Nanobiomaterials
for stem cell and tissue engineering
화학생물공학부 김병수 교수
Medical Approach
Pharmacol.
Management
Organ
Transplantation
Kidney, BM, Liver Heart, Pancreas, etc.
Regeneration of Organ
Stem Cell Therapy
& Tissue Engineering
미래의학 = 재생의학 (再生醫學) Regenerative medicine
Prof. S. Yamanaka (2012 Nobel Prize)
• Human disease model
• Drug screening
Research is presently being conducted on several different types of tissues and organs, including:
Skin
Cartilage
Blood Vessels
Bone
Muscle
Nerves
Liver
Kidney
etc. etc. etc.
Tissue Engineering
is thein vitro
development of tissues or organs to replace or support the function of defective or injured body parts, or the directed management of the repair of tissues within the body.출처 : Regenerative medicine market, Trimark, 2013
Injection of MSCs to infarction area through catheter
(Intracoronary injection) Autologous
bone marrow aspirate
MSCs Infarction
area In vitro culture
Limitations :
5% increase in ejection fraction (heart function)
Likely due to Low possibility of stem cell differentiation into cardiomyocyte
Wash-out of transplanted stem cells from the injection area
FCB-Pharmicell, Inc (Korea)Approved clinical use of bone marrow stem cells
for myocardial infarction treatment
Cardiac stem cell sheets
for myocardial infarction treatment
Prof. Teruo Okano
(Tokyo Women’s Medical University)
Proarrhythmic (부정맥) potential of MSC transplantation to myocardial infarction heart
Due to no electric coupling of inexcitable MSCs to cardiomyocytes (심근세포)
Overexpression of connexin 43 in skeletal myoblasts
prevents ventricular arrhythmias following transplantation
S. Fernandes et al., J. Cell. Mol. Med. 13; 3703-3712, 2009
Connexin 43 overexpression in myoblasts improved intercellular electrical coupling between transplanted myoblasts and cardiomyocytes in infarcted heart
VT : ventricular tachycardia (심실성빈맥)
Ventricular hyperexcitability of the myocardium after myoblast transplantation
Connexin 43
(a gap junction protein)
• control : myoblasts
• null : myoblasts + empty vector
• Cx43 : myoblasts + connexin43 gene
M. Shevach et al., J. Mater. Chem. B, 2013, 1, 5210 (A) Fabrication of PCL–gelatin fiber scaffolds by electrospinning. (B) Evaporation of gold NPs onto the fibers to create nanocomposite scaffolds. (C) Cardiac cells are seeded in the scaffolds for (D) engineering a functional cardiac tissue
To increase the electrical conductivity and enhance the electrical signal transfer between cardiac cells
Gold nanoparticle-incorporated cardiac patch
M. Shevach et al., J. Mater. Chem. B, 2013, 1, 5210
Cardiac sarcomeric actinin immunostaining
scaffolds without gold Gold NP scaffolds
Engineered cardiac tissue function
Normal crdiomyocytes
Gold nanoparticle-incorporated cardiac patch
Au - - + +
Electric
stimulation - + - +
Cx43 Intensity
J.O. You, et al., Nano Lett. 2011, 11, 3643–3648
Conductive scaffolds enhance connexin 43 expression
D.H. Kim et al., Integr. Biol., 2012, 4, 1019–1033
well-aligned myocardium
Nanopatterned cardiac patches promote myocardial
regeneration by cardiosphere-derived cells
D = 283 nm D = 749 nm
Christopherson GT, et al. Biomaterials (2009)
Control of neural stem cell differentiation
with nanofiber diameter
Nestin+ : progenitor Tuj1+ : neuron
RIP+ : oligodendrocyte GFAP+ : astrocyte
Fiber D = 749nm
283nm
Control of neural stem cell differentiation with nanofiber diameter
Christopherson GT, et al. Biomaterials (2009)
Human fetal tendon stem cell
seeding
Aligned Random
Nanofibers
CFDA
6 weeks in vivo
Yin Z, et al. Biomaterials (2010)
teno-lineage differentiation
Effects of nano-fiber alignment on cell
alignment and tendon differentiation
Within bone, type I collagen has parallel array of collagen fibrils 10 - 300 nm in size.
Pitch = 400 nm 1400 nm 4000 nm planar control
Nanotopographical pattern modulates osteogenic differentiation of MSCs
17 Watari S, et al. Biomaterials (2012)
B.K.K. Teo et al., ACS Nano 2013, 7: 4785
Nanotopography modulates focal adhesion and
differentiation of stem cells
# of focal adhesion per cell
3D AFM height images of Ti surfaces
8 nm 15 nm
MSCs stained for actin and osteocalcin
planar Ti 8 nm 15 nm nanodots
T. Sjöström et al., Adv. Healthcare Mater. 2013, 2, 1285–1293
Nanopatterning of titanium implants for enhancing
osteointegration
Topographically defined implant for enhanced osteoconduction
DAPI F-actin
Guided osteoblast migration in vitro
Tissue Engineering 22: 654-664 (2016) Implants with different topography
Width of pits = 5µm
Enhanced bone regeneration by topographically defined implant
Topographed implant (diameter = 5 mm)
calvarial defect (diameter = 4 mm)
Regenerated bone tissue
7 weeks Osteoblast
migration
Mouse calvaria
Implant fixation with fibrin gel
Tissue Engineering 22: 654-664 (2016)
FDA approved for spine fusion, bone fracture, and dental bone grafting
InFUSE® (USA)/ InductOS ® (Europe) : BMP-2 soaked in bovine type I
collagen sponge
Source: www.infusebonegraft.com
OP-1 ® : BMP-7 mixed with bovine bone collagen and sterile saline solution to form a paste
Clinical use of BMP-2 and -7
Sustained release of BMP-2 for effective bone regeneration
OH
O
O
O
C O OH C C
O O
O O O
O O
OH
O C O
HO O
OH OH OH OH
OH O
O
O
OO OO O
O
O • Hydrophobic amino acids (A, F, G, I, L, M, P, V, W, Y)
• Positively charged amino acids (K, R) Bone Morphogenetic Protein 2
Hydrophobic π domain Negatively charged
oxygenated group
Graphen Oxide Surface
“Hydrophobic Interaction”
“Electrostatic Interaction”
Ti/GO- (Outer-most Layer: GO-)
GO-COO- GO-NH3+
Ti
GO-COO- (GO-) GO-NH3+ (GO+)
Layer-by-Layer (LbL) Assembly
Delivery of BMP-2 protein for osteointegration of dental implants
La WG et al., Small 2013, 9(23): 4051-4060
0 2 4 6 8 10 12 14 16 0
20 40 60 80 100
Ti Ti/GO+
Ti/GO-
Cumulative release (%)
Time (Days)
*
*
*
*
Release of BMP-2
Lower Loading Amount
& Higher Initial Burst
Sustained Release Ti
Ti/GO-
With BMP-2 Without BMP-2
Ti or Ti/GO- rings with or without BMP-2
Screws
La WG et al., Small 2013, 9(23): 4051-4060
Delivery of BMP-2 protein for osteointegration of
dental implants
Microfracture ACT Matrix assisted ACT
Current techniques for cartilage repair
E.A. Makris et. al. Nature Reviews 2014
Strengths
Regeneration of hyaline-like cartilage
Limitations
Multiple surgery procedures
Expensive cost due to in vitro cell expansion step
Patients’ waiting during the in vitro cell expansion period
Dedifferentiation of chondrocytes occurs during in vitro culture
Invasive surgery
Autologous chondrocyte transplantation (ACT)
(세원셀론텍)
ACT using scaffold (Bioseed-C, 독일 Biotissue 사)
• Autogenous chondrocytes seeded on PLGA-poly-p- dioxanone scaffold
• Advantages over ACT :
- No harvesting of periosteum
- Arthroscopical implantation possible ; less invasive
• Limitations - 2번 수술
- 세포배양하므로 가격 비쌈
3x2x0.2cm
9 months later
Transplantation of cells harboring therapeutic gene (코오롱생명과학, 미국에서 임상 3상 시험)
TGF-ß Gene
치료 유전자 (TGF-beta)를 바이러스벡터를 이용하여
타가 연골세포에 삽입
Cell + therapeutic gene 관절내 이식
1 주후 6 주후
TGF-β1
Control
Transplantation of cells harboring therapeutic
gene (토끼 동물시험)
돼지연골세포를 배양한 후 세포를 제거하고
남은 세포외기질
Biofilm to enhance marrow stimulation- mediated cartilage regeneration
㈜리젠프라임, ArtiFilm
TM줄기세포의 연골분화 유도 안됨
→ 섬유연골 재생 가능성
Scaffold to enhance marrow stimulation-mediated cartilage regeneration
(미국 Cartilix사, ChonDuxTM)
3D printing of organ
• Cell spheres are fused to form vessels
• Advantages
- Precise control of robotic bio- fabrication of organs and vessels - Fabrication of patient-specific
shaped organs
www.explainingthefuture.com
생체고분자 틀 INJECTION
3D bioprinting
• 3D프린팅 기술로 만든 기도 부목이 생후 2개월이었던 아기의 생명을 구했다.
• 이 아기는 숨을 쉬는 동안에도 기도가 약해서 기도벽이 무너져 내리는 증상 을 가졌다.
• 기도 부목은 3년 동안 아기의 기도가 정상적으로 자라나도록 도운 뒤 자연스
럽게 몸 속에서 생분해 됐다.
3D bioprinting of airway splint (기도 부목)
N Engl J Med 2013; 368:2043-2045
3D bioprinting of airway splint
A : the airway before placement of the splint
B : the patient-specific computed tomography–based design of the splint (red)
C : an image-based 3D printed cast of the patient's airway without the splint in place D : the splint in place
E : intraoperative placement of the splint (green arrow) F : the airway after placement of the splint
G : the airway 1 year after placement of the splint
N Engl J Med 2013; 368:2043-2045
얼굴뼈 종양을 제거한 후, 눈 선상이 일직선이지 않은 상태의 CT를 찍어 임플란트를 디자인하고 이를 3D 프린터로 출력하여 생분해성 임플란트를 제작함. 이를 이식하고 2주 경과 후 모습.
http://ims.postech.ac.kr/
3D bioprinting of bone
3D 프린팅 기술로 맞춤형 치아 교정기와 보청기 제조
3D bioprinting of absorbable stent
S.A Park etal. Materials Letters141(2015)355–358
신약 개발에 있어서, 동물실험에서 확인하기 어려운 부분을 인체유래 세포에서 생성된 인공 조직이나 인공기관으로 대체할 수 있음
유럽은 동물실험을 이용한 화장품 및 화장품원료 판매금지
피부와 관련된 독성시험이 동물실험 대신 세포실험 등으로 표준화되는 추세
인공피부를 이용한 피부자극성 및 독성 실험
동물대체시험법으로서 인공조직 및 인공기관모델 개발
동물대체시험법으로서 인공조직 및 인공기관모델 개발
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