Studies on effect of graphene-based materials on chondrogenic differentiation of human adipose-derived stromal cells

Abstract

Cartilage is a tissue consisting of chondrocytes and extracellular matrix (ECM), which is highly elastic, buffers against a given force, and the friction coefficient of joint cartilage is very low, helping the joints to move in a state of little friction. But cartilage is a kind of expendable body part that wears out as much as it is used, and it is also damaged by inflammation, trauma, and aging. Cartilage has very limited self-renewal because there are no blood vessels, nerves, or lymphatic vessels. Thus, cartilage cannot easily stop when it starts to damage, and damaged cartilage has many limitations about regenerating into cartilage with normal function and structure. Also, the damaged cartilage area becomes more vulnerable to mechanical pressure, so it is easily broken and worn, resulting in larger defects. As such, a number of cartilage-related diseases develop and progresses faster than other tissues. Some typical diseases include degenerative arthritis, which causes pain due to cartilage wear, and others include rheumatoid arthritis, achondroplasia, pyogenic arthritis, and chondrosarcoma. Furthermore, these cartilage-related diseases can lead to bone-related complications if they persist for a long time. These diseases can lead to a reduction in the quality of life and life expectancy of patients, and the loss of substantial medical costs. Therefore, it is very important to regenerate damaged cartilage early, but there are many deficiencies in current cartilage treatment. Although many researchers continue to suggest ways to treat cartilage-related diseases, they are still far from normal and perfect cartilage regeneration.

Up until now, studies have been actively conducted to regenerate damaged cartilage tissue using an ideal biomaterial that can mimic and replace cartilage tissue with various cells. Among them, cells that are widely used in the field of regenerative medicine are human adipose-derived stromal cells (hASCs). This cell has a great advantage that it can be easily obtained from any tissue of the human body, and it is suitable as a material for a cell therapy agent because it does not take much time for cell proliferation and can be repeatedly collected. And recently, graphene (G), a carbon-based material, is emerging as a biomaterial in tissue engineering and regenerative medicine. In addition, a variety of graphene-based materials (GBMs) have also been recognized for their research value as biomaterials. GBMs are derivatives of G. Most GBMs are made through the process of oxidation. GBMs that have the advantage of oxidation are more applied in various fields than G. In particular, GBMs play a role as biomaterials due to their various functional groups, biocompatibility, mechanical stability, and other characteristics. And GBMs have been extensively studied in the field of tissue regeneration and repair. However, since G was discovered in 2004 and is a new material only about 20 years old, there is a limit to the lack of prior research related to cartilage. Therefore, cartilage regeneration studies related to GBMs are essential. And because accurate standard indicators for GBMs have not yet been created, characterization and comparison of various GBMs is critical. Based on an understanding of the characteristics of GBMs, this study was conducted to apply to cartilage regeneration studies.

The GBMs used in this study were graphene oxide (GO), nano-graphene oxide (nGO) and graphene quantum dot (GQD). All are oxidized GBMs. These are the forms in which parts of the surface have been replaced with oxygen as graphene is oxidized, and the great advantage is that the binding force of each layer of graphite is reduced and distributed well in the solution. Since it can be synthesized in the solution phase, it can be mass-produced and overcome the disadvantages of graphene, which is a very expensive material and has a very high production cost. And because it is easier to attach new functional groups to oxygen than to carbon, it is possible to make more functionalized graphene derivatives. As hydrophobic graphene turns into hydrophilic, oxidized GBMs, affinity with cells increases, and it has the advantage of being easily mixed into culture medium.

Therefore, since these advantages allow me to study GBMs as biomaterials, I analyzed the characteristics of each GBMs using Fourier Transform Infra-Red (FT-IR), X-ray Photoelectron Spectroscopy (XPS), and Electrophoretic Light Scattering (ELS). Functional group analysis confirmed that all GBMs were oxidized by showing binding to carboxyl groups, hydroxyl groups and epoxy groups common to surface of GBMs. Through C1s spectra analysis, the degree of oxidation can be determined by the binding and binding ratio of carbon and oxygen. It was found that oxidation is high in the order of nGO, GO, and GQD. Hydrodynamic radius analysis was used to confirm the hydrodynamic radius of each material. This means the radius of the particles in the solution. Therefore, the size of each particle in the solution can be known, and the order in which it is large was confirmed to be GO, nGO and GQD. Finally, Zeta potential analysis was used to confirm the dispersion stability of the particles in solution. The larger the negative value, the higher the dispersion stability. Generally, over –30 means having good stability, so I found that GO and nGO had better dispersion stability than GQD. Therefore, the dispersion stability is high in the order of nGO, GO, and GQD. These data show that all three GBMs are oxidized and help to understand and compare the physical and chemical properties of each particle.

Based on the unique properties of these GBMs, I applied them to chondrogenic differentiation of hASCs. Although studies on cartilage regeneration using various cells and biomaterials have been actively studied, there are still no standardized therapies related to cartilage as various problems such as potential side effects on regenerative therapy and verification of effects. So the study was conducted to investigate the effect on the cartilage differentiation of hASCs by applying GBMs, which have recently emerged as biomaterials in the field of regenerative medicine, to cartilage research. First, I conducted a study on the characterization of hASCs. Microscopic photographs showed that hASCs had fibroblastic morphology, and the ability to differentiate into osteocyte, adipocyte and chondrocyte was confirmed through three different dyeing reagents. And based on the GO with the largest particle size, I confirmed the concentration suitable for chondrogenic differentiation of hASCs. As a result, it was confirmed that a concentration of 10 μg / ml or less was suitable. Thus, GO, nGO, and GQD at concentrations of 1 and 10 μg / ml were applied to hASCs. As a result, the size of the chondrocyte pellet was not different from the induction group. However, it was confirmed that Glycosaminoglycans (GAGs) were significantly increased in the 1 μg / ml group of GO and 1 μg / ml of nGO group compared with the induction group through alcian blue staining and toluidine blue staining. These results suggest that GO and nGO, the oxidized graphene, support Chondrogenic differentiation of hASCs.

The main purpose of this study was to determine the effect of GBMs on chondrogenic differentiation of hASCs. Particularly, it was confirmed that the effect of chondrogenic differentiation is different according to the particle size and degree of oxidation of GBMs. These studies will help to understand GBMs, which are bio-new materials, and will greatly contribute to the development of new cartilage therapies using these materials.

본 논문에서는 3가지의 다른 종류의 그래핀 기반 물질들의 물리적, 화학적 특성을 분석하고 비교를 통해 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점에 대한 이해에 대한 연구와 그래핀 기반 물질들을 생체 소재로 활용한 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 대한 조직 공학 연구를 진행하였다.

Chapter 3에서는 그래핀 기반 물질들의 특성을 이해하기 위해 물리적, 화학적 분석에 대한 연구를 진행한 후, 분석된 그래핀 기반 물질들을 이용하여 인간 지방유래 기질세포의 연골 분화에 대한 효과를 확인하였다. 그래핀은 발견된 지 20년이 채 되지 않은 바이오신소재로, 참고할 만한 이전 연구가 많이 없다. 그래서 그래핀 유도체들의 물리, 화학적 표준 지표가 마련되어 있지 않기 때문에 각 각의 특성들을 정확하게 분석하고 이해하는 것이 매우 중요하다. 먼저, Fourier Transform Infra-Red (FT-IR)를 이용하여 각 물질들의 표면에 어떤 작용기들로 구성되어 있는지 확인하였다. 3 종류의 그래핀의 표면에서 공통적으로 카르복실기, 하이드록실기, 에폭시기를 가진다는 것을 확인하였다. 이는 3종류의 그래핀 기반 물질들이 모두 산화되었음을 보여준다. X-ray Photoelectron Spectroscopy (XPS)를 이용해서는 C1s spectra 분석을 하였다. 이를 통해 탄소와 산소의 결합과 비율로 산화 정도를 확인할 수 있었고, 그 결과, 산화 정도는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. Electrophoretic light scattering (ELS)를 이용해서는 각 물질들의 유체역학적 반경을 측정하였다. 이는 용액 속에서 입자가 영향을 주는 반경을 의미하기 때문에, 입자의 크기를 알 수 있다. 그 결과, 입자의 크기는 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. 마지막으로, ELS를 이용하여 zeta potential 값을 측정하였다. 제타 포텐셜 값이 높은 음의 값을 가질수록 용액에서의 분산 안정성이 높으며, 일반적으로 –40이 넘어가면 안정성이 좋다는 것을 의미한다. 그 결과, 분산 안정성이 높은 순서는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서임을 확인하였다. 이러한 결과들을 통해 그래핀 기반 물질들의 표면 작용기, 산화 정도, 입자의 크기, 분산안정성에 대해 이해할 수 있었고, 이는 그래핀 기반 물질들을 다양한 분야에서 적용시킬 때 많은 도움을 줄 것으로 생각한다. 그리고 분석한 그래핀 기반 물질들을 바이오소재로서 이용하여 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 관한 연골 재생 연구를 하였다. 소수성인 그래핀을 산화시켜 만든 그래핀 기반 물질들은 친수성으로 성질이 바뀌면서 세포와의 친화성이나 배양 배지에 쉽게 분산될 수 있는 장점을 가져 조직공학 분야에 접목시키기 유리하다. 그래서 나는 먼저, 인간 지방유래 기질세포의 형태학적인 분석과 골, 지방, 연골로의 분화능에 대한 분석을 하였다. 인간 지방유래 기질세포는 섬유아세포와 같은 모양을 가졌으며, 골, 지방, 연골로의 분화능을 가지는 것으로 확인하였다. 그리고 나서 인간 지방유래 기질세포의 연골 분화에 적합한 산화 그래핀의 농도를 확인하였다. 산화 그래핀을 기준으로 한 이유는 입자의 크기가 가장 크고, 조직 공학과 관련된 참고문헌이 가장 많기 때문이다. 그 결과, 10 ㎍/㎖의 이하의 농도에서 적합한 것을 확인하였다. 그래서 1과 10 ㎍/㎖의 농도의 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점을 이용해 인간 지방유래 기질세포의 연골 분화 영향을 확인하였을 때, 콘드로사이트의 펠렛 크기에는 영향을 주지 않았다. 하지만 alcian blue staining과 toluidine blue staining으로 조직학적 분석을 하였을 때, 1 ㎍/㎖의 농도의 산화 그래핀과 나노 산화 그래핀 군에서 양성대조군에 비해 GAGs의 함량이 유의적으로 증가함을 확인하였다. 그 결과, 100 nm 이상의 크기를 가진 산화 그래핀 기반 물질들이 인간 지방유래 기질세포의 연골분화에 효과가 있음을 확인하였다. 그러므로 이 연구를 통해 재생 의학이나 조직공학 분야에서 떠오르는 바이오 신소재로 자리매김을 할 수 있는 가능성을 확인하였다.

앞으로 그래핀 기반 물질들이 세포와 세포 외 기질 등 주변 환경에 미치는 영향이나 세포 독성, 적합한 배양 조건에 관한 깊이 있는 연구가 더 진행된다면 충분히 조직 공학적 응용이 가능해 질 것이다