Preclinical reporton 

Decellularised bovine pericardium for therapeutic implantation2011

Biomedical technology WingSree Chitra Tirunal Institute for Medical Sciences and Technology

ThiruvananthapurampProject funded by DBT

D ll l i d b i i diDecellularised bovine pericardium

Decellularised bovine pericardiumBSE freeBSE free0.25 to 0.35mm thickness8cmx8cm size piecesP i ll li k d d R d l blPartially crosslinked and Remodelable

Hydrophilic surface- contact angle 350

Hydrated- 76% water contentBiodegradablePorous- 37% is 6-18m size pores pMade up of collagen 92% and

Elastin 8%Stored in 70% ethanolStored in 70% ethanol

Sourcing of animal tissue and its collection

BSE free Bovine pericardium is sourcedpericardium is sourced from Kerala Livestock Development Board (KLDB), a Government ( ),of Kerala Organization which has modern cattle farms with ISO 9001 certification.

KLDB: Animal tissue collection is based on international standard

The bovine tissue is sourced from a ‘low risk herd or ‘well monitored herd’ in which for at least the previous six yearsleast the previous six years

– There is documented veterinary monitoring

– There is no case of Bovine spongiform encephalitis (BSE)spongiform encephalitis (BSE)

– There is no feeding of mammalian derived protein

– There is a fully documented breeding history

– Each animal is traceable– Genetic material is introduced

only from herds with the same BSE free status and No brain penetrating stunning– No brain penetrating stunning methods are used for slaughter.

KLDB: Tissue collection based on ISO22442 (part 2)(p )

A i l ti i ll t d d k d dAnimal tissue is collected and packed under clean environment with minimum bioburden and care is taken to avoid cross contamination at all stages

Quality assurance

SOP for tissue collection and transport is SOP for tissue collection and transport is followed

Periodic audit is done to check Periodic audit is done to check conformance to standards and proceduresprocedures

Traceability of each tissue is maintained Brain and blood samples are tested for Brain and blood samples are tested for

BSE and other relevant bovine diseases.

Decellularisation of bovine pericardiumDecellularisation is done using a proprietary process and confirmed using standard proceduresR l d i ‘U h k l (2011) J Bi d M R A J 1 9 (3) 311 320’Results are reported in ‘Umashankar et al., (2011) J Biomed Mater Res A Jun 1;97(3): 311-320’

A B

A Normal bovine pericardium withA. Normal bovine pericardium with nucleus

B. Decellularised bovine pericardium showing absence of nuclear remnantsremnants

Residual DNA after decellularisation

Lane 1 marker

Lane 2 Decel BP

Figures showing reduction in DNA content And fragmentation of DNA following decellularisation

Lane 3 Decel BPdecellularisation.

Minimal criteria of decellularisation are <50ng/mg tissue DNA content, <200bp DNA fragment length and

Lane 4 Fresh BP<200bp DNA fragment length and Absence of nucleus demonstrated by light microscopyThe decellularised BP produce by this technique meets this requirement

Residual DNA

120 technique meets this requirement.

40

60

80

100

120

g/m

g tis

sue

0

20

40

Fresh BP 0.2DclBP

ng

Removal of non-structural proteins following decellularisation

Extractable protein:BP

0.25

0.3

e

0 05

0.1

0.15

0.2

mg/

100m

g tis

su

BPF

DclBP

DclBPW

0

0.05

1

m

BPF- Fresh bovine pericardium0.2Dcl: decellularised BP0.2DclPW: decellularised BP after wash

Non-structural proteins were removed by decellularisation and extensive wash. Residual proteins were rendered less i i b ild h i l li kiimmunogenic by mild chemical crosslinking

Electron microscopic analysis

A: Normal bovine pericardium showing intact corrugated surface B: Decellularised bovine pericardium showing eroded surface with exposure of collage fibers.

Decellularisation has resulted in retention of intact collagen and elastin which will provide adequatemechanical strength needed for surgical treatment

In vitro degradation

In vitro Collagenase (type2) resistance

60Commercially available BP

30

40

50

60

g

10

20

30mg

Decellularised BP

00 1 3 7

Days

Decellularised bovine pericardium is amenable to in vivo remodeling as it will be susceptible to tissue collagenases

Macrophage activation & cytotoxicity

Decellularised pericardium was seen no-cytotoxic (B) compared to commerciallyavailable gluteraldehyde treated pericardium (A)

Cytokine release from activated macrophage

400

available gluteraldehyde treated pericardium (A)

LPS20mic-lipopolysaccharide 20 mic gGlut cntr: glutaraldehyde

200

300

400

pg

LPS20mic

Glut cntr

0.2 DCL

Glut cntr: glutaraldehyde Treated commercially available BP0.2Dcl: decellularised BP

0

100

IL1beta TNFalpha

cytokines

Inflammatory cytokine release was noticed more in commercially available BP than decellularised BP

Immunogenicity testing based on ASTM F1906 98; (Re approved 2003)ASTM F1906-98; (Re-approved 2003)In vivo immune response was studied by rat sc implantation for 60 days followed by evaluation of antibody response and cell mediated immune response

D ll l i d BP (0 2D l) l i i d t l t ld h d t t d BP

Antibody response against Gal epitopes Antibody resposne (IgG,IgM,IgA) against BP proteins

Decellularised BP (0.2Dcl) was seen less immunogenic compared to glutaraldehyde treated BP

2.6

2.65

2.7

OD

BP proteins

0.250.3

0.350.4

D

2.45

2.5

2.55

0.2Dcl GlutBP

O

00.05

0.10.15

0.2

0.2Dcl Glut

OD

DHSR

7

Lymphocyte transformation test

3(Delayed hypersensitivity reaction)

23

4

56

pad

thic

knes

s

0.2Dcl

GlutBP0

0.51

1.52

2.53

OD

0

1

2

0h 1h 24h 48h

Foot

0BP 50BP 0BP 50BP 0BP 50BP 0BP 50BP

0.2Dcl GlutBP GlutBP 0.2Dcl

21D 90D

C l ifi i 60D i l iCalcification response: 60D rat sc implantation

In vivo Calcification: rat s/c model

30

35ht

15

20

2530

mg

dry

wei

gh

0

510

mic

g/m

Gl tBP GlutBP 0.2DclGlutBP 0.2Dcl

Decellularised BP produced lessDecellularised BP produced less calcification compared to commercially available BP

Tissue response: 60D rat SC implantation

Decellularised bovine pericardium showedless inflammation, better host cell incorporation, newcollagen laying and neo-angiogenesis within graftIndicating remodeling of graft

Gluteraldehyde treated bovine pericardium showed inflammation at graft –tissue interphase (arrow), graft devoid of host cell incorporation and angiogenesis limited to graft- tissue interphase. Graft remodeling was not visible

Preclinical testing

– Heavy metal and trace element content √– Mechanical testing √

Tensile strength

Burst strength Suture retention

– Cytotoxicity (Direct and on extract): √– Haemocompatibility √Haemocompatibility √– Systemic toxicity √– Intracutaneous irritation √

√– Sensitization: √– Endotoxin testing√– Genotoxicity (AMES test): ongoingGe oto c ty ( S test) o go g– Sterility testing: passed √– Validation of sterilization: √

V lid ti f i ti ti f t i ibl t i– Validation of inactivation of transmissible agent: ongoing

Heavy metals and trace elements:5g sample

No Element Result0.2Dcl (ppm)

Remarks (data from Hazardous substance data bank, NLM, USA)

1 Iron (Fe) 38.42 LD50 of Iron oxide= 5500mg/Kg (PO)2 Lead (Pb) 0.25 LD50 of Lead nitrate (iv) =93mg/kg (IV)3 Arsenic (As) BDL Detection limit is 0 053 LD50=26mg/kg3 Arsenic (As) BDL Detection limit is 0.053, LD50 26mg/kg4 Nickel (Ni) 0.37 LD50 of Nickel Chloride (IM)= 71mg/Kg (IM)5 Manganese (Mn) 0.25 Manganese chloride LD50: 250mg/Kg6 Cadmium (Cd) 0.06 LD50 Cadmium Chloride 3.5mg/Kg (iv)7 Mercury (Hg) BDL Detection limit is 0.0610, LD50=5mg/Kg

8 Ch i (C ) 2 38 Chromium (Cr) 2.3 LD50 of Chromium iv oxide= 80mg/kg (IV)9 Copper (Cu) 0.425 LD50 of copper oxide = 470mg/Kg (PO)

10 Zinc(Zn) 1.875 LD50 of Zinc Chloride= 1100mg/Kg (PO)

11 Magnesium (Mg) 84.28 LD50 of MgSO4=1200mg/Kg (sc)

The values are within safe limits12 Calcium (Ca) 141.05 LD50 CaCl2= 1000mg/Kg (PO)

Burst Strengthg5

3.5

4

4.5

pa)

2.5

3

t  strength (M

1

1.5

2

 Burs

0

0.5

0.2 DCL Fresh Unprocessed BP PETp

Decellularised BP has mechanical strength comparable to fresh BP

Tensile strengthg Mechanical Characteristics

8

4

6

tren

gth (M

Pa)

2

Tensile

 st

00.2 DCL  Fresh Unprocessed BP

Decellularised BP has mechanical strength comparable to fresh BP

Elongation at breakg100

80

(%)

40

60

gatio

n at Breakk 

20

40

Elon

00.2 DCL  Fresh Unprocessed BP

Decellularised BP has mechanical strength comparable to fresh BP

Summary of test results: yMechanical continued

Name of test Result Remarks

Porosity evaluation using Micro CT 0.2Dcl BP has a pore The test material has y gexamination.

psize ranging from 6 to 30 micron size with nearly 24 to 37% of pore volume

adequate porosity for the specified application.

37% of pore volume constituted by 18 to 6 micron size pores.

Suture retention: test is done using 0 2Dcl BP showed suture The suture retentionSuture retention: test is done using Instron 3365, Universal testing machine with 100N load cell. Decellularised BP (0.2Dcl) strips

0.2Dcl BP showed suture retention strength of 5.76±1.73N.

Fresh bovine

The suture retention strength was observed adequate for the specified

of 10x50mm size (n=6) were used. 5/0Prolene suture with taper needle was used to take bite at 2mm distance from the

pericardium has a suture retention strength of 6 24±2 65N

application.

bite at 2mm distance from the edge.

6.24±2.65N

Summary of test results: in vitro Toxicity

In vitro cytotoxicity: by direct contact (ISO 10993-5,1999)

Test Non-cytotoxic to L929 fibroblast cells (figure B). Commercially available BP

Decellularised BP is non-cytotoxic

was moderately cytotoxic (figure A)

MTT assay: extract of the i l diff

50% extract of D ll l i d BP h d

Decellularised BP extract is t itest material at different

concentration is exposed to L929 cells. The effect on metabolic activity of these

Decellularised BP showed 96% metabolic activity, whereas commercially available BP showed 14%

non- toxic.

metabolic activity of these cells is measured by studying the reduction of yellow colored tetrazolium

lt 3 (4 5 Di th l thi l

available BP showed 14% activity

salt 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide to purple colored formazan.to purple colored formazan.

Summary of test results: in vivo toxicity

Acute Systemic toxicity:

Physiological saline extract and cotton seed oil extract of the test sample injected animals (i/v and i/p respectively)

Decellularised BP meets the requirement of test as per ISO 10993-11:2006(E)

ISO 10993-11:2006(E)

did not show any weight loss or abnormalities during the observationperiod of 7 days

S i i i C d il f l i h C d il fSensitization ISO 10993-10:2002/Amd.1:2006(E)

Cotton seed oil extract of test sample with Freunds’ adjuvant is injected intra dermally for induction. Seven days after this, induction is repeated by topical

Cotton seed oil extract of Decellularised BP meets requirement of the test as per ISO 10993-1:2006(E) this, induction is repeated by topical

application of test sample extract for 48h. 14 days after this, challenge dose is applied topically for 24h and the animals

b d d d t 24 h 48h

per ISO 1099310:2002/Amd.1:2006(E)-Biological evaluation of medical devices: Part 10. T t f i it ti dwere observed and scored at 24 h, 48h

and 72 h for erythema and oedema. A reaction of grade 1 or above is positive.

Test for irritation and delayed type hypersensitivity: Clause 7.4 Maximization test forMaximization test for delayed hypersensitivity

Summary of test results: in vivo toxicityIntracutaneous Reactivity test (ISO 10993-10:2002 (E) Amd 1:2006(E) In this test

The physiological saline and cotton seed oil extract of the test material meet the

Decellularised BP did not cause intra cutaneous irritation.

Amd.1:2006(E). In this test cotton seed oil extract andphysiological saline extract ofthe test material is injected intracutaneously into rabbit to

test material meet the requirement of this test.

intracutaneously into rabbit to assess the potential of the material under test to produce irritation

Endotoxin testing Kinetic Chromogenic method using Endosafe PTS endotoxin. Less than 0.5EUis considered non-pyrogenic

Test sample has <0.1EU/mL. Hence meets requirement of USP23NF21<85>, bacterial endotoxin testnon pyrogenic endotoxin test

Sterility test: USP 31/NF The test material do not Sterilization method is 26<No.71> contain any viable

microorganism and hence meet the requirements of sterility

adequate., Report on validation of sterilization is awaited.

Summary of test results: in vitro Haemocompatibility

In-vitro Haemocompatibility ( SO 0993 2002 ( )

Platelet consumption:6 31 3 24%

PET has platelet i 18 64 2 28%

Summary of test results: in vitro Haemocompatibility

(ISO 10993-4:2002 (E): This test consists of exposing the test materialto anticoagulated human

6.31±3.24%Leukocyte consumption: 4.14±1.05%. Erythrocyte consumption:

consumption 18.64±2.28%, Leukocyte consumption 18.36±1.67%, PTTdecrease 26.15±2.57to anticoagulated human

blood and platelet, RBC, WBC counts are taken. Percentage hemolysis, l k t dh i

Erythrocyte consumption:1.03±0.7%Increase in clottable fibrinogen: 4 9% 4%

decrease 26.15±2.57

Roy Joseph et al J.Mater Sci. Mater Med (2009)20 S153 S159leukocyte adhesion,

activation of coagulation system by studying PTT, fibrinogen assay and

4.9%±4%Decrease in PTT:24.8±2.4%% hemolysis:

(2009)20:S153-S159

PET is clinically approved material. Hence the valuesfibrinogen assay and

platelet activation study are conducted.

% hemolysis:0.12±0.025%Platelet activation: 0.13±0.005%

material. Hence the values reported for decellularised pericardium is assumed safe for human clinical use

Decellularised BP as cardiacDecellularised BP as cardiac patch

Preclinical trial in pig aortic/ LA patch model2010-2011

Test device: 0 2Dcl of following batches Test device: 0.2Dcl of following batches– Sample ID: BPCKA10760PATCH.06.12.001, Mar 2010

Sample ID: BPCKA10661PATCH.10.10.002, Jan 2010

Sample ID: BPCKA10207PATCH.08.12.002, Mar2010

Sample ID: BPCKA10742PATCH.10.16.001, Mar2010

• Control Device: SJM Biocor™ Bovine pericardium

Experimental design: Animal model adult pigExperimental design: Animal model adult pigOne month Test device 6

Control device 3Control device 36 months Test device 6

Control device 3Control device 312 months Test device 2

Control device 2

Methodology: graft implanted as aortic/LA patchMethodology: graft implanted as aortic/LA patch

Evaluation endpointsEvaluation endpoints•Clinical•Biochemical/HematologicalBiochemical/Hematological•Gross & Histopathological evaluation

Clinical, biochemical and hematological observationg

All test and control animals survived the All test and control animals survived the period of study.

Planned autopsy were conducted at durations of 1month, 6 months and 12 months.

Test and control animals showed normal hematological and biochemical parameters at the end of the study

Test 1month implant/explant

Sample ID: BPCKA10742PATCH.10.16.001, Mar2010

Control 1monthControl 1monthSJM Biocor BP implant/explant:p p

Test 6 months i l t/ l timplant/explant

Sample ID: BPCKA10661PATCH.10.10.002, Jan 2010

C t l SJM Bi BPControl SJM Biocor BP 6 months implant/explant p p

Test 12 monthsTest 12 monthsimplant/explant

Sample ID: BPCKA10207PATCH.08.12.002, Mar2010Sample ID: BPCKA10207PATCH.08.12.002, Mar2010

Control SJM Biocor BP 12 months explant

Histopathology of graft healing

Graft degradation at one month: aortic positionGraft degradation at one month: aortic position

Decellularised BP showing initiation of graft degradation at one month, whereas SJM Biocor Sh i N f d d i ll hi i dShowing No graft degradation at all at this period.

Test : 0.2Dcl Control: SJM Biocor

Graft healing and inflammation at one month: aortic positiong p

Decellularised BP showing early endothelialization and less inflammation at one month, whereas SJM Bi h i l d h li li i d i fl i hi i dSJM Biocor showing les endothelialization and more inflammation at this period.

Test: 0 2DclControl: SJM Biocor

Test: 0.2Dcl

Graft healing at six months: aorta

Decellularised BP showing thin neointima and integration of graft into native aorta. whereas SJM Biocor showing thicker neointima, no graft degradation and no integration of graft

into native vessel.

0 2Dcl SJM Biocor0.2Dcl SJM Biocor

Graft healing at six months: aortic position

A B

A: Decellularised BP showing excellent host cell incorporation in to the graftA: Decellularised BP showing excellent host cell incorporation in to the graft B: SJM Biocor showing very less host cell incorporation in to the graft.

Graft healing at six months: aortic positionGraft healing at six months: aortic position

A: Test device showing α smooth muscle actin + cells in graft regionB: Control device (SJM Biocor) showing absence of α smooth muscle actin + cells

Graft endothelialization: 6months

Decellularised BP showing compact neointima Control: SJM Biocor showing loose neointima

Graft healing at 12 monthsgA B

A: Decellularised BP showing integration of graft into native aorta (arrow) at 12 monthsB SJM Bi h i ll l ft ( ) ith t i t ti i t ti t t 12 thB: SJM Biocor showing acellular graft (arrow) without integrating into native aorta at 12 monthsMassons Trichrome

Graft healing at 12 monthsA B

A: Test graft showing no calcification and cellularity B: Control graft (SJM Biocor) showing areasA: Test graft showing no calcification and cellularityof the graft region (arrow) with structural integrity.

B: Control graft (SJM Biocor) showing areas of acellularity (arrow) with regions ofcalcification (open arrow)

Graft calcification at 12 monthsVon Kosa stainingg

A BA B

A: SJM Biocor BP showing graft calcification (arrow) at 12 monthsA: SJM Biocor BP showing graft calcification (arrow) at 12 monthsB: Decellularised BP showing owing no calcification of graft.

Animal study: summaryy y

Graft failure was not noticed in both test and control graft at all Graft failure was not noticed in both test and control graft at all periods.

All animals completed the implantation period un-eventfully. Bl d t ithi l i b th t t d Blood parameters were within normal range in both test and control animals at all periods

Test graft showed biodegradation, less graft calcification, early d th li li ti h t ll i ti i t ff ld dendothelialization, host cell incorporation into scaffold and

integration in to native aorta. Control graft failed to degrade, calcification of graft, no host cell

incorporation and no integration into native aorta was observed.

Decellularised BP as dura substitute

Preclinical studies animal studies are ongoing in rabbit modelrabbit model.

Thank youThank you