2. Introduction
Regeneration is a complex process by which animals restore the shape,
structure and function of injured part.
Regeneration may occur through different modes including the
rearrangement of preexisting tissues, activation of resident stem cells and
regression of specialized cells or tissues to simpler form by the process
known as dedifferentiation
Owing to the lack of organ donors and complications associated with
immune suppressive treatments ,scientists are continuously looking for
new strategies to regenerate the injured heart.
Mammals, including human, form scar tissue after cardiac damage like that
caused by a heart attack. This scarring permanently impairs heart function.
But certain model amphibians can regrow heart tissue after injury.
3. The high regeneration ability of amphibians provides a valuable
model system to gain basic information on regeneration that may be
transferable to human trauma and diseases that cause damage to such
structures.
Vitamin A was found to be good model to accelerate regenerative
ability in anuran amphibians.
We decided to explore whether anuran amphibians(frogs & toads)
could regenerate heart tissue in different modes like insitu, in
transplantation setup and in culture medium under the influence of
Vitamin A, Emblica and Arjuna.
Transplantation technique will open new doors in the field of cardiac
tissue engineering.
In the present study three parameters will be discussed i.e.
heart regeneration in vivo,in transplantation and invitro.
4. Objectives
To enhance myocardial regeneration/ repair and
preserving cardiac contractile function after injury.
To develop cardiac patches from dedifferentiated
cardiac cells and to study their functional activity.
For this purpose cardiac patches will be developed
into ex-vivo culture medium and at ectopic site to
study their normal contractile functional activity.
To study how various drugs like, Emblica, Arjuna
and Vitamin A affect the heart regeneration.
5. Materials & Methods
Young and mature tadpoles of the toad, Bufo
melanostictus were employed as experimental animals.
Experiments were completed in three phases-
In the first phase a small cut was made in skin on
anterior ventral surface of young tadpole to expose the
heart and then the tip of ventricle was incised.(Fig.1)
Operated animals were reared in tap water (controls)
and in Emblica (0.01ml Emblica/ml tap water),
Arjuna (0.01ml Arjuna/ml tap water) and vitamin A
solutions (15 IU/ml) for first three days and then
transferred into water.
7. In second phase of experiment-meshed heart tissue was
implanted into a pit made on mid-lateral position of tail of
mature host tadpoles (Fig.2).Half (30) of the operated tadpoles
with implants were reared in water(control) and remaining (30)
were reared in vitamin A solution(15 IU/ml) for first three days
and then transferred into water. Experiment was terminated on
day 20 after operation.
8. Fig. 2 Photograph showing the site of implantation(SI) of meshed
cardiac tissue into a pit made on mid lateral position of tail
of the host tadpole (20X).
9. In third phase of experiment-the ventricle tip from ten young
tadpoles were incised and pooled and meshed in Leibovitz {L-
15} culture medium. Vitamin A was supplemented to the culture
medium for treated group. Cultures were terminated after 5, 10
15 and 40 days of inoculation {Fig-3}.
10. Fig 3: Schematic diagram showing the process of meshed cardiac tissue regeneration
in culture medium
a) Figure showing showing level of amputation through ventricle.
(b) Incised ventricle tip inoculated in culture medium.
(c) Preparation of cellular meshed extract of cardiac tissue (ventricle part) as explants.
(d) Formation of undifferentiated cells from meshed cardiac tissue into the culture
medium.
(e) Differentiation of newly formed cells into cardiomyocytes.
(f) Differentiation of cardiomyofibrils.
(g) Differentiation of cardiomyocytes into functional cardiac muscle
11. Results & Observation
Results obtained are presented in the table-1
Table1 : Influence of Vitamin A on heart regeneration in tadpoles
of the toad, Bufo melanostictus
The results presented in this study clearly demonstrate that vitamin A induced and accelerated
heart regeneration in all three modes of experiment wiz in situ, in transplantation setup and invitro.
The percentage of heart regeneration in vivo was 70% in Vit-A treated cases in comparison to
untreated control tadpoles it was 40%. The similar pattern of the percentage was found in second
mode of experiment i.e high in vitamin A treated cases (60%) and low in untreated control group
animals (30%). Where as meshed cardiac explant tissue in culture medium supplemented with
vitamin A showed similar sequential events of cardiac tissue regeneration. In culture medium
some of the undifferentiated cells found to aggregate on certain foci and showing further
differentiation (Fig 12). Consequently by day 20 cardiomyocytes differentiated into a network of
cardiomyofibrils. By day 40 of inoculation well developed cardiac muscles patches were reported
in vitamin A treated explant. The newly developed regenerated tissue were having normal cardiac
muscles architecture (Fig 13). Some of vitamin A treated cardiac patches showing normal
rhythmic beating (Fig 14).
12. Mode of Experiment Group Day of
presservation
No. of
operated
preserved
animals/
No. of explant
culture
examined
No of cardiac tissue Regeneration Percentage of
cardiac tissue
re
generation
Re
generated lost
part/Explant
with differen
tiated cardiac
muscles
Non
regenerated
un identified
tissue
I. Heart regeneration in situ (in
vivo)
C1
(control)
5 5 12 18 40%
10 5
15 10
20 10
V1
(vitamin A
treated)
5 5 21 09 70%
10 5
15 10
20 10
II. Ectopic cardiac tissue
regeneration(Transplantation
technique)
C2
(control)
5 5 09 21 30%
10 5
15 10
20 10
V2
(vitamin A
treated)
5 5 18 12 60%
10 5
15 10
20 10
III. In vitro cardiac tissue
regeneration
C3
(control
culture
medium)
5 30 42 78 35%
10 30
20 30
40 30
V3
(vitamin A
supplimented
culture
medium)
5 30 66 54 55%
10 30
20 30
40 30
13. •For the study of sequential events occurred during heart
regeneration, operated animals were preserved at different
time intervals (Day 5,10, 15 and 20 ).
By day 5 ,the wound showed proper healing (Fig.4) where
as on day 10 the injury site showed generation of new
cells (blastema cells) from neighboring healthy cardiac
tissue (Fig.5). By day 20 in vitamin A treated cases
complete regrowth of the amputated region, resulting in
functional heart. (Fig. 6 ,7 and 8).
14. Fig. 4
Photograph showing proper healing of injured
(amputated) heart on day 5.
WH = Wound healing (30×)
15. Fig.5
Microphotograph of a section passing through the
amputated heart of vitamin A treated young tadpoles
(5 days old ) showing dedifferentiated blastemal cells at the
site of amputation (100X)
H - Healing
DBC - Dedifferentiated blastemal cells
16. Fig. 6 Photograph of operated heart of 20 days old
vitamin A treated tadpole showing complete
regeneration of lost ventricular part. (40X)
RCTF –Regenerated cardiac tissue fiber,
RH- Regenerated heart
17. Photographs of regenerated heart of Vit-A treated tadpoles
(20×):showing complete heart regeneration on day 15 after
operation
( Figs. 7 & 8)
18. Promising results obtained in the second phase of experiment.
The mode of experiment was ectopic transplantation of meshed
cardiac tissue. The cardiac patches could survive and beat for up
to 15 days after engraftment on the tail.(Figs.9,10 and 11). The
pattern of regeneration was found similar to in vivo study
(phase first). It was high in vitamin A treated cases (60%) and
low in untreated control group animals (30%).
19. Figure 9. Photograph of cardiac tissue implant at ectopic site (mid
lateral position of tail).Figure shows development and growth of
cardiac implant (20X).VTR-Ventriclar tissue regenerate at ectopic
site, T-Tail
20. Fig10. Photograph of cardiac
ventricular tissue implant on the
tail of recipient. Vitamin A treated
tadpole showing normal growth
and cardiac beating on day 20
after implantation. (20X)
Fig 11. Microphotograph of a section
passing through the regenerated implant
(ventricular tissue) on recipient vitamin A
treated tadpole’s tail showing normal
differentiation of cardiac tissue. (100 X) .
RvTI - Regenerated ventricular tissue
implant,
RCTF - Regenerated cardiac tissue fiber
21. The survival of cardiac patches(meshed cardiac tissue)
under transplantation conditions-is a major challenge that
will have to be addressed. Therefore, the creation of
engineered tissue that not only assembles cardiac cells but
which also includes factors and /or cells favoring
revascularization.
The transplanted cardiac tissue differentiates like
miniature ectopic heart on tail . It appears to function
normally .(Figs.10 and11)
That’s amazing ………………….
24. Fig 13 : Microphotograph of a
section passing through the 40
days old explant after inoculation
in culture medium supplemented
with Vitamin A showing complete
regeneration of cardiac tissue
(ventricular part) (100x)
Fig 12 : Microphotograph of
sections passing through the 15 days
old explants after inoculation in
culture medium supplemented
with Vitamin A showing
differentiation of cardiomyocytes
(100x)
25. Fig 14 : Photograph of a regenerated explant (ventricular tissue) on day 40
after inoculation in culture medium supplemented with vitamin A (40X) .
Note - Regenerated cardiac tissue showing rhythmic beating.
RExVT- Regenerated explant ventricular tissue.
26. Conclusion
In light of the above results obtained in the present study
,amphibian system can make a substantial contribution to our
understanding of heart regeneration.
These important areas for research have the potential to provide
basic information that could be used to induce and control heart
repair in mammals.
The findings also suggest that all vertebrates including human,
might possess this regenerative capability and that methods could
be developed to tap it.
Transplantation technique opens new doors in the
field of cardiac tissue engineering.
