Development of nanofibrous membranes with chemical and physical modifications for guided bone regeneration

Abstract

As increasing aging population, demands of alveolar bone regeneration are increasing and various commercialized membranes for guided bone regeneration (GBR) had been used for effective regeneration of alveolar bone. GBR membranes prevent infiltration of surrounding fibrous tissue which interrupt bone regeneration by block bone defect site, as a result facilitate bone regeneration. Existing commercialized GBR membranes have limitations such as required secondary surgery for non-resorbable membranes or lack of mechanical stability occurred by its acute degradation for resorbable membranes. Furthermore, those of membranes did not have ability for regulation of cell behavior which resulted in limited bone regeneration. In this thesis, I fabricated highly porous nanofibers by using electrospinning method and developed biomimetic GBR membranes by various chemical physical modifications which can regulate cell behaviors. First, to induce guidance effect of cell migration on nanofibers, I fabricated nanofibers with different fiber orientation and confirmed that the fiber orientation modulated direction of adhesion and migration of hMSCs. Moreover, the orientation of nanofibers guided in vivo bone regeneration, the effect seems occurred by regulation of collagen matrix direction. Next, I fabricated inorganics such as biphasic calcium phosphate or hydroxyapatite incorporated PLGA biodegradable nanofibers to mimic microenvironments in natural bone tissue. Both of two nanofibers facilitated adhesion and osteogenic differentiation of preosteoblasts or hMSCs due to incorporation of inorganics. Gelatin was incorporated with different ratio into synthetic polymer nanofibers to increase bioactivity, and the increase of gelatin incorporation ratio, increased bioactivity of nanofibers such as adhesion, proliferation and osteogenic differentiation of hMSCs. Heparin was chemically immobilized on gelatin and synthetic polymer composite nanofibers to immobilze growth factors. The incorporated growth factors mediated by heparin were released out and induced cell proliferation, migration and neovascularization. Furthermore, growth factor immobilized nanofibers facilitated in vivo angiogenesis and bone regeneration. From these results, we can expect effective bone regeneration of developed biomimetic nanofibers can regulate cell functions by proper chemical and physical modification as advanced membranes for GBR.|전 세계적으로 고령화가 가속화 됨에 따라 치조골 재생의 수요가 증가하고, 효과적인 치조골 재생을 위하여 골재생유도막이 다양하게 상용화되어 사용되어 왔다. 골재생 유도막은 골결손부위를 차단함으로서 외부의 섬유조직이 안쪽으로 침투하여 골재생을 방해하는 것을 방지하고 결과적으로 골재생을 촉진시키는 역할을 한다. 기존의 상용화 되어있는 골재생유도막은 비흡수성일 경우 제거를 위한 2차적 수술을 필요로 하거나 흡수성일 경우 급속한 분해로 인한 물리적 안정성 결여 등의 한계점을 가지고 있다. 게다가 이러한 상용화된 막은 세포의 거동을 조절할 수 있는 능력이 없기 때문에 골조직 재생에 있어서 한계를 가지고 있다. 때문에 본 논문에서는 전기방사법을 이용하여 다공성구조를 가지는 나노섬유를 제작하였고 다양한 화학적 물리적 변형을 통하여 세포의 거동을 조절할 수 있는 생체모사형 골재생유도용 차폐막을 개발하였다. 먼저, 나노섬유 상에서 세포의 이동 방향성을 유도하기 위하여 각기 다른 방향성을 가지는 나노섬유를 제작하였으며, 이러한 나노섬유의 방향성은 중간엽 줄기세포의 부착 방향뿐만 아니라 이동방향까지도 조절할 수 있음을 확인하였다. 또한 나노섬유의 방향성은 생체 내 골재생의 방향성까지도 조절하였으며 그 효과는 콜라겐 기질의 방향성을 조절하는 것에 기인한 것으로 보인다. 체내에 존재하는 골조직의 미세환경을 모사하기 위하여 PLGA 생분해성 고분자 나노섬유에 무기물질인 biphasic calcium phosphate 또는 하이드록시아파타이트가 도입된 나노섬유를 제작하였다. 두종류의 나노섬유 모두 무기물질의 도입으로 줄기세포의 부착 및 골분화를 촉진하였으며, 생체 내에 이식되었을 때 골재생을 촉진하였다. 합성고분자 나노섬유의 세포친화성을 증가시키기 위하여 젤라틴을 비율별로 도입하였고 젤라틴의 비율이 증가할수록 줄기세포의 부착 및 증식 그리고 골분화가 증가되어 나노섬유의 세포친화성을 증가시킴을 확인하였다. 성장인자를 도입하기 위하여 젤라틴과 합성고분자가 혼합된 나노섬유에 화학적인 방법으로 헤파린을 도입하였고, 헤파린을 통해 도입된 성장인자는 나노섬유로부터 방출되어 세포의 증식, 이동 및 혈관신생을 유도하였다. 뿐만아니라 성장인자가 도입된 나노섬유는 체내에서의 혈관신생과 골재생도 촉진하였다. 이들 결과를 종합하여 볼 때, 개발된 생체모방형 나노섬유는 적절한 화학적 물리적 변형을 통하여 세포의 기능을 조절할 수 있기 때문에, 기존에 사용되었던 골재생유도용 차폐막에 비하여 진보된 차폐막으로서 효과적으로 골재생을 유도할 수 있을 것으로 기대된다.; As increasing aging population, demands of alveolar bone regeneration are increasing and various commercialized membranes for guided bone regeneration (GBR) had been used for effective regeneration of alveolar bone. GBR membranes prevent infiltration of surrounding fibrous tissue which interrupt bone regeneration by block bone defect site, as a result facilitate bone regeneration. Existing commercialized GBR membranes have limitations such as required secondary surgery for non-resorbable membranes or lack of mechanical stability occurred by its acute degradation for resorbable membranes. Furthermore, those of membranes did not have ability for regulation of cell behavior which resulted in limited bone regeneration. In this thesis, I fabricated highly porous nanofibers by using electrospinning method and developed biomimetic GBR membranes by various chemical physical modifications which can regulate cell behaviors. First, to induce guidance effect of cell migration on nanofibers, I fabricated nanofibers with different fiber orientation and confirmed that the fiber orientation modulated direction of adhesion and migration of hMSCs. Moreover, the orientation of nanofibers guided in vivo bone regeneration, the effect seems occurred by regulation of collagen matrix direction. Next, I fabricated inorganics such as biphasic calcium phosphate or hydroxyapatite incorporated PLGA biodegradable nanofibers to mimic microenvironments in natural bone tissue. Both of two nanofibers facilitated adhesion and osteogenic differentiation of preosteoblasts or hMSCs due to incorporation of inorganics. Gelatin was incorporated with different ratio into synthetic polymer nanofibers to increase bioactivity, and the increase of gelatin incorporation ratio, increased bioactivity of nanofibers such as adhesion, proliferation and osteogenic differentiation of hMSCs. Heparin was chemically immobilized on gelatin and synthetic polymer composite nanofibers to immobilze growth factors. The incorporated growth factors mediated by heparin were released out and induced cell proliferation, migration and neovascularization. Furthermore, growth factor immobilized nanofibers facilitated in vivo angiogenesis and bone regeneration. From these results, we can expect effective bone regeneration of developed biomimetic nanofibers can regulate cell functions by proper chemical and physical modification as advanced membranes for GBR.