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(1)

“Tissue Engineering Tissue Engineering

C til R ti

- Cartilage Regeneration –“

Takashi USHIDA

(Mechano-bioengineering) (Mechano bioengineering)

(2)

ES Cell Bone Marrow Stem Cell Matured Cell ES Cell Bone Marrow Stem Cell Matured Cell

◯ △

×

Tissue transplantation

×

Cell transplantation Organ transplantation

(3)

1. Cell sources

2. 3 dimensional scaffolds

(4)

C ll

Cell source

From who?

1. Autologous 2 Allogeneic

2 . Allogeneic

3. Heterologous

(5)

A t l

1. Autologous

it

・No immuno-rejection merit

No immuno rejection

No problem concerning virus

・No problem concerning virus demerit

・Limitation of cell sources

・Low activity of cells

(6)

2. Allogeneic

merit

・Higher activity of cells

・Capable of virus check demerit

・Immuno rejection

・Immuno-rejection Ethi l bl i

・Ethical problems in some cases

(7)

H l

3. Heterologous

merit

・No limitation of cell sources

・Higher activity of cells demerit

demerit

・Immuno-rejectionImmuno rejection

・Risk of unknown virus

・Risk of unknown virus

(8)

C ll

Cell source

From where?

1. ES cell

2. Bone marrow cell 3. Somatic stem cellSo a c s e ce 4 Matured cell

4. Matured cell

(9)

1 ES cell 1. ES cell

ES (Embryonic Stem) cell

Stem cell ・Self renewal

・Multipotential ES cell ・Self renewal

p

・Pluripotential

・Allogenic

・Ethical problem by brokingEthical problem by broking zygocyte

(10)

Bone Marrow Derived Stem Cells

1 Hematopoetic Stem Cell 1.Hematopoetic Stem Cell

2.Mesenchymal Stem Cell

(11)

Differentiation of Hematopoetic Stem Cell

(12)

S i S C ll 3.Somatic Stem Cell

Stem cells located in tissues Stem cells located in tissues

1)N l St C ll 1)Neural Stem Cell

2)Epidermal Stem Cell 3)Hepatic Stem Cell

4)Small Intestine Epithelial Stem C 5)Hair Root Stem Cell

(13)

Small Intestine Epithelial Stem Cell

(14)

Epidermal Stem Cell

(15)

ES Cell Bone Marrow Stem Cell Matured Cell ES Cell Bone Marrow Stem Cell Matured Cell

◯ △

×

Tissue transplantation

×

Cell transplantation Organ transplantation

(16)

1. Cell sources

2. 3 dimensional scaffolds

(17)

Biomaterial

Collagen, Hyaluronan, Laminin, Elastin

Biodegradable polymer

PLLA, PGA, PLGA PLLA, PGA, PLGA

Biopolymers Used in the Field of Tissue Engineeri

(18)

Cell - cell interaction C ll t i i t ti

Cell - matrix interaction

(19)

Collagen Type I

(20)

Types of Collagens yp g

(21)

Basement membrane Basement membrane

Structure of basement membrane

(22)

Proteoglycan Structure of proteoglycan Proteoglycan Structure of proteoglycan

(23)

Biodegradable pol mer Biodegradable polymer

・ PLLA (Polylactic acid) year

・ PLGA ( Polylactic-acid-polyglycolic-acid-copo month

・ PGA polyglycolic acid ) week

(24)

Biomaterial

Merit

・good biocompatibility

・good biocompatibility h i ll dh i it

・having cell adhesion sites Demerit

Demerit

・difficult for forming

・difficult for forming

・weak mechanical properties

(25)

Biodegradable pol mer Biodegradable polymer

Merit

・easy for forming

・enough mechanical properties

D it

Demerit

・hydrophobic

・hydrophobic

ll dh i it

・no cell adhesion site

(26)

PLGA mesh

Collagen sponge

PLGA-collagen mesh at original magnification × 60 PLGA-collagen mesh at original magnification × 20

SEM photomicrographs of PLGA knitted mesh SEM photomicrographs of PLGA knitted mesh

& its composite with collagen

(27)

Calcium Phosphates Calcium Phosphates

Ca/P formula

0 5 Calcium diphosphate Ca(H2PO4)2・H2O MCPM

0.5 α,β,γ-Ca(PO3)2 MTCP

1 0

Calcium

monophosphate

CaHPO4・2H2O CaHPO

DCPD DCP 1.0 monophosphate CaHPO4

α,β,γ-Ca2P2O7

DCP

α,β,γ-CPP 1.34 Octa-calcium phosphatep p Ca88 2H2(PO( 44))66・5H22O OCP

1.5 Caicium triphosphate α,β,γ-Ca3(PO4)2 α,β,γ-TCP 1.67 Hydroxyapatite Ca10(PO4)6・(OH)2 HAp,HA

2.0 Tetra-calciumphosphate Ca4P2O9 TTCP

(28)

3 major diseases of articular cartilage

sport injuries

osteoarthritis (OA)

rheumatoid arthritis (RA)( )

knee joint Articular cartilage

Type II collagen

(Type I collagen fibrous cartilage)

no blood vessel no nerve

Lateral femoral groove stained with S-O low regeneration activity

(29)

Cell Transplantation

Genzyme system

Cell Transplantation

Clinical application of autologous chondrocyte implantation

Regenerated Tissue Transplantation Transplantation

(30)

SEM Photomicrographs g p

PLGA-collagen hybrid mesh Polylactin 910 (PLGA) kitted mesh

Hybridization

PLGA PLGA Collagen

microsponges

(31)

Gross Appearance Gross Appearance

Single Sheet Laminated (5-sheet) Rolled

12 weeks

12 weeks 12 weeks12 weeks 12 12

weeks weeks

8 mm 8 mm 1 mm

1 mm Thickness: 200 µm

Thickness: 200 µm

(32)

Histological Results of 5

Histological Results of 5--Sheet ImplantSheet Implant

4 w

4 w 4 w4 w 8 w8 w 12 w12 w

HE

8 8

4 w

4 w 44 88 12 w12 w

4 w

4 w 4 4

w w

8 8 w w

12 w 12 w

Safranin-O/fast greeng 4 w4 w4 w4 w 8 w8 w8 w8 w 12 w12 w12 w12 w

Toluidine blue Toluidine blue

(33)

Proteoglycan Proteoglycan

chondrocyte

Type II Collagen

Hydrostatic pressure loading to chondrocytes Hydrostatic pressure loading to chondrocytes

by weight or moving

(34)

1985、Hodge et al

Hardingham, Glycoforum 1998 Hodge et al. 1985 Proteoglycan

Direct measurement of loaded stress to hip joint

Cartilage model with collagen, chondrocyte

g g ,

proteoglycans & water

weight, movement hydrostatic pressure

Type II Collagen

articular cartilage chondrocyte

Chondrocytes physiologically loaded with hydrostatic pressure

(35)

Effect of Hydrostatic Pressure Loading

On Matrices Production in Chondrocyte Pellets

Hydrostatic pressure+ Hydrostatic pressure -

y

H-E staining of the pellet

Alcian-Blue staining of the pellet

Safranin-O staining of the pellet

Bar=200μm

Safranin O staining of the pellet

(36)

Physical Biochemical

Cell Differentiation

shear stretch growth factor Cell Differentiation

Tissue Regeneration

hydrostatic pressure

shear, stretch cytokine

Material (Scaffold)

collagen, hyaluronan, laminin

3 Essential Factors

for in vitro Tissue Regeneration for in vitro Tissue Regeneration

(37)

幹細胞

Culture Medium

M h l t ll C Me

Mesenchymal stem cell

Cell selection Cell differentiation

多孔性セラミック 高分子ゲル ゲルコート繊維束

基盤材料

Cell selection Cell differentiation

Physical Biochemical

growth factors growth factors cytokines

stretch shear pressure

幹細胞

骨折大腿骨インプラントbone

膝関節軟骨再建

cartilage

アキレス腱再建

tendon pressure

幹細胞

骨細胞 軟骨細胞 腱/靭帯

線維芽細胞

Tissue Engineering

Tissue Engineering

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