Tissue engineering using nonwoven:nonwoven: Limitations and futuristic thoughts

Dr. Sourabh Ghosh

Professor Department of Textile and Fibre Engineering Institute Chair ProfessorInstitute Chair Professor Indian Institute of Technology (IIT) Delhi

Basic principles of Tissue engineering

Monolaye

Cells from a biopsy

Monolayer culture

Culture on a 3D scaffold

Expanded cells

Generation of a graft

Load transmission,shock absorption

Articular cartilage

shock absorption, maintenance of functionalintegrity of the jointNo blood vesselsNo blood vesselsNo nervesCells of low mitotic activityThickness ONLY : ~ 0.5 to 3 mmVery poor regeneration capacityVery poor regeneration capacity

Ann Biomed Eng , 1999PNAS , 1997

‘bioactive’ scaffolds

should provide environmental signals to the llcells

to better control proliferationenhance the synthesis of ECMassembly of a functional neo-tissueassembly of a functional neo tissue.

PLAPLAHyalograft-C

Mesenchymal condensation during tissue formation

Day 4Day 4

Day 8

Day 15

Day 20

Diameter distribution for 13 cm  mat

7A

Electrospun Silk matrix13 cm  mat

01234567

100150200250300350400450500550600650700i ( )

coun

t

Std dev= 47.8Counts = 30

Diameter (nanometers)

Diameter distribution for 22cm  mat

45678

ount

S d d 02 9

B 22 cm  mat

01234

100150200250300350400450500550600650700diameter (nanometers)

co

Diameter distribution for 30 cm mat

Std dev= 102.9Counts = 30

30 cm matDiameter distribution for 30 cm  mat

01234567

coun

t Std dev= 139.1Counts = 30

C 30 cm  mat

0160 180 200 220 240 260 280 300 320 340 360 380

Diameter (nanometers)

Biomaterials, 2009, 30, 6530-6540

A B C

YMYMGPa

µm

Stem cells can sense difference in matrix stiffness

Stiffest electrospg matrix- day 1 day 10day 5

loose electrospg matrix- day 1 day 10day 5

Biomaterials, 2009, 30, 6530-6540

13 cm 30 cmGAG accumulationGAG accumulationA

2020μμmm 2020μμmmA B

Collagen type II mRNACollagen type II mRNAB

2020μμmm 2020μμmmC D

AGGRECAN GENE EXPRESSION

1 82

sion

Sox‐9 GENE EXPRESSION

1.5

on

Col 2 GENE EXPRESSION

3.0

ION

Cartilage specific gene expression

00.20.40.60.811.21.41.61.8

A B C D E F Gl

Relative gen

e express

‐0 3

‐0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

A B C D E F G

Relative gen

e expressio

0.0

0.5

1.0

1.5

2.0

2.5

A B C D E F GSamples

Relativ

e GEN

E EXPR

ESS

Samples 0.3Samples

Samples

Alpha5 GENE EXPRESSION

0.50.60.70.80.91

 gen

e expression

Alpha V GENE EXPRESSION

0.15

0.2

0.25

0.3

gene

 expression

Beta1 GENE EXPRESSION

0 4

0.6

0.8

1

gene

 expression

00.10.20.30.4

A B C D E F GSamples

Relative

0

0.05

0.1

0.15

A B C D E F GSamples

Relative 

‐0.2

0

0.2

0.4

A B C D E F G

Samples

Relative g

Rho‐A GENE EXPRESSION CDC‐42 GENE EXPRESSION FN‐1 GENE EXPRESSION

0.10.20.30.40.50.60.70.80.91

Relative gen

e expression

0.1

0.3

0.5

0.7

0.9

Relative gen

e expression

00.20.40.60.811.21.41.61.82

Relative gen

e expression

A=13‐DAY5 : B= 13‐DAY21 : C= 22‐DAY5 : D= 22‐DAY21 : E= 30‐DAY5 : F= 30‐DAY14 : G= 30‐DAY21

0

A B C D E F GSamples‐0.1 A B C D E F G

Samples

0A B C D E F G

Samples

TEM pictures of hMSCs grown over flexible nanofibrous mats

Rough ER

Gap junctionTight junction

1 μm 0.5 μm

5 μm 2 μm1 μm 5 μm 2 μm1 μm

0.2 μm0.5 μm 0.5 μm 0.4 μm

A B

TEM pictures of human embryonic stem cells on flexible mat

1 μm0 5 μm μ0.5 μm

C  D

1 μm2 μm

Prof Donald Ingber’s tensegrity model

on flexible-puckering surface –Rounded cell morphology

rounded nucleus

on rigid surface –flattened nucleus

rounded nucleus

Fig from, D Ingber, Cellular mechanotransduction: putting all the piecesn: putting all the pieces together again, FASEB J, Vol. 20 , 2006

Limitations: Poor cell infiltrationInadequate mechanical propertiesInadequate mechanical propertiesInconvenient handling issuesScale up

50 µmµm

Day 1

Day 14

Unstable cellular phenotypes

Transient Bone

JointCartilage

ORBone

PermanentCartilage

cartilage

JointCartilage

ORTransient cartilage OR

articularcartilage

TransientCartilage

ORArticular cartilage

cartilage

l10 1 col1a1

Late embryo andAdult

Mid‐early to late embryo

Very earlyembryo

sox9 col2a1

Chondrogenesis

col10a1

Osteogenesis

col1a1

sTransient cartilage cells undergo hypertrophy, angiogenesis and eventual replacement by conversion to bone while joint cartilage does not.

3D bioprinting

BMSCs cultured in 3D bioprinted construct in absence of TGF-β1resist hypertrophic differentiation and undergo articular cartilage yp p g gdifferentiation.

silk-gelatin bioink: direct negative regulatory role of on IHH signaling pathway

Bioprinting, 2017

signaling pathway

Massive upregulation of Wnt signaling pathway in minimizing hypertrophy d i h d i diff i i f BMSCduring chondrogenic differentiation of BMSC

Media coverage:28th May, 2017: The Hindu highlighted our work on 3D bioprintedcartilage;http://www.thehindu.com/todays-paper/tp-features/tp-sci-tech-and-agri/indian-researchers-develop-3d-bioprinted-cartilage/article18592836.eceg11-12th June, 2017: NDTV highlighted this work on 3D bioprintedcartilage;http://doctor.ndtv.com/bones-joints/knee-cartilage-recreated-by-iit-delhi-team-1711471; 4th June, 2017: The Asomiya Pratidin highlighted this work on 3D bioprinted cartilage; http://web.iitd.ac.in/~sghosh08/media.php30th May, 2017: 3dprint.com;y, p ;https://3dprint.com/176176/3d-bioprinted-knee-cartilage29th May, 2017: 3ders.org;http://www.3ders.org/articles/20170529-iit-delhi-3d-bioprinted-knee-cartilage-india-first-lab-grown-printed-tissue.html30th May, 2017: thebetterindia.com;http://www.thebetterindia.com/102746/iit-delhi-3d-bioprinted-catilage/1st May , 2017: indiatodayy , 0 7: d atodayhttp://indiatoday.intoday.in/story/3d-printed-cartilage-may-help-treat-osteoarthritis/1/942737.html25th June , 2016: indianexpress.com;http://indianexpress.com/article/technology/science/scientists-develop-new-bio-ink-for-3d-printing-287490830th May , 2017:3dprintingindustry.com; https://3dprintingindustry.com/news/indian-institute-technologys-3d-https://3dprintingindustry.com/news/indian institute technologys 3dprinted-silk-cartilage-helps-activate-organ-growth-114508/3rd May , 2017: iascurrentaffairsweb.wordpress.com; https://iascurrentaffairsweb.wordpress.com/2017/05/03/3d-printed-cartilage-to-treat-osteoarthritis-current-affairs-cor-civil-service30th May, 2017: www.fashionatingworld.com;https://www.fashionatingworld.com/new1-2/3d-bioprinted-knee-cartilage-with-printed-tissue-developedcartilage with printed tissue developedDBT website; http://www.dbtindia.nic.in/scientists-develop-joint-cartilage-in-the-lab-for-the-first-time/

I t t i t f fib ff ld t iIntertwinement of nanofibrous scaffold matrix compliance, cell shape, cytoskeletal mechanics, and developmental processes

1. Nanofibrous scaffold matrix compliance… human b i t ll d d lt h l tembryonic stem cells and adult mesenchymal stem

cells

2 Lack of cell infiltration2. Lack of cell infiltration

3. Effect of matrix chemistry: controlling cellular signaling and phenotypeg g p yp

AcknowledgementAcknowledgement

I d S i l dIndo-USAIndo-SwitzerlandIndo-IrelandIndo-KoreaIndo-Egypt

Kusuma Trust Outstanding Young Faculty Fellowship (2008 12)

Indo-PolandIndo-Italy

Kusuma Trust Outstanding Young Faculty Fellowship (2008-12)

High Impact Research Project – IIT Delhi

ITCDRL

BIRAC