Designing of cornea tissue-specific bioink and

3D bioprinting of corneal stromal equivalent

 Introduction
Eye- external view Cornea – Anatomy and Ultrastructure

Cornea Sclera

O.5 mm

Transparent 11.5 – 12 mm
Avascular
Barrier
Superior refractive surface
Steeper centrally
Anterior corneal rigidity – maintains curvature
Consist of 5 layers

Dere et al., 2011, * photo courtesy - The National Eye Institute http://www.nei.nih.gov/health/cornealdisease
 Introduction

Anatomical factors
Tear film
Epithelium – homogeneity
Lamellar arrangement
Transparency
No vascularization
Lamellar arrangement of collagen fibrils

Physiological factors
Hydration Collagen fibrils
• Regular lattice
• Separated by less than wavelength of
light, i.e., 400 – 700 nm
• Scattered light destroyed

3
 Need for corneal graft
Corneal blindness – 4th leading cause of global
blindness

India - 6.8 million people who have poor

vision in at least one eye due to corneal
diseases – KC, wound scar, infection,
chemical burn, inflammation etc.

Corneal transplantation from cadaver - gold Only 1 in 70 of the needs are covered -
standard
worldwide
Clear need to develop alternative procedures
to restore corneal function.
12.7 million people waiting for a
For bridging gap - fully functional corneal corneal transplantation
tissue with a fair amount of accuracy

Gain et al., 2015; Fagerholm et al., 2010

 Preparation and characterization of the
decellularized corneal matrix (DCM) hydrogel

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 Decellularization of Cornea

C Native
150 Native
Decellularized D 150
Decellularized
125 125
* # * * * #
Content (%)

Content (%)
100 100

75 75

50 50

25 25

0 0
DNA sGAG Collagen DNA sGAG Collagen

hDCM bDCM
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Chameettachal et al., ACS Applied Biomaterials 2020
 Rheological Characterization
hDCMH bDCMH hDCMH bDCMH
A 10 6 B 10 5 Storage Modulus
o
29 C 8000 F 10 4 Storage Modulus 7000
Loss Modulus 7000 Loss Modulus 6000
10 5 10 4
Viscosity (mPa.s)

Time 6000 10 3 Time

Modulus (Pa)
5000

Modulus (Pa)
10 4 5000

Time (s)
10 3

Time (s)
4000
10 3 4000 10 2
10 2 3000 3000
10 2
2000 10 1 2000
10 1 10 1
1000 1000
10 0 10 0 0 10 0 0
10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 0 5 10 15 20 25 30 35 40 15 20 25 30 35 40
Shear rate(s-¹)
Temperature ( oC) Temperature ( oC)

hDCMH
hDCMH bDCMH
G 2% 3% H
1% 10 6 10 3
10 7
10 5 10 2

Viscosity (mPa.s)

Shear rate (s-1)

10 6
Viscosity (mPa s)

10 1
10 5 10 4
10 0
10 4 10 3
10 -1
10 3
10 2 10 -2
10 2
10 1 10 -3
10 1 0 100 200 300
10 -2 10 -1 10 0 10 1 10 2 10 3
Shear rate(s-¹) Time action (s) 7
Chameettachal et al., ACS Applied Biomaterials 2020
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 Biological Characterization
Cells are viable and active in hydrogel

DCM ***
2 Collagen ***

Absorbance (A U)
2D

*** ***
ns ns
1 ns
ns

0
1 7 14 21
Days

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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization

bDCMH hDCMH

100 *
**

Ki67 Positive cells (%)

80
#
60 *
40
**
# #
20

0
0 2 4 6 8 10 12 14
Days

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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization
DCM hydrogel exhibits wound healing property and cell migration in vitro

1h 24 h 30 h 30 h
With out

200 μm
With

200 μm

A. Wound healing assay B. Stained with DAPI

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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization
DCM hydrogel supports cell migration through the gel
A B
30 h

200 μm

80% of the cells were migrating

Confocal image - Depth analysis at a gel height between 200-250
µm from the bottom
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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization
DCM Hydrogel prevent differentiation to myofibroblast – Phenotypical analysis

W/O DCM
Myofibroblastic

Keratocytic
With DCM

Fibroblastic

Human corneal keratocytes cultured in DCM hydrogel – day 14

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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization

DCM hydrogel maintains keratocyte genotype – Gene expression analysis

DCM Hydrogel ***

6 Collagen

Fold change
4 ***
2
*

0
1

KS1
ST
SM
L
CO

CH
a

(*) indicates p≤0.05, (**) indicates p≤0.01, (***) indicates p≤0.001 and # indicates p>0.05 or non- significance (ns)
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Chameettachal et al., ACS Applied Biomaterials 2020
 Biological Characterization

DCM hydrogel maintains keratocytic genotype – Protein expression analysis

Human corneal keratocytes encapsulted in DCM hydrogel – day 14

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Chameettachal et al., ACS Applied Biomaterials 2020
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 In vivo evaluation of DCM hydrogel as a
prophylactic and therapeutic strategy for blinding
corneal scarring

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 Overview
• Scarring – Keratocytes Myofibroblast – Excessive ECM production – Opacity -
blindness
• Causes - Injury, infection, post surgical intervention, etc.
• Current treatment strategy – Cadaveric cornea grafting, full thickness transplantation
• No preventive measures
• No data on, prophylactic approach, treatment for existing scar other than donor cornea
grafting
• No data on application of hydrogel, injectable hydrogel or injectable corneal hydrogel
for scarring

Photo courtesy: Nagpal et al., 2020 18

 Assessment of Prophylactic potential of dCMHs:
Efficacy analysis in corneal injury model

Surgical plan and procedure

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Chameettachal et al., Manuscript under communication
 Prophylactic study
Corneal hydrogel accelerates epithelialization

Fluorescein staining 20
Chameettachal et al., Manuscript under communication
 Prophylactic study
Regaining of transparency with no visible scar

Follow up study 21
Chameettachal et al., Manuscript under communication
 Prophylactic study
Structural comparison – OCT analysis

22
Chameettachal et al., Manuscript under communication
 Prophylactic study
Thickness recovery at different
. zones - Pachymetry

23
Chameettachal et al., Manuscript under communication
 Prophylactic study
Microarchitecture evaluation
A B

PAS Staining

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Chameettachal et al., Manuscript under communication
3D Bioprinting of Corneal Stroma

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 Printing of support molds using stereolithography (SLA)

CAD Model SLA 3D printed construct

Average size of human cornea: 11.2 ( Vertical)X 11.8 (Horizontal) X 3.7 mm (Sagittal depth)
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Chameettachal et al., Manuscript in preparation
 Optimization of Process Parameters

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Bera et al., Macromolecular Bioscience, 2022, in press
 Bioprinting of Corneal Stroma

Print Parameters
Needle- 200 µm
Temperature – 29oC
Pressure – 0.2 bar
Speed - 30 mm/s
Cells used – Human corneal keratocytes (P3)
Cell density - 1× 106 cells/ ml of hydrogel
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Chameettachal et al., Manuscript in preparation 3D Bioprinting process
 Human-Sized Corneal Stroma

Diameter – 13.56 ± 0.22

Diameter after 14 days of
incubation - 12.16 ± 0.20 mm
Sagittal height - 3.65±0.21 mm

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Chameettachal et al., Manuscript in preparation
 Biological Characterization
A B C
Bioprinted Non Bioprinted Bioprinted Non-Bioprinted
150 # # ***
**
** ** 1.0 # #
# **

Absorbance (AU)
** #

Viability (%)
100

0.5
50

0 0.0
Day 1 Day 7 Day 14 Day 1 Day 7 Day 14

Bioprinted Non-Bioprinted
D 80
#
60
Deswelling (%)

*** ** *** ***

40

20 #

0
Day 1 Day 2 Day 4 Day 8

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Chameettachal et al., Manuscript in preparation
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 Mechanical Characterization

Compression analysis at day 14

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Chameettachal et al., Manuscript in preparation
In vivo evaluation of the Bioprinted cornea

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 Evaluation of the Implanted Bioprinted Cornea Structure
PRE OP DAY 08
Pre-op Day 03 Day 8

3-DP1

3-DP1

3-DP2
3-DP2

3-DP3 3-DP3

DSLR Images Fluorescein staining

 Regeneration of Epithelium - Fluorescent Staining

PRE OP DAY 08

3-DP1

3-DP2

3-DP3
 Evaluation of the implanted structure by OCT

PRE-OP Day-0 Day-3 Day-08

3-DP1

3-DP2

3-DP3
Conclusion

• DCM hydrogel has many advantages over existing materials including easy
availability, simple formulation procedures, and biocompatibility
• It prevents the trans-differentiation of corneal stromal keratocytes to
myofibroblasts and its potential as a prophylaxis against blinding scar formation
and also to treat existing scar.
• We have bioprinted a human-sized corneal stroma and characterized it

Future scope
• Toxicology study of hydrogel as per FDA norms
• Feasibility study on human corneal scar patients
• Application of DCM hydrogel for Ectasia - Pre clinical evaluation with emphasis on
keratoconus
• Feasibility study on human keratoconus patients
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