A heat and light sensitive tissue scaffold

Abstract

The invention relates to a heat and light sensitive tissue scaffold suitable for the growth, proliferation and organization of the cells in the technical field of tissue engineering, which is endowed with intelligent behavior and thus has a dynamic structure, and a method for producing the same.

Description

[0001] A HEAT AND LIGHT SENSITIVE TISSUE SCAFFOLD

[0002] TECHNICAL FIELD

[0003] The invention relates to a heat and light sensitive tissue scaffold suitable for the growth, proliferation and organization of the cells in the technical field of tissue engineering, which is endowed with intelligent behavior and thus has a dynamic structure, and a method for producing the same.

[0004] PRIOR ART

[0005] Tissue engineering is an interdisciplinary field of study that uses the principles of engineering, medicine and life sciences to develop biological systems designed to fulfill or support the functions of tissues and organs, or to replace them completely. With the tissue engineering method, researchers aim to eliminate waiting lists for organ transplants and to produce tissues and organs suitable for patients from the patient’s own stem and primary cells in the laboratory.

[0006] Tissue engineering combines the principles of cell transplantation, materials science and engineering to develop biological substitutes that can restore and maintain the normal function of diseased or injured tissues / organs. Despite the technical progress in recent decades, the use of these approaches has been limited to research applications and few have been used in the clinic. Many clinicians still use biodegradable polyesters, which were first approved for use in humans 30 years ago. This is a serious concern because morphogenesis is strongly influenced by the interactions between cells and the extracellular environment during normal tissue development. While simple synthetic polymers currently in use provide support for neo-tissue development, they fail to successfully mimic the complex interactions between tissue-specific cells and tissuespecific extracellular matrices (ECMs) that promote functional tissue regeneration.

[0007] In tissue engineering, three strategies are used to modify or stimulate targeted tissues: (1 ) the use of cells alone, (2) the use of biocompatible biomaterials, (3) the use of a combination of both cells and biomaterials. These cells and biomaterials are assembled into scaffolds through a variety of processes that can generally be classified as top-to- bottom or bottom-to-top. In recent years, the demand for the synthetic light-sensitive tissue scaffolds has been increasing. The synthetic light-sensitive tissue scaffolds often comprise materials with phototropic and photoreactive properties. Such materials can change shape or exhibit other properties by showing certain reactions when exposed to light. However, it is seen that the smart materials to be used as tissue scaffolds have not been sufficiently investigated in the relevant technical field and there are deficiencies in the production of the smart biomaterials that can actively participate in tissue regeneration.

[0008] Therefore, the smart biomaterials that actively participate in functional tissue regeneration should be developed and utilized for future applications.

[0009] As a result, it has been determined that a tissue scaffold which can show high biological performance in the field of tissue engineering and have a form that can be compatible for the patients after transplantation, and the innovations for the production thereof should be provided.

[0010] SUMMARY OF THE INVENTION

[0011] In the technical field of tissue engineering, the appropriately manufactured / patterned tissue scaffolds are needed to enable cells to form target tissues in an organized manner in tissue transplantation for patients.

[0012] The fabrication of the tissue scaffolds from smart materials is a recent research and development field. The development of new materials in this technical field will contribute to technological development for tissue engineering.

[0013] The inventors have the aim of providing the use of the material that can be used frequently and generate relatively low costs as light-sensitive biomaterial by providing P(NIPAm) material known to be heat sensitive in the art to be light sensitive. In this way, unlike many studies in the literature, in which P(NIPAm), which is sensitive to heat but not to light, is used by direct heating, in the present invention, the present inventors have achieved temperature increase in specific desired regions with light energy. Compared to the direct heating, the heat generation by light illumination not only allowed for an adjustable temperature-dependent phase transition, but also enabled a much faster folding rate for the platforms.

[0014] Accordingly, the primary object of the present invention relates to the production of smart materials with shape memory or self-activating properties for use in the technical field of tissue engineering.

[0015] Another object of the invention is to provide a tissue that changes physical or chemical properties in a controlled and functional manner when exposed to light and heat.

[0016] Another object of the invention is to provide a tissue production method that eliminates the need to use extra devices.

[0017] Another object of the invention is to provide a tissue production method for maximizing cell viability.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] In this detailed description, the subject of the invention relates to a tissue scaffold comprising smart materials with shape memory or self-activating properties as biomaterials to be used in the technical field of tissue engineering, and the production thereof, and is described only with the examples that will not create any limiting effect for a better understanding of the subject matter.

[0020] In the invention, “complex tissue” means a biological structure in which different cell types and components are combined to fulfill a unique function.

[0021] In the invention, “smart material” refers to materials that react to environmental conditions or applied stimuli. Said smart materials show changes in their physical or chemical properties when exposed to a specific stimulus. These changes can occur in response to various stimuli such as temperature, pH, light, electric field, magnetic field or chemical compounds.

[0022] The biomaterial of the present invention is essentially sensitive to heat. With the embodiment realized in this invention, it can also become sensitive to light. To achieve this, the biomaterial consists of at least two layers. Accordingly, at least one first layer, which is the top layer in the biomaterial, is sensitive to light, while at least one second layer in its lower neighborhood is a heat sensitive layer.

[0023] In the present invention, a self-folding hydrogel platform with adjustable folding capability is designed and produces using P(NIPAm) and P(AAm) hydrogels. Unlike many studies study the literature, in which P(NIPAm), which is sensitive to heat but not to light, is used by direct heating, in this study, the temperature increase is achieved in specific desired regions with light energy. The folding of the platforms was manipulated in a controlled manner with an optimal combination of different acrylic dye colors, pattern design and light illumination. Compared to the direct heating, the heat generation by light illumination not only allowed for an adjustable temperature-dependent phase transition, but also enabled a much faster folding rate for the platforms.

[0024] In a preferred embodiment, at least one substrate may be added to the lowest neighborhood of at least one first layer and at least one second layer. This substrate can provide mechanical strength for these structures or act as an auxiliary component in the processes that the tissue scaffold will perform.

[0025] In a preferred embodiment, said substrate is obtained with three-dimensional material.

[0026] Based on the technical teachings mentioned herein, the method for producing the tissue scaffold of the invention is characterized by the following process steps.

[0027] Obtaining guide molds with a 3D printer

[0028] Said molds are designed by means of design software known in the art and preferably produced on 3D printers using PLA filaments. Here, PLA may change in the future. But today, it is preferred because it is the most well-known and frequently used material. The scope of protection of the invention is not limited thereto. Likewise, design and software used for design are not decisive for the subject matter of the invention. What is desired to be achieved here for the relevant technical field is to obtain tissue scaffolds that are more effective than the products to be provided by 3D printing, that can be compatible with the environment and the patient and, if preferred, have a more complex design. Accordingly, a mold is created with a 3D printer. And in this invention, the term guide mold for mold is preferred to avoid ambiguity. In this invention, there are existing inventors’ embodiments for obtaining the mold, but since the novelty of the invention is not included here, a detailed description is not necessary.

[0029] Obtaining a light sensitive first layer

[0030] The product desired to be produced as a tissue scaffold in this invention is essentially a hydrogel. The hydrogel consists essentially of at least one first layer and at least one second layer.

[0031] The polymerization reaction is carried out to obtain the light sensitive first layer. In a preferred embodiment of the invention, the hydrogel comprises poly-N- isopropylacrylamide (abbreviated as P(NIPAm)) compound as a heat sensitive layer. Said P(NIPAm) compound is obtained in the guide mold obtained in the previous process step. To achieve this, NIPA is used as monomer, MBAm is used as crosslinker, APS and TEMED are used as initiator and accelerator respectively. In the guide mold, this process is carried out by free radical polymerization.

[0032] The following process steps are followed to obtain a light sensitive first layer in the hydrogel: obtaining the first solution by adding NIPA and MBAm compounds into water, obtaining the second solution by dissolving APS in water independently, obtaining the third solution by combining and mixing the first and second solutions,

[0033] - adding at least one acrylic dye to the third solution and stirring, adding TEMED to the resulting mixture and pouring the resulting mixture into the guide molds.

[0034] The first solution comprises NIPA / water at a value in the range of 1 :1 to 2:1 g / ml.

[0035] The first solution comprises MBAm / water at a value in the range of 2:1 to 3:1 mg / ml. Said second solution comprises APS / water at a value in the range of 20:1 to 30:1 mg / ml.

[0036] Said third solution comprises the first solution / the second solution at a value in the range of 1 :1 to 2:1 ml / ml.

[0037] The third solution comprises at least one acrylic dye in a value in the range of 1 % to 5% by volume. Said acrylic dye is at least one of the colors yellow, red or green.

[0038] The acrylic dye is added, then TEMED compound is added to the resulting mixture at a value in the range of 0.05% to 0.5% by volume.

[0039] The reaction in the guide mold takes place at room temperature for at least 15 minutes.

[0040] As a result of these processes, a light sensitive layer is obtained. This layer is located in the hydrogel as the first layer.

[0041] Obtaining a heat sensitive second layer

[0042] The following process steps are followed to obtain a heat sensitive second layer in the hydrogel: obtaining the first solution by adding NIPA and MBAm compounds into water, obtaining the second solution by dissolving APS in water independently, obtaining the third solution by combining and mixing the first and second solutions, adding TEMED to the resulting third solution and pouring the resulting mixture over the first layer.

[0043] The first solution comprises NIPA / water at a value in the range of 1 :1 to 2:1 g / ml.

[0044] The first solution comprises MBAm / water at a value in the range of 2:1 to 3:1 mg / ml.

[0045] Said second solution comprises APS / water at a value in the range of 20:1 to 30:1 mg / ml. Said third solution comprises the first solution / the second solution at a value in the range of 1 :1 to 2:1 ml / ml.

[0046] TEMED compound is added to the third solution in a value in the range of 0.05% to 0.5% by volume.

[0047] The reaction in the guide mold takes place at room temperature for at least 15 minutes.

[0048] Obtaining the passive layer substrate

[0049] In a preferred embodiment of the invention, the hydrogel comprises polyacrylamide (abbreviated as P(AAm)) compound as a third layer which is not sensitive to heat or light. Said P(AAm) compound is obtained such that it is positioned above the second layer in the guide mold obtained in the previous process step. To achieve this, P(AAm) is used as monomer, MBAm (N,N'-methylenebis(acrylamide)) is used as crosslinker, APS (ammonium persulfate) and TEMED (N,N,N,N',N'-tetramethylethylenediamine) are used as initiator and accelerator respectively. In the guide mold, this process is carried out by free radical polymerization.

[0050] In a preferred embodiment, a substrate is positioned in the lowest neighborhood of the hydrogel.

[0051] The following process steps are followed to obtain a non-heat sensitive third layer in the hydrogel: obtaining the first solution by adding AAm, MBAm and APS compounds to water,

[0052] - obtaining the second solution by adding TEMED to the first solution,

[0053] - adding the second solution over the first layer and performing reactions in the guide mold.

[0054] The first solution comprises AAm / water at a value in the range of 1 :2 to 1 :5 g / ml.

[0055] The first solution comprises MBAm / water at a value in the range of 5:1 to 1 :1 mg / ml.

[0056] Said first solution comprises APS / water at a value in the range of 10:1 to 5:1 mg / ml. The second solution comprises a TEMED compound in a value in the range of 0.05% to 0.5% by volume.

[0057] The reaction in the guide mold takes place at room temperature for at least 30 minutes.

[0058] As a result of all the processes mentioned here, hydrogel suitable for use as tissue scaffold can be obtained.

[0059] In a preferred embodiment, the resulting hydrogel is soaked in water at room temperature for at least 30 minutes for easy demolding.

[0060] The hydrogel obtained in the invention will be suitable for use as a tissue scaffold that is sensitive to light through the first layer and to heat through the second layer. With the light energy of the first layer containing P(NIPAm), the temperature increase was achieved in the desired specific regions. The folding of the hydrogel can be manipulated in a controlled manner with an optimal combination of different acrylic dye colors, pattern design and light illumination. Compared to the direct heating, the heat generation by light illumination not only allowed for an adjustable temperature-dependent phase transition, but also enabled a much faster folding rate for the hydrogels.

[0061] The hydrogel obtained in the invention can be effective when the light source has a wavelength in the range of 400 to 480 nm. This makes it possible for the hydrogel to function as a smart material at low temperatures. Said low temperature is around 35 □.

[0062] On the other hand, the obtained tissue scaffold is expected to be biocompatible. The produced hydrogel was seeded with C2C12 cells on a V5 model sample to examine cell interactions. After the cell proliferation procedure was performed, the cells were detached from the flasks with the help of trypsin, redistributed and counted in fresh medium and then seeded onto the samples with a micropipette at a rate of 200,000 cells per sample. These samples were then transferred into 6-well cell culture plates and incubated (37eC, 5% CO2) for 1 hour to allow the cells to adhere to the surface. At the end of 1 hour, 4 ml of proliferation medium was applied on them and they were placed back in the incubator (37eC, 5% CO2). The cell culture was maintained for 7 days. The medium was replaced with fresh medium every 48 hours. According to the results obtained, it was determined that the tissue scaffold was biocompatible.

[0063] The Phalloidin and cell nuclei (DAPI) staining was performed on Day 7 of the culture to examine the morphology of the cells seeded on the hydrogel platform. According to the results obtained, it was observed that the tissue scaffold was biocompatible and the cells adhered, proliferated and survived on the platform. It is predicted that more effective results can be obtained if additional surface treatments are performed to increase cell adhesion and diffusion.

[0064] The scope of protection of the invention is described in the attached claims and cannot be limited to what is explained in this detailed description for the exemplary purposes. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

CLAIMS1 . A heat and light sensitive tissue scaffold as a biomaterial for use in the technical field of tissue engineering, characterized in that it is a hydrogel containing at least two layers, and it comprises- a light sensitive poly-N-isopropylacrylamide polymer containing at least one acrylic dye as the first layer,- a heat sensitive transparent poly-N-isopropylacrylamide polymer located in the lower neighborhood of the first layer.

2. A tissue scaffold according to claim 1 , wherein it comprises at least one of the colors yellow, red or green as acrylic dye.

3. A tissue scaffold according to one of the preceding claims, wherein it comprises a third layer, insensitive to heat and light, located in the lowest neighborhood of the first and second layers.

4. A tissue scaffold according to claim 3, wherein it comprises polyacrylamide polymer.

5. A method for producing a heat and light sensitive tissue scaffold as a biomaterial for use in the technical field of tissue engineering, characterized in that it comprises the following process steps:- obtaining a light sensitive first layer, o obtaining the first solution by adding NIPA and MBAm compounds into water, o obtaining the second solution by dissolving APS in water independently, o obtaining the third solution by combining and mixing the first and second solutions, o adding at least one acrylic dye to the third solution and stirring,o adding TEMED to the resulting mixture and pouring the resulting mixture into the guide molds,- obtaining a heat sensitive second layer, o obtaining the first solution by adding NIPA and MBAm compounds into water, o obtaining the second solution by dissolving APS in water independently, o obtaining the third solution by combining and mixing the first and second solutions, o adding TEMED to the resulting third solution and pouring the resulting mixture over the first layer.

6. A method according to claim 5, wherein the first solution comprises NIPA / water at a value in the range of 1 :1 to 2:1 g / ml to obtain the first layer.

7. A method according to claim 5 or claim 6, wherein the first solution comprises MBAm / water at a value in the range of 2:1 to 3:1 mg / ml to obtain the first layer.

8. A method according to one of claims 5-7, wherein the first solution and the second solution comprise APS / water at a value in the range of 20:1 to 30:1 mg / ml to obtain the first layer.

9. A method according to one of claims 5-8, wherein the third solution comprises the first solution / the second solution at a value in the range of 1 :1 to 2:1 ml / ml to obtain the first layer.

10. A method according to one of claims 5-9, wherein the third solution comprises at least one acrylic dye at a value in the range of 1 % to 5% by volume to obtain the first layer.11 . A method according to one of claims 5-10, wherein the acrylic dye is added, then TEMED compound is added to the resulting mixture at a value in the range of 0.05% to 0.5% by volume to obtain the first layer.

12. A method according to one of claims 5-11 , wherein the first solution comprises NIPA / water at a value of 1 :1 to 2:1 g / ml to obtain the second layer.

13. A method according to one of claims 5-12, wherein the first solution comprises MBAm / water at a value in the range of 2:1 to 3:1 mg / ml to obtain the second layer.

14. A method according to one of claims 5-13, wherein the first solution and the second solution comprise APS / water at a value in the range of 20:1 to 30:1 mg / ml to obtain the second layer.

15. A method according to one of claims 5-14, wherein the third solution comprises the first solution / the second solution at a value in the range of 1 :1 to 2:1 ml / ml to obtain the second layer.

16. A method according to one of claims 5-15, wherein TEMED compound is added at a value in the range of 0.05% to 0.5% by volume to obtain the second layer.

17. A method according to one of claims 5-16, characterized in that it comprises the following process steps to obtain a third layer insensitive to heat and light: o obtaining the first solution by adding AAm, MBAm and APS compounds to water, o obtaining the second solution by adding TEMED to the first solution, o adding the second solution over the first layer and performing reactions in the guide mold.

18. A method according to claim 17, wherein the first solution comprises AAm / water at a value in the range of 1 :2 to 1 :5 g / ml to obtain the third layer.

19. A method according to claim 17 or claim 18, wherein the first solution comprises MBAm / water at a value in the range of 5:1 to 1 :1 mg / ml to obtain the third layer.

20. A method according to one of claims 17-19, wherein the first solution comprises APS / water at a value in the range of 10:1 to 5:1 mg / ml to obtain the third layer.

21. A method according to one of claims 17-20, wherein the second solution comprises TEMED compound at a value in the range of 0.05% to 0.5% by volume to obtain the third layer.

22. A method according to one of claims 5-21 , wherein the resulting hydrogel is soaked in water at room temperature for at least 30 minutes for easy demolding.