학술논문
골 재생을 위한 지지체 기반 트립신 프리 세포 스탬핑 시스템의 개발 / Trypsin free cell stamping system using scaffolds for bone regeneration
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
척추유합술은 디스크가 마모되었을 때 발생하는 척추 통증과 압력을 완화하기 위해 가장 일반적으로 수행되는 치료 방법이다. 현재 척추 고정기기 개발을 통해 유합률을 높이기 위한 많은 시도가 이루어지고 있지만, 56%의 불유합이 보고되고 있다. 이를 극복하기 위해 거부반응이 적고 골 결손 부위가 클 때도 치료가능한 세포 지지체 시스템이 필수 적이다.세포 지지체 시스템에서, 세포가 지지체로 이식되기 전에 세포는 증식되고 계대배양 되어야 한다. 그러나 이 과정은 트립신 처리를 포함해 세포의 생산성과 품질을 저하시키는 여러 과정이 요구된다. 따라서 이 연구에서는 세포의 손상없이 세포를 나노 섬유 지지체로 이동시키는 트립신 프리 세포 스탬핑 시스템을 개발하고 스탬핑 할 때의 최적화 조건을 확립하였다.이 시스템을 도입함으로써, 세포가 세포배양접시에서 일반적인 방법으로 분리될 때 발생하는 계대수 증가를 막을 수 있다. 또한, 세포에게 인공적인 자극을 주는 것을 최소화해 외부의 다른 자극없이 세포를 고분자 기반의 나노 섬유 지지체로 옮길 수 있고 이로 인해 줄기 세포의 분화능을 높일 수 있다.실험적으로 살펴보면, 지방유래 줄기세포를 골분화 배양액에 배양하고 전기방사로 제작된 젤라틴 기반 나노 섬유지지체로 스탬핑했다. 그 결과, 세포 스탬핑 방법으로 옮겨진 세포가 일반적으로 트립신을 처리해 세포를 옮기는 세포 씨딩 방법보다 더 좋은 분화능을 가지는 것을 확인할 수 있었다. 또한, 골 분화된 세포를 세포 스탬핑으로 옮겼을 때, 옮기기 전 세포와 유사한 골 분화정도를 보였다. 이러한 결과는 세포스탬핑 시스템으로 인해 세포를 지지체로 효과적이고 쉽게 옮길 수 있음을 보여준다. 따라서, 세포 스탬핑 시스템은 세포치료제로써 척추유합술 치료 및 재생의학과 조직공학의 전반적인 분야에 활용 가능하다.
Spinal fusion is most commonly performed treatment to relieve the pain and pressure on the spinal cord that results when a disc wears out. Currently, attempts are made to increase the spine fusion rate with the development of spinal fixation devices, but 56% of nonunion are reported. Therefore, it is necessary to overcome these problems by using cell scaffold system, which has low rejection and can be treated even with large bone defect sites.In cell scaffold system, cells should be expanded and subcultured to be seeded on the scaffolds before implantation. However, the process requires several steps including trypsin treatment leading to a possible decrease in productivity and quality of cells. In this study, we examined a trypsin-free cell stamping system that transfers cells to scaffolds by using nanofibers without cell damages. Also, an increase in the passage number of stem cells can be prevented compared with conventional methods of isolating cells from a cell culture dish, while providing a scaffold, which is an essential condition of cell growth, by introducing the trypsin free cell stamp system. In addition, the artificial effects on cells can be minimized since the cells migrate to a polymer-based nanofiber scaffold without other external stimulation. Thus, the potency of stem cells is increased, thereby inducing more effective differentiation.For experiments, adipose-derived stem cells were cultured with osteogenic differentiation media and stamped onto gelatin-based nanofibers fabricated by electrospinning. Cell stamping method has better ability of osteogenic differentiation than cell seeding method that used generally to transplanting cells. And we found out that all the differentiated cells were viable after cell stamping and showed similar osteogenic differentiation as compared with cells seeded and cultured in a conventional way. These results indicate that cell stamping system could be used as an easy and effective way of cell transfer onto scaffold in tissue engineering applications. And thus, cell stamping system can be applied cell therapeutic agent, can be utilized in general fields of regenerative medicine for bone regeneration.
Spinal fusion is most commonly performed treatment to relieve the pain and pressure on the spinal cord that results when a disc wears out. Currently, attempts are made to increase the spine fusion rate with the development of spinal fixation devices, but 56% of nonunion are reported. Therefore, it is necessary to overcome these problems by using cell scaffold system, which has low rejection and can be treated even with large bone defect sites.In cell scaffold system, cells should be expanded and subcultured to be seeded on the scaffolds before implantation. However, the process requires several steps including trypsin treatment leading to a possible decrease in productivity and quality of cells. In this study, we examined a trypsin-free cell stamping system that transfers cells to scaffolds by using nanofibers without cell damages. Also, an increase in the passage number of stem cells can be prevented compared with conventional methods of isolating cells from a cell culture dish, while providing a scaffold, which is an essential condition of cell growth, by introducing the trypsin free cell stamp system. In addition, the artificial effects on cells can be minimized since the cells migrate to a polymer-based nanofiber scaffold without other external stimulation. Thus, the potency of stem cells is increased, thereby inducing more effective differentiation.For experiments, adipose-derived stem cells were cultured with osteogenic differentiation media and stamped onto gelatin-based nanofibers fabricated by electrospinning. Cell stamping method has better ability of osteogenic differentiation than cell seeding method that used generally to transplanting cells. And we found out that all the differentiated cells were viable after cell stamping and showed similar osteogenic differentiation as compared with cells seeded and cultured in a conventional way. These results indicate that cell stamping system could be used as an easy and effective way of cell transfer onto scaffold in tissue engineering applications. And thus, cell stamping system can be applied cell therapeutic agent, can be utilized in general fields of regenerative medicine for bone regeneration.