodiment, the length of the cross section of the circular line may be 100 to 1,000 um or 200 to 500 um. If the length is less than 100 um, the structure of the structure cannot be maintained, and physical properties such as strength are too low, which is not suitable for use as a graft material. In addition, if it exceeds 1,000 um, there is a fear that the elasticity will be reduced.

In one embodiment, an interval between one circular line and an adjacent circular line in two or more circular lines may be 500 to 10,000 um or 500 to 5,000 um. Both strength and elasticity are excellent in the spacing range. When two or more intervals between the circular lines exist, the intervals may be the same, and an error of the interval may be 20% or less, 10% or less, or 5% or less. Those having the same spacing may have uniform properties (physical properties and cellular reactivity when applied to the body) throughout the 3D polymer composite structure.

In the present invention, the circular grid may have one or more straight lines arranged in a radial shape based on the center of the circular grid. The straight line helps maintain the structure of the polymer support and may play a role in controlling physical properties such as strength and elasticity.

In one embodiment, the straight line may be formed of 2 to 12, or 4 to 10. The straight lines may be formed in an even number, and the straight lines may be arranged radially symmetrically. In addition, when n straight lines are formed, an angle between the radially formed straight lines may be 360 degrees/n degrees.

In one embodiment, the length of the straight cross section may be 100 to 1,000 um or 200 to 500 um. If the length is less than 100 um, the structure of the structure cannot be maintained, and physical properties such as strength are too low, which is not suitable for use as a graft material. In addition, if it exceeds 1,000 um, there is a fear that the elasticity will be reduced.

In addition, in one embodiment, the average diameter of the 3D polymer composite may be 10 to 15 cm, and the value may be the same as the 3D polymer composite structure.

In addition, the thickness of the three-dimensional polymer composite may be 0.5 to 10 mm. In the above thickness range, physical properties as an implantable material can be retained, and stable formation and attachment of the nanofiber layer is possible.

In the present invention, the nanofiber layer is formed on one side of the three-dimensional polymer composite.

The nanofiber layer may include a biodegradable polymer, and specifically may be a biodegradable synthetic polymer, and polycaprolactone, poly(l-lactic acid), polyglucolic acid (Poly( glycolic acid)), polylactic-glucolic acid copolymer (Poly(lactic-co-glycolic acid)), polylactic-caprolactone copolymer (Poly(L-Lactide-co-ε-Caprolactone)) and their It may include one or more selected from the group consisting of mixtures. In the present invention, the same polymer as the biodegradable polymer included in the 3D polymer composite may be used as the biodegradable polymer, and adhesion or adhesion between the nanofiber layer and the 3D polymer composite may be improved.

In one embodiment, the diameter of the nanofiber layer may be the same as the diameter of the three-dimensional polymer composite structure and the diameter of the three-dimensional polymer composite described above. In addition, the thickness of the nanofiber layer may be 100 um to 500 um. It can be easily used as a graft material for breast reconstruction within the above thickness range, and has excellent cell reactivity.

In addition, in one embodiment, the average diameter of the cross section of the nanofibers forming the nanofiber layer may be 300 to 600 nm. That is, the nanofiber layer has a nonwoven fabric form in which nanofibers having an average diameter of 300 to 600 nm are entangled.

In addition, the three-dimensional polymer composite structure according to the present invention may include adipose tissue. In one embodiment, the adipose tissue may be located on one side of the three-dimensional polymer composite in which the nanofiber layer is not formed. The adipose tissue is implanted into the body together with the three-dimensional polymer composite structure to maintain the activity of the transplanted adipose tissue, has excellent affinity with surrounding tissues, and no inflammatory reaction. In addition, it can withstand the movement and load of the breast implant used for breast reconstruction.

The three-dimensional polymer composite structure manufactured according to the present invention can be used as an internal implant, and specifically, can be used as a medical device as an implant for breast reconstruction.

Medical devices using biodegradable polymers 1) must not cause inflammation when inserted into the body, 2) have excellent biocompatibility and fusion with surrounding adipose tissue, and 3) mechanical properties when enclosing a breast implant or suturing the surrounding muscles. Should be maintained. The three-dimensional polymer composite structure according to the present invention satisfies all the above conditions and can be easily used as a medical device.

In one embodiment, when the three-dimensional polymer composite structure is used as an implant material for breast reconstruction, the nanofiber layer of the three-dimensional polymer composite structure may contact the breast implant.

In one embodiment, the puncture strength of the 3D polymer composite structure may be 1.5 MPa or more or 1.7 MPa or more. In the above range, both strength and elasticity are excellent and can be easily applied as a living body implant. At this time, the transmittance intensity represents a value measured using a texture analyzer (TA XT Plus, UK).

In addition, the present invention may further include an additional configuration for fixing the three-dimensional polymer composite structure to the human body.

In addition, the three-dimensional polymer composite structure according to the present invention can be applied for delivery/fixation to local parts of the human body for cosmetic/plastic purposes in addition to the aforementioned implant for breast reconstruction.

In addition, the present invention relates to a method of manufacturing the above-described three-dimensional polymer composite structure.

The manufacturing method of the present invention comprises the steps of (S1) 3D printing a first polymer solution containing a biodegradable polymer to prepare a three-dimensional polymer composite; And

(S2) electrospinning a second polymer solution containing a biodegradable polymer on one surface of the three-dimensional polymer composite to prepare a nanofiber layer.

In the present invention, step (S1) is a step of preparing a three-dimensional polymer composite by 3d printing a first polymer solution containing a biodegradable polymer.

Through the above steps, a three-dimensional polymer composite structure having a circular grid structure including two or more circular lines, and in which one or more straight lines are radially arranged based on the center of the circular grid can be prepared. The circular grid structure may be the circular grid described above in the three-dimensional polymer composite structure.

In one embodiment, the first polymer solution may be used by preparing the first polymer in a liquid state by heat without using a separate solvent.

In one embodiment, 3d printing can be used without limitation, a method generally used in the art.

Specifically, during 3d printing, a filament is ejected from a nozzle, and the filament may be manufactured as a three-dimensional polymer composite according to a preset structure. That is, the three-dimensional polymer composite is a laminate of filaments. The height of the 3D polymer composite may be determined according to the number of stacked filaments, and the number of stacking may be determined according to the desired physical properties and application purpose.

In addition, the 3d printing conditions can be appropriately adjusted according to the shape and size of the 3D polymer composite to be manufactured. For example, the nozzle diameter may be 0.1 to 0.5 mm, the nozzle temperature may be 80 to 90 °C, The discharge pressure may be 10 to 100 psi or 50 to 100 psi, and the nozzle movement speed may be 1 to 5 mm/s or 2 to 5 mm/s. It is possible to prepare a three-dimensional polymer composite and a composite structure having the desired physical properties within the above range.

In the present invention, step (S2) is a step of preparing a nanofiber layer by electrospinning a second polymer solution containing a biodegradable polymer on one surface of the three-dimensional polymer composite prepared in step (S1).

In one embodiment, the solvent of the second polymer solution is acetone (acetone), acetic acid (acetic acid), dichloromethane (dichloromethane), chloroform (chloroform), dimethylformamide (dimethylformamide), hexafluoroisopropyl alcohol (1,1,1,3,3,3-hexafluoro-2-isopropanol), formic acid, or a mixed solvent thereof.

In one embodiment, electrospinning can be used without limitation, a method generally used in the art.

In one embodiment, the electrospinning conditions, that is, the applied voltage, the concentration of the polymer solution, and the discharge rate may be appropriately adjusted according to the thickness of the nanofiber layer to be prepared and the diameter of the nanofiber, and for example, the applied voltage is 5 to It may be 20 kV or 10 to 15 kv, the discharge rate may be 0.1 to 5 ml/hr or 0.5 to 2 ml/hr, and the distance between the syringe and the nanofiber layer collector may be 10 to 30 cm.

Through the above step, a nanofiber layer is formed on one surface of the 3D polymer composite (preparation of a 3D polymer composite structure), and the 3D polymer composite and the nanofiber layer may have excellent adhesion and adhesion.

In addition, the present invention may further include a step of supporting adipose tissue on the three-dimensional polymer composite structure prepared in step (S2). The adipose tissue may be supported on a three-dimensional polymer composite in which the nanofiber layer of the three-dimensional polymer composite structure is not formed.

In the above step, the adipose tissue may be collected by liposuction. By carrying the adipose tissue, when transplanted into the body, the activity of the transplanted adipose tissue is maintained, the affinity with the surrounding tissue is excellent, and the inflammatory reaction can be reduced.

The present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and those skilled in the art can understand that various modifications, modifications, or applications are possible without departing from the technical matters derived by the matters described in the appended claims. will be.

Example

Example 1. Preparation of three-dimensional polymer composite structure

(1) Preparation of 3D polymer composite using 3d printing

A three-dimensional (3d) polymer composite was prepared using a 3d printer (Biobot, USA).

Polycaprolactone (Sigma Aldrich, USA) was used as a polymer, and the nozzle diameter was 0.1~0.5 mm, the nozzle temperature was 80~90℃, the discharge pressure was 50~100 psi, and the nozzle movement speed was 2~5 mm/s. A three-dimensional polymer composite having the structure as shown in FIG. 1 was prepared. The prepared three-dimensional polymer composite had a structure in which eight straight lines were radially formed based on the center of a circular grid (an angle of 45° between the straight lines).

The prepared 3D polymer composite was washed with 70% ethanol for about 30 minutes to remove foreign substances and dried.

(2) Fabrication of 3D polymer composite structure using electrospinning method

The polycaprolactone nanofibers were collected and adhered to one side of the 3D polymer composite prepared by 3d printing in (1) by electrospinning as shown in FIG. 1 to prepare a 3D polymer composite structure.

Specifically, a polymer solution was prepared by dissolving 6% by weight of polycaprolactone in hexafluoroisopropyl alcohol ((1,1,1,3,3,3-hexafluoro-2-isopropanol, TCI, Japan). , A three-dimensional polymer composite structure was prepared by electrospinning a polymer solution onto a three-dimensional polymer composite under conditions of an applied voltage of 12 kv, a discharge rate of 1 ml/hr, and a distance of 10-20 cm between the syringe and the nanofiber layer collector.

The residual solvent was removed by drying at room temperature for 12 hours, and then foreign substances were additionally removed for 6 hours using a 70% ethanol solution.

The surface shape, physical properties, cell reactivity, and lipodifferentiation induction ability of the prepared three-dimensional polymer composite structure were evaluated, and biocompatibility and applicability to breast reconstruction were analyzed through animal experiments.

Comparative Example 1. Preparation of a 3D polymer composite using 3d printing

A three-dimensional polymer composite was prepared using the method of Example 1 (1).

At this time, the three-dimensional polymer composite is based on the center of the circular grid, each of 4 straight lines (angle between straight lines 90°), 6 straight lines (angle between straight lines 60°) and 8 straight lines (angle between straight lines) 45°). These were expressed as 3D PCL (90°), 3D PCL (60°), and 3D PCL (45°), respectively, and were used as a control group.

Comparative Example 2. Preparation of polycaprolactone nanofibers using electrospinning

Polycaprolactone nanofibers were prepared by the electrospinning method of Example 1 (2).

Comparative Example 3. Preparation of silicone film for simulating breast implants

Silicone (Polydimethylsiloxane, SYLGARD® 184 SILICONE ELASTOMER, Dow Corning, USA), a raw material for breast implants, was mixed with a curing agent in a weight ratio of 10:1, and thermally polymerized at 60°C for 12 hours to prepare a silicone film.

Comparative Example 4. Cell-free allogeneic dermis

A commercially available cell-free alloderm (ALLODERM, Allergan, USA) was used.

Experimental example. Characterization of 3D polymer composite structure

(1) Analysis of the physical properties of the 3D polymer composite structure

In order to analyze the physical properties of the three-dimensional polymer composite structure, transmission strength was measured.

To this end, as shown in Figure 2, the three-dimensional polymer composite structure (3D PCL / NFs or 3D PCL (45 °) / NFs) prepared in Example 1, the three-dimensional polymer composite prepared in Comparative Example 1 (3D PCL (90°), 3D PCL (60°), 3D PCL (45°)), polycaprolactone nanofibers (PCL NFs) prepared in Comparative Example 2, silicone film prepared in Comparative Example 3 (Silicone), and Comparative Example Cell-free allogeneic dermis (ADM) of 4 was used as each group, and permeation intensity was measured using a texture analyzer (TA XT Plus, UK) for 5 samples for each group.

The transmission strength measurement method and calculation formula are shown in FIG. 3.

The measurement results are shown in FIG. 4 and Table 1 below.

4 and Table 1, polycaprolactone nanofibers (PCL NFs) of Comparative Example 2 had a transmittance of about 0.4 MPa, whereas the three-dimensional polymer composite of Comparative Example 1 transmitted as the number of straight lines increased. It can be seen that the strength increased from 2 to 3 times.

In addition, when comparing the three-dimensional polymer composite structure (3D PCL/NFs) and 3D PCL (45°) of Example 1 having the same number of straight lines, the example in which the nanofibers were additionally bonded showed about 17% or more physical properties. It could be confirmed that it increased.

In particular, as a result of measuring the permeation strength after soaking the cell-free allodermis on the market in physiological saline, it showed a permeation strength of about 63% compared to silicone, whereas the three-dimensional polymer composite structure of the present invention was about 150% than that of silicone. Above showed an increased transmission strength. Through this, it was confirmed that the three-dimensional polymer composite structure of the present invention has suitable physical properties to physically support the breast implant.

(2) Analysis of microscopic shape of 3D polymer composite structure

In order to analyze the shape of the three-dimensional polymer composite structure prepared in Example 1, an electron microscope (GeminiSEM 500, ZEISS, Germany) was used to photograph the upper, lower, and 45-degree distorted side images as shown in FIG. 5. In FIG. 5, (A) to (C) are upper, (D) to (F) are lower, and (G) to (I) are side images.

As shown in FIG. 5, the three-dimensional polymer composite structure can be confirmed that the nanofibers and the three-dimensional polymer composite are combined. As a result of taking pictures at a magnification of 10,000 times by specifying the two upper and lower portions, polycaprolactone nanofibers formed by electrospinning can be observed, and it can be seen that the diameter of the nanofibers is 300 to 600 nm. (Fig. 5 (A) to (F)).

In addition, if you look at the side image that is distorted by 45 degrees, you can see the bonding of the 3D polymer composite structure. Specifically, it can be seen that the porous polycaprolactone nanofibers are stably adhered to the three-dimensional polymer composite (FIGS. 5 (G) to (I)).

(3) Evaluation of cellular reactivity of the three-dimensional polymer composite structure

In order to analyze the cellular effect of the 3D polymer complex structure, adipose tissue-derived stem cells were cultured on the sample surfaces of Example 1 and Comparative Example 1 to evaluate the reactivity of the cells.

Specifically, after fixing a medical silicone film (Comparative Example 3, silicone) simulating a breast implant on the bottom surface of a tissue culture plate (TCP, Corning, USA), the three-dimensional polymer composite structure of Example 1 (3D PCL ( 45°)/NFs) and the three-dimensional polymer complex (3D PCL (45°)) of Comparative Example 1 were placed, and 5000 adipose-derived stem cells (ADSCs, number of passages: No. 4, Lonza, USA) were cultured. The effect of each sample on adipose-derived mesenchymal stem cells over the next 7 days was analyzed using a cell counting kit (CCK-8, Dojindo Molecular Technologies, JAPAN) and a confocal fluorescence microscope (LSM700, Zeiss, Germany). At this time, 10 v/v% Fetal bovine serum (FBS, Welgene, Korea) and 1 v/v% antibiotics (Welgene, Korea) are mixed in Dulbecco's Modified Eagle Medium (DMEM, low glucose, Invitrogen, USA) as a cell culture medium. The cultured medium was used, and the cells were cultured under conditions of 5% carbon dioxide at 37°C, and the cells were observed and analyzed over 1, 4, and 7 days after the start of the culture.

In addition, adipose-derived stem cells were cultured on TCP or silicon film as a control group.

The analysis results are shown in FIGS. 6 and 7.

As shown in FIG. 6, on the first day of culture, compared to the activity of the adipose-derived stem cells cultured in TCP, the activity of the silicon film and the cells cultured in the samples of Example 1 and Comparative Example 1 was about 30%, A statistical difference was found between the silicone film and the three-dimensional polymer composite structure of Example 1 (3d PCL(45°)/NFs on silicone) and the three-dimensional polymer composite of Comparative Example 1 (3d PCL(45°) on silicone). I couldn't. This trend was maintained until the 7th day of culture.

In addition, as shown in Fig.7, similar between the silicone film and the three-dimensional polymer composite structure of Example 1 (3d PCL (45 °) / NFs) and the three-dimensional polymer composite of Comparative Example 1 (3d PCL (45 °)) It can be confirmed that it has cell affinity.

(4) Evaluation of the ability to induce fat differentiation of the three-dimensional polymer composite structure

To analyze the cellular effect of the 3D polymer complex structure, adipose tissue-derived stem cells were cultured on the surface of the sample for 2 weeks to evaluate the ability to differentiate lipophilicity.

Specifically, after fixing the silicone film on the bottom of TCPs and TCPs, the three-dimensional polymer composite structure of Example 1 (3D PCL (45°)/NFs) and the three-dimensional polymer composite of Comparative Example 1 (3D PCL (45°) )) respectively, and 5000 adipose-derived stem cells were cultured. For stable attachment of cells, cells were cultured in a cell proliferation medium (StemPro® MSC SFM XenoFree, Thermo, USA) for 1 day and then cultured in an adipose differentiation medium (StemPro Adipogenesis Differentiation Kit, Thermo, USA) for 14 days.

In addition, adipose-derived stem cells were cultured on TCP or silicon film as a control group.

The fat differentiation ability of the samples was compared and analyzed and shown in FIGS. 8, 9, 10, 11 and 12.

First, FIG. 8 shows the results of observing the degree of fat differentiation through Oil Red O staining on the 7th and 14th days of induction of fat differentiation of adipose-derived stem cells, respectively. As shown in FIG. 8, a difference in lipodifferentiation ability between each group could not be observed until the 7th day of induction of fat differentiation. However, on the 14th day, adipose-derived stem cells cultured in a silicone film and 3D PCL (45°) were found to have fewer cells than TCPs, but significantly more differentiated into fat.

Quantitatively examining these results, as shown in FIG. 9, it can be seen that the number of cells decreased by about 40% in all groups during the culture period for 2 weeks after induction of lipodifferentiation, and the number of adipose-derived stem cells cultured in TCPs was It can be seen that it is higher than other groups. However, when analyzing the degree of differentiation through Oil Red O, the number of adipose-derived stem cells decreases, but compared to TCPs, the fat of cells cultured in silicone film and 3D PCL (45°) and 3D PCL (45°)/NFs Differentiation ability was higher than about 300%. In particular, it was confirmed that the lipodifferentiation capacity of cells cultured in 3D PCL (45°)/NFs combined with nanofibers increased by more than 400% than that of the TCPs group.Through this, the nanostructure of the nanofibers is the fat of the fat-derived stem cells. It can be seen that it actively induces differentiation capacity.

Meanwhile, FIG. 10 shows the results of a quantitative polymerase chain reaction (qPCR, StepOnePlus Real-time PCR system, USA) for relatively analyzing the lipodifferentiation ability of adipose-derived stem cells cultured in each sample at the mRNA level.

For this, RNA was extracted by treating adipose-derived stem cells after induction of lipodifferentiation with Trizol reagent (sigma, USA). After synthesizing the extracted RNA using AccuPower® CycleScript RT Premix (Bioneer, Korea), stable cDNA is synthesized and then the genes expressed during fat differentiation: Lipoprotein lipase (LPL), Fatty acid binding protein 4 (FABP4), CCAAT/enhancer-binding protein To quantify the primers for alpha (CEBP-a) and sterol regulatory element-binding protein 1 (SREBP-1), PCR was performed using Power SYBR Green PCR Master Mix (Applied biosytem by life, USA).

As shown in FIG. 10, it can be seen that LPL and CEBP-a expressed in mature adipocytes were 300% and 500% higher in 3D PCL (45°) and 3D PCL (45°)/NFs, respectively, compared to the TCPs group.

In addition, in order to analyze the adipogenic differentiation ability of adipose-derived stem cells cultured for 14 days in each sample using a fluorescence microscope, the degree of differentiation after staining with a fluorescent sample (Nucles: Blue, Actin filament: Red, lipid: green) was compared/analyzed. I did.

As shown in FIG. 11, it can be seen that more lipids formed by adipose-derived stem cells cultured in silicon film, 3D PCL (45°) and 3D PCL (45°)/NFs than TCPs. In particular, 3D PCL (45°)/NFs to which nanofibers are bonded formed the most lipids. In addition, as shown in Fig. 12, which quantitatively analyzed this, it can be confirmed that the amount of Lipid in the PCL (45°)/NFs group is approximately 100% higher than that of TCPs, and through this, the presence of nanofibers is determined by adipose-derived stem cells. It can be seen that it effectively induces the differentiation of fat.

(5) Evaluation of biosafety of 3D polymer composite structures through animal testing

To analyze the biosafety of the three-dimensional polymer composite structure, a breast implant for animal testing (Hans Biomed, Korea) (hereinafter, Silicone) and the three-dimensional polymer composite structure (3D PCL (45°)/NFs) of Example 1 and comparison The three-dimensional polymer complex of Example 1 (3D PCL (45°)) was inserted into the dorsal side of SD rats (6 weeks old, male, Orient Bio, Korea), respectively, and tissues obtained through euthanasia at intervals of 1 to 3 weeks were H&E. Staining was performed.

The results are shown in FIG. 13.

As shown in FIG. 13, a group in which only breast implants for animal experiments were inserted (Silicone) and 3D PCL (45°) or 3D PCL (45°)/NFs were inserted at the 1st and 3rd weeks. In both cases, no inflammatory reaction was observed around the graft.

As a result of measuring the thickness of the capusule formed around the implanted implant, as shown in FIG. 14, it was not possible to observe a difference in the thickness of the capsule between each group at week 1 and week 3, as shown in FIG. From the results of these animal experiments, it can be seen that the 3D polymer composite structure according to the present invention exhibits similar biosafety results compared to the silicone breast implant.

(6) Evaluation of the applicability of 3D polymeric composite structures to breast reconstruction through animal experiments

The applicability to breast reconstruction combined with implantation (ie, silicone implant implantation) and autologous fat grafting of the 3D polymer composite structure was evaluated. That is, the possibility of simultaneous application of silicone implant implantation and autologous fat graft was evaluated.

3 g (grams) of fat collected from the groin of SD rats (8 weeks old, male) was used in (5) for animal testing and breast implants (Silicone) and the 3D polymer composite structure of Example 1 (3D PCL (45°)/ NFs) and the three-dimensional polymer complex (3D PCL (45°)) of Comparative Example 1 by dividing 1 g each and inserting it into the back of SD rats, and performing H&E staining on tissues obtained through euthanasia at intervals of 1 and 4 weeks. .

The results are shown in FIG. 15.

As shown in FIG. 15, at week 1, autologous fat transplanted in the group (Silicone) in which only breast implants for animal experiments were inserted and all groups in which 3D PCL (45°) or 3D PCL (45°)/NFs were inserted together. There was no difference in the activity of. At the 4th week, it was confirmed that only the breast implant and 3D PCL (45°)/NFs were inserted together in the animal group maintained the shape of fat and maintained the activity.

Through this, it can be confirmed that the three-dimensional polymer composite structure of the present invention can be applied to breast reconstruction.

Claims (17)

3D polymer composite; And
Including a nanofiber layer formed on one surface of the three-dimensional polymer composite,
The three-dimensional polymer composite and the nanofiber layer each contain a biodegradable polymer,
The three-dimensional polymer composite has a circular grating structure including two or more circular lines, and at least one straight line is arranged in a radial shape based on the center of the circular grid. 3D polymer composite structure.
The method of claim 1,
The three-dimensional polymer composite structure has an average diameter of 10 to 15 cm.
The method of claim 1,
The three-dimensional polymer composite structure has a thickness of 1.0 to 5.0 mm.
The method of claim 1,
Biodegradable polymers include polycaprolactone, poly (l-lactic acid), poly(glycolic acid), polylactic-glucolic acid copolymer (Poly( lactic-co-glycolic acid)), polylactic-caprolactone copolymer (Poly(L-Lactide-co-ε-Caprolactone)), and a three-dimensional polymer composite structure comprising at least one selected from the group consisting of a mixture thereof .
The method of claim 1,
A three-dimensional polymer composite structure in which 20 to 30 circular lines are formed in a circular grid structure.
The method of claim 1,
The length of the cross section of the circular line is 100 to 1,000 um, a three-dimensional polymer composite structure.
The method of claim 1,
A three-dimensional polymer composite structure with a spacing between the circular lines of 500 to 10,000 um.
The method of claim 1,
A three-dimensional polymer composite structure in which 2 to 12 straight lines are formed based on the center of the circular grid in the circular grid structure.
The method of claim 8,
The straight line is formed in an even number, and the straight line is a three-dimensional polymer composite structure that is arranged radially symmetrically.
The method of claim 1,
The length of the cross section of the straight line is 100 to 1,000 um, a three-dimensional polymer composite structure.
The method of claim 1,
The average particle diameter of the cross-section of the nanofibers forming the nanofiber layer is 300 to 600 nm, a three-dimensional polymer composite structure.
The method of claim 1,
The three-dimensional polymer composite structure has a puncture strength of 1.5 Mpa or more.
The method of claim 1,
A three-dimensional polymer composite structure used as an implant for breast reconstruction.
(S1) 3d printing a first polymer solution containing a biodegradable polymer to prepare a three-dimensional polymer composite; And
(S2) comprising the step of electrospinning a second polymer solution containing a biodegradable polymer on one surface of the three-dimensional polymer composite to prepare a nanofiber layer,
The three-dimensional polymer composite has a circular grating structure including two or more circular lines, and at least one straight line is arranged in a radial shape based on the center of the circular grid. Method for producing a three-dimensional polymer composite structure.
The method of claim 14,
The solvent of the second polymer solution is acetone, acetic acid, dichloromethane, chloroform, dimethylformamide, hexafluoroisopropyl alcohol (1,1, 1,3,3,3-hexafluoro-2-isopropanol), formic acid (formic acid), or a method for producing a three-dimensional polymer composite structure as a mixed solvent thereof.
The method of claim 14,
In 3d printing, the nozzle diameter is 0.1 to 0.5 mm,
The nozzle temperature is 80 to 90°C,
The discharge pressure is 10 to 100 psi
The nozzle movement speed is 1 to 5 mm / s, a method of manufacturing a three-dimensional polymer composite structure.
The method of claim 14,
The applied voltage in electrospinning is 5 to 20 kv,
The discharge rate is 0.1 to 5 ml/hr,
A method of manufacturing a three-dimensional polymer composite structure in which the distance between the syringe and the nanofiber layer collector is 10 to 30 cm.

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