PROGRESS REPORT

CENTRE OF EXCELLENCE IN

ORTHOPEDIC TISSUE ENGINEERING

AND REHABILITATION

FUNDED BY TEQIP-II

National Institute of Technology, Rourkela

Sanction order No: F.No. 16-16/2013-TS. VII (General), (SC), (ST) Date: 13th June 2013

Name of the

Investigator

Department Designation

1 Dr. K. Pramanik Dept. of Biotechnology & Medical Engg. Professor

2 Dr. Subrat Panda Dept. of Mechanical Engg. Assistant Professor

3 Dr. A. Thirunanam Dept. of Biotechnology & Medical Engg Assistant Professor

4 Dr. S.K Sarangi Dept.of Mechanical Engineering Professor

5 Dr. Mukesh K. Gupta Dept. of Biotechnology & Medical Engg Associate Professor

6 Dr. Amit Biswas Dept. of Biotechnology & Medical Engg Assistant Professor

7 Dr S. Dasgupta Dept. of Ceramic Engineering Associate Professor

8 Dr. I. Banerjee Dept. of Biotechnology & Medical Engg Assistant Professor

9 Dr. D.P Mohapatra Dept. of Computer Engineering Associate Professor

10 Dr. Kunal Pal Dept. of Biotechnology & Medical Engg Assistant Professor

11 Dr. B.C. Roy Dept. of Metallurgical &Material Engg. Professor

Name of Investigators

Procurement

Sl no. Name of Equipment Present Status

1 RT-PCR

Equipment received

2 Force Plate 3D Motion Analysis

Equipment received

3 Hypermesh Equipment received

4 Environmental SEM

Process is initiated

5 Mimics

Process is initiated

•Research Scholar Recruitment-

Name of the student PhD/M.Tech by Reseach Darte of joining

1 Sudhanshu S. Behera PhD Dec.2013

2 Shreesan Jena PhD

Dec.2013

3 Gourishankar Saw M.Tech (R) 6th Dec.2013

4 G Gurimruti PhD July 2014

5 Tanushree Sahu PhD July 2014

Activities under CoE

Aim of the research

Development of load bearing orthopedic implants including knee & hip joints

Development of neovascularised Bone Tissue Construct

Osteochondral Tissue engineering

Design of rapid prototyping device for fabrication of 3D scaffold

Development of orthotic solution for patient having abnormal Gait pattern

cellular &

molecular

level

tissue and

organ level

Physiological

Functional

level

ORTHOPEDIC TISSUE ENGINEERING AND REHABILITATION

Our Approach…

WORK DONE SO FAR

Surface modification of load bearing titanium implants for

orthopedic application

Objective

Surface modification of Titanium alloy for improving the wear resistance femur

Surface modification of titanium stem material to reduce the stress shielding

effect and improving the osteoconductive

Development of porous titanium scaffold for load bearing bone defects

Fig 1: SEM micrographs of Thermally oxidized Ti6Al4V at 700°C for (a) 12 hrs. (b) 24

hrs and (c) 36 hrs

•The thermal oxidation of Ti6Al4V oxidized at 700°C suggests the

formation crack free oxide film

•The amount of anatase and rutile phase depends on the temperature &

duration of thermal oxidation

Fig 2: SEM micrographs showing the thickness of oxide formed at

the surface of thermally oxidized at 750°C for (a) 24 hrs (b) 36 hrs.

• The oxide formation of titanium has been studied earlier on the surface

that prevents it from further oxidation or oxygen diffusion at lower

temperature. The occurrence of mainly rutile phase on Ti6Al4V oxidized

at 700°C for 24hrs and 36 hrs suggests the formation of a thick oxide film.

Development of Neovascularised bone tissue construct

Objectives

Preparation & characterization of cobalt (Co+2) doped hydroxyapatite

in-vitro evaluation of its pro angiogenic and osteogenic properties

Generation of bone tissue construct by in vitro growth of cell-seeded scaffold

in vivo biocompatility of tissue construct by animal model test

-

S.No. Sample Notation Color Yield

(g)

Theoretical

%of doping

Experimental

% of doping

1 STD HAp HA1 White - 0

0

2 Pure HAp HA2 White 0.79 0

0

3 0.5% CoCl2-HAp HAC1 White 0.94 0.5 0.175

4 0.5% Co(NO3)2-HA HAN1 White 0.8 0.5 0.175

5 1% CoCl2-HAp HAC2 Light ash 0.79 1 0.33

6 1% Co(NO3)2-HAp HAN2 Light ash 0.83 1 0.37

7 5% CoCl2-HAp HAC3 Dark green 0.85 5 1.19

8 5% Co(NO3)2-HAp HAN3 Dark ask 0.8 5 1.2

Work done so far…

Synthesis of doped hydroxy apatite and characterization of doping

XRD Analysis FTIR Analysis

Physico-chemical Characterization….

Biological Characterization……

Cell proliferation study Cell Cycle analysis

Biological Characterization……

Study of Osteoblast Differentiation

Ru

nx 2

Ostr

ex

SE

M s

tud

y

Biological Characterization……

Study of Angiogenesis

VEGF expression

OBJECTIVES

To develop natural gum modified bio-polymeric hydrogel & tissue engineered scaffold

To study physico-chemical and mechanical properties of the hydrogel and scaffold

To study invivo and invitro biocompatibility of the hydrogel and scaffold

DEVELOPMENT OF CARBOXY-METHYL TAMARIND & TAMARIND

GUM MODIFIED HYDROGELS AND TISSUE ENGINEERED SCAFFOLD

FOR BONE TISSUE REGENERATION

Sample Gelatin(20% soln)

(ml added)

Carboxymethyl

Tamarind gum (20%

soln) (ml added)

Tamarind gum(20%

soln)(ml added)

Glutaraldehyde

(25%) Reagent

(ml)

C1 16 4 - 1

C2 12 8 - 1

C3 8 12 - 1

T1 16 - 4 1

T2 12 - 8 1

T3 8 - 12 1

C1 C2 C3 T1 T2 T3

0.5h 1h 2h 3h 4h 5h 6h 7h 8h

0.0

0.1

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1.0

% s

we

llin

g r

atio

time

CMT-1

CMT-2

CMT-3

0.5h 1h 2h 3h 4h 5h 6h 7h 8h

0.0

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% s

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llin

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atio

time

CMT-1

CMT-2

CMT-3

Sw

elli

ng

ra

tio

Time

0 200 400

0 200 400

A (##Temp./ّ C)

T1

T2

T3

PG

0.5h 1h 2h 3h 4h 5h 6h 7h 8h

0.0

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% s

we

llin

g r

atio

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TG-1

TG-2

TG-3

Sw

elli

ng

ra

tio

Time

0.5h 1h 2h 3h 4h 5h 6h 7h 8h

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% s

we

llin

g r

atio

time

TG-1

TG-2

TG-3

0 200 400

0 200 400

A (##Temp./ّ C)

C1

C2

C3

PG

DSC Thermogram DSC Thermogram

Swelling study Swelling study

Physico-chemical characterization of the hydrogel

0 10 20 30 40 50 60 70 80

0

10

20

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90

Fo

rce

Time(sec)

Force(T1)

Force(T2)

Force(T3)

0 10 20 30 40 50 60 70 80

0

10

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Fo

rce

(gm

)

Time(sec)

c(1)

c(2)

c(3)

pg(4)

0 2 4 6 8 10 12

0

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Fo

rce

Time

c(1)

c(2)

c(3)

pg(4)

0 2 4 6 8 10 12

0

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Fo

rce

Time

c(1)

c(2)

c(3)

pg(4)

0 2 4 6 8 10 12

0.00

0.05

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Fo

rce

Time(sec)

T(1)

T(2)

T(3)

0 2 4 6 8 10 12

0.00

0.05

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0.30

Fo

rce

Time(sec)

T(1)

T(2)

T(3)

Stress Relaxation Stress Relaxation

Compression Compression

Physico-chemical characterization of the hydrogel (continued…)

Hemocompatibility

Cytocompatibility

60 min 210 min 360 min 510 min 660 min

-5

0

5

10

15

20

25

30

35

40

CP

DR

time(min)

c1

c2

c3

60 min 210 min 360 min 510 min 660 min

-5

0

5

10

15

20

25

30

35

40

CP

DR

time(min)

c1

c2

c3

60 min 210 min 360 min 510 min 660 min

-5

0

5

10

15

20

25

30

35

40

CP

DR

TIME(Min)

T(1)

T(2)

T(3)

60 min 210 min 360 min 510 min 660 min

-5

0

5

10

15

20

25

30

35

40

CP

DR

TIME(Min)

T(1)

T(2)

T(3)

Cell viability of MG-63

Biological characterization of the hydrogel

In vitro drug release study

In vitro drug release study

OBJECTIVES

Development of orthotic solution for people having

abnormal Gait pattern

Studying gait parameters of control group

Designing computational model of orthotic solution

Fabrication of orthotic solution

Gait Analysis of Human Locomotion

Gait Analysis refers to the study of various motions executed by the human

body during day-to-day movement.

A normal gait cycle can be divided into the following phases, as shown

below:

Force plate system

for Gait Analysis

Ground reaction force curve

Ground Reaction Force

(GRF) is the reaction force

offered by the surface on

which the human executes

the act of locomotion.

GRF is the force which

propulses the body

forward.

Representative ground reaction force curve of a normal

Representative ground reaction force curve of a pathological gait

Representative centre of pressure curve of

a) normal and (b) pathological gait

Centre of Pressure graph

Line of action upon which the

gravitational force is acting.

The Torque Curve

Variation of the torque acting about

the ankle-foot joint with respect to

time.

Resultant force acting at a specific

point on the surface of the force

plate and a torque about the

vertical axis.

Representative torque curve of

a) normal and (b) pathological gait

Power expenditure curve of

a) normal and b) pathological gait

The power expenditure curve

Variation of power with time.

(-) shows energy stored during

swing phase. (+) value implies

release of energy (enabling the

forward motion)

The coefficient of friction curve

plots the variation of COF with

time.

Coefficient of friction curve of

a) normal and b) pathological gait

Impulse curve of a) normal and b) pathological gait

Impulse is the change in linear

momentum of the body.

May be defined as the product

of average force multiplied by

the time over which the force

is exerted.

GRF curve in slow gait

Slow-speed gait does not

produce sharp peaks at heel-

strike and toe-off phases of

gait as compared to fast and

normal walking speeds.

Slower gait patterns of

a) normal and b) physically challenged subject

Steps involved in the solid modeling process

Design of an Orthotic foot ware using Solidworks

Orthotic design for the individual

with ankle-foot joint distortion -

(a) three dimensional view (b) front

view and (c) side view.

OTHER ACTIVITIES DONE DURING THE PERIOD

MoU signed:-

•Ispat General Hospital, Rourkela

•University of South Carolina, Columbia, USA (www.usc.edu)

•Mondragon Unibertsitatea, Mondragon, Spain

(www.mondragon.edu)

•Konkuk University, Seoul, South Korea (www.konkuk.ac.kr).

Starting of PG Degree Course:-

A PG Degree course “M. Tech in Biotechnology & Medical

engineering (specialization: Tissue Engineering) has been

planned to be started from the coming academic year

•Publication-

List of journal paper (2013-2014)

•Nadeem Siddiqui, Pramanik, K. (2014), Effects of micro and nano β-TCP fillers in

freeze gelled chitosan scaffold for bone tissue engineering. Journal of applied polymer

science (in Press)-2014

•Senthilguru K, Pramanik K, Pal Kunal, Maiti T. K. & I. Banerjee, Development of

proangiogenic hydroxyapatite for bone tissue engineering communicated to Acta

Biomaterila (2014)

•Panda, N. N., Jonnalagadda, S., & Pramanik, K. (2013). Development and evaluation

of cross-linked collagen-hydroxyapatite scaffolds for tissue engineering. Journal of

Biomaterials Science, Polymer Edition, 24(18), 2031-2044.

•Panda, N. N., Pramanik, K., & Sukla, L. B. (2013). Extraction and characterization of

biocompatible hydroxyapatite from fresh water fish scales for tissue engineering

scaffold. Bioprocess and biosystems engineering, 1-8.

•Kaur, R., Pramanik, K., & Sarangi, S. K. (2013). Cryopreservation-induced stress on

long-term preserved articular cartilage. ISRN Tissue

Engineering,.http://dx.doi.org/10.1155/2013/973542

Publication in conferences

•PrajnaKabiraj, Indranil Banerjee(2014) “Alginate Bead Based Implant

For Drug Release And Tissue Engineering Application” oral paper

presentation national conference on Bio-mechanical science(NCBMS-

2014)

• Parinita Agrawal1, K. Pramanik, “Preparation Of Non-Woven Silk

Fibroin, Chitosan And Poly Ethylene Oxide Nanofibers By Free-Surface

Electrospinning For Cartilage Tissue Engineering” at second

International conference on Tissue Engineering and Regenerative

Medicine (ICTERM-13), NIT Rourkela, Nov-2013

Thank You

OBJECTIVE

• Development of EMG based wireless control system.

• Application of above control system in rehabilitation devices such as

wheel chairs.

Development of wireless EMG based control system

for rehabilitative device (wheel chair)

Basic Block Diagram of EMG based control system

CIRCUIT DESIGN IN MULTISIM

PCB DESIGNING AND DEVELOPMENT

BOTTOM VIEW TOP VIEW

PCB

Index

flexion

Middle

flexion

Thumb

flexion

All finger

abduction

EMG signal for

different finger movement

Signal classification

in Labview Glowing of LED as

per the signal

Classification of EMG signals for controlling the device

TESTING OF THE CLASSIFICATION

EFFICIENCY

PROTOTYPE WHEELCHAIR MOVEMENTS

(a) initial position (b) Forward (c) left and (d) right

COMPLETE SETUP FOR THE WIRELESS CONTROL OF

WHEELCHAIR MOVEMENT USING EMG SIGNAL

CONCLUSION

The ultimate target of the rehabilitation research work to be carried out

under CoE at National Institute of Technology Rourkela in the next three

years are as follows:

• To develop well-researched and optimized orthotic remedies for

individuals with altered gait patterns.

• To fabricate such product(s) and make it viable enough to be made

available in the market.

• To lay the foundation of research for a world-class facility for designing

and fabrication of orthotic and prosthetic solutions.

Methodology

MSC isolation and characterization

• Isolation, culture and sub culture of mesenchymal stem cells from umbilical cord blood

• Cell Characterization using MSC-specific surface markers such as CD105, CD73, and CD90, non specific markers such as CD 44, CD 45 and HLA-DR.

Cryopreservation Experiment

• Preparation of a less toxic cryopreservation solution

• To optimize the biological freezing procedure, hCBMSCs were control-rate frozen in different concentrations of the new cryopreservation solution at different freezing rate

• Thawing at 37°C and determining cell viability by Trypan Blue exclusion test.

Methodology • To investigate the effect of control-rate freezing on

cell metabolism, MTT assay was performed and control-rate frozen hCBMSCs were thawed, cultured and differentiated into chodrocytes and osteocytes.

• Development of tissue engineered construct using

hCBMSCs seed on silk chitosan scaffold

• Development of tissue engineered cartilage

construct(TECC)

• Cryopreservation of TECC

• Verify desirable characteristics of cryopreserved

chondrocytes and cartilaginous tissue constructs-

survival and proliferation study, Type I and II

Collagen and Gag Content by

immunocytochemistry, DNA content by

Expected outcome

• A clinical grade, non toxic freezing solution is expected to be formulated using biological cryoprotectants and natural osmoprotectants.

• A cryopreservation protocol that shall serve as a standard for the preservation of tissue engineered cartilage construct.

Up-dated progress achieved vis-à-vis time schedule of objectives proposed.

Duration Progress of Work Done

Dec 2013- Feb 2014

MSCs were isolated from

umbilical cord blood and

characterized.

Less toxic cryopreservation

solution was prepared and

its potential was verified by

cryopreserving hCBMSCs.

The desirable

characteristics of

cryopreserved cells were

also verified in terms of cell

viability and cell

differentiation potential

Mar –May 2014 Development of TEC

Cell Differentiation Assessment

To construct an instrumented staircase with adjustable step height for recording ground reaction force and moment about ankle data during stair ascent or descent

Use of a 3-D motion analysis system to find out the dynamic reactions at other parts of the body (equipment to be acquired shortly).

Improvements in the existing orthotic solution.

Applying the model solution to a larger control group with similar disability or restriction in motion

Neural and myoelectric signals need to be studied in order to develop powered orthotics for amputees. This is an emerging sector in powered prosthetic limb design with the potential to revolutionize prosthetic development.

FUTURE WORK PLAN

SCHEMATICS REPRESENTATION OF

THE EMG ACQUISITION SYSTEM

SCHEMATICS REPRESENTATION OF

THE DEVELOPED WIRELESS CONTROL

SYSTEM

Conclusion

Easy implementation

Wireless control system

Easy to use

Low cost

Development of scaffold for bone tissue engineering using Curcuma longa extract

Objectives:

i. To extract turmeric essential oil from turmeric plant (Curcuma

longa L.) and their characterization for desired properties.

ii. To develop the scaffolds using essential oil for bone tissue

engineering applications.

Methodology 1. Extraction of turmeric essential oil using steam

distillation method

2. Characterization of extracted turmeric oil by Gas Chromatography Mass Spectroscopy (GC-MS)

3. Preparation of phytochemical scaffold (alginate/CS/CL-E scaffolds) using alginate, chitosan and curcumin extract

4. Characterization of prepared scaffold using XRD, FTIR, SEM, TGA-DSC methods

5. To study swelling property, porosity, cell proliferation, cell viability, cell attachment assay, protein adsorption, in-vitro degradation assay of the prepared scaffolds for bone tissue engineering applications.

Preparation of alginate/CS/CL-E scaffold

Future work plan

• Morphology, porosity and swelling properties of the scaffold will be investigated

• Protein adsorption on the scaffold will be measured and compared with that of control.

• Degradation of scaffold under physiological conditions will be determined.

• The adhesion and spreading of hMSCs on the scaffolds will be analysed by SEM (Cell attachment study).

• The viability of hMSCs on the scaffold will be tested using Alamar blue assay (cell viability assay).

• The cell proliferation assay will be carried out using Alamar blue test (Cell proliferation assay)

• Differentiation of hMSC by alkaline phosphatase (ALP) activity will be analysed.