The Ultra Structural Study of Blastema in Pinna Tissues of Rabbits with Transmission Electron Microscope
Nasser Mahdavi Shahri,
Fereshteh Sadeghie Shakib
The aim of this study is to introduce an experimental
model to produce blastema tissue and to prepare samples for distinction
studies with Transmission Electron Microscope (TEM). Blastema tissue is
a group of undifferentiated cells that are able to divide and differentiate
in some parts of the body. New Zealand White male rabbits with body weight
of less than 2.5 kg and age of nearly 6 months were used in this study.
At first with the help of punching techniques 4.5 mL holes were produced
in rabbits` pinnas and then in 4, 5, 6, 7, 11, 21 and 24 days after regeneration
the tissues around the punching holes were biopsied, using a grasp with
more diameter than the one which the holes had been caused by and then
samples were prepared for histological studies with electron and objective
microscopes. Qualitative and quantitative investigations of electron micrographs
demonstrated that in punching site dedifferentiation of blastema cells
was obvious. The number of cells of each
type were counted and they were compared
with each other. The surface of organelles such as cytoplasm, nucleus,
endoplasmic reticulum, golgi sacs, mitochondrions, lysosoms and light
and dark vacuoles were also measured and compared and then the related
data were studied statistically. According to the
results obtained from morphologic (cytological) and quantitative studies
(comparison between different cellular organelles), The development of
Blastema tissue cells regarding the time of study is obvious, so that
the existence of chondroblast cells in chondrogenesis and endothelial
cells in angiogenesis in 11 and 24 days after regeneration can be seen.
So, considering the importance of understanding the blastema cells ultrastructure
in mammals, morphology of their development and the fact that it has not
been reported so far, this study is the first step and can be continued
by other researchers.
to cite this article:
Nasser Mahdavi Shahri, Fatemeh Naseri, Masoumeh Kheirabadi, Sakineh Babaie, Fereshteh Sadeghie Shakib and Mahnaz Azarniya, 2008. The Ultra Structural Study of Blastema in Pinna Tissues of Rabbits with Transmission Electron Microscope. Journal of Biological Sciences, 8: 993-1000.
Regeneration is new growth of the cells similar to the injured cells
structurally and functionally, instead of them (Kawamura et al.,
2005). Restoration and healing up is a kind of regeneration in which epithelium
and connective tissues will grow again (Alves, 2001; Pauwelyn and Verfaillie,
2006; Clark et al., 1998).
Lately, a lot of progress has been made in the information about physiology
of chronic wounds and functions and unique characteristics of fetal wounds
and the ability of healing by peptide growth factors (Down and White,
2003; Okamoto et al., 2003; Metcalfe and Ferguson, 2005; Mahdavi,
The histologic structure of rabbit pinna is made up of a single very
unordered plate of elastic cartilage with a kind of lenient perichondrium
on it which contains lots of elastic fibers. This cartilaginous layer
is covered by skin on the dorsal and ventral surfaces (Ten Koppel et
al., 2001; Mahdavi, 2007; Grimes, 2005).
Rabbit pinna is an effective experimental model for doing regeneration
and restoration process because after making a hole in the middle of rabbit
pinna, all of the lost tissues will be reconstructed (Ten Koppel et
al., 2001; Williams-Boyce and Daniel, 2005).
In this research, rabbit pinna was used as a live experimental model.
According to the references, after wound making in rabbit pinna, restoration,
pigmentation patterns, cellular differentiation and similar processes
to fetal periods or histogenesis can be well shown in these organs during
regeneration steps (Yotsuyanagi et al., 1999; Mahdavi, 2007; Sardari
et al., 2007).
Among the mammals rabbits are very unique, since the rabbit pinna regenerates
the injured or lost tissues from the edges of thickness of holes (Goss
and Grimes, 1975). So, the purpose of this research is studying morphological
processes of developing cells of blastema tissues during 5 to 24 days
after the wound appearance with TEM and in the period of rabbit pinna
healing. Blastema tissue is a group of undifferentiated cells which are
able to divide and differentiate in some parts of the organism body (Suzuki
et al., 2005; Abbott et al., 1981; Bauduin et al.,
2000). The findings of this research and expanding the studies on blastema
tissue development can also be applied in tissue engineering biotechnology
(Juncosa et al., 2003).
Since the basis of tissue engineering is using the cells and live substances
or extra cellular compounds together, each of which naturally or synthetically
and expanding the inducted parts or methods for reestablishment or function
replacements. The importance of considering these situation, not only
provides a type of new strategy in healing, but also some new ideas for
making tissues or organs (Zhiming et al., 2000; Haverich, 2004;
Gawronska kozak, 2004).
The other aim of this research is getting some information about morphologic
processes of blastema development in mammals. Morphologic studies of blastema
tissues with electron microscope have not been reported so far and this
makes our study new. In morphologic (cytological) assessments and ultra
structural and quantitative investigations (organelle surfaces comparisons),
the development of blastema cells is studied on the basis of time. A prospect
of this research can be the usage of the rabbit pinna for
injured tissue regeneration. More over, the cells after blastema culturing
can be placed in biologic scaffolding then can be grafted in the injured
tissue and regeneration process can be fallowed.
MATERIALS AND METHODS
Five rabbits were provided from Ghaem Hospital of Mashhad, which they
weighed about 2.5 kg and aged about 6 months. The considered organ in
this study was pinna.
The method of rabbit pinna punching: First of all hairs of the
pinna were removed with hair removing cream and then local anesthesia
was caused by Lidocain solution. Then 3 holes with 4.5 mm diameter were
made in three different anatomical regions of both pinnas of each animal
with punching apparatus.
Tissue preparation for transmission electron microscopy: In 4,
5, 6, 7, 8, 11 and 24 days after regeneration, those localities were punched
with a punch of more diameter than the one which the holes were made by
and then the specimens were quickly fixed in 2.5% gluteraldehide solution
for half an hour in room temperature, and were then kept 24 h in the
refrigerator. After that the specimens were at least washed four times
in phosphate buffer (pH = 7.2) during 18 h. Next fixation in 2% Osmium
Tetroxide was done for 1-2 h in room temperature. Then the tissues were
again washed once in phosphate buffer and twice in deionized very rapidly.
Dehydration steps were done completely with Merck ethanol solutions of
different degrees: 50 and 70% (each of them for 5-7 min). The samples
were then soaked in uranyl acetate for an hour in order to dye better
in the end. Dehydration processes were then continued as fallows ethanol
70% (5-7 min), 95% (7-10 min) and 100% three times (each one 5 min); dehydration
in Propylene oxide Solvent (5-15 min).
The samples were then infiltered during 4 steps:
Step 1:Epon paraldehyde (1 part)+Propylene oxide (2 parts) 30
Step 2:Epon paraldehyde (1 part)+Propylene oxide (1 part) 30 min
Step 3:Epon paraldehyde (2 parts)+Propylene oxide (1 part) 1-2
Step 4:Epon paraldehyde (3 parts): over-night
After complete passage of the samples and resin curing by placing the
capsules in 60 °C for 2 days, the blocks were first trimmed and then
cut by ultramicrotome. At last they were stained with 5% lead citrate
and 3% Uranyl Acetate, to be prepared for electron Microscopy. TEM model,
with which the images were captured, was LEO 912 AB with 1 nm resolution
and 80-500000 times magnification.
Sample preparation for light microscopy: After punching the regeneration
locality, just like electron microscopic preparation, the samples were
fixed at first, but in bouin`s fixative this time and then washed in 70%
ethanol. Other steps of tissue passaging were also done. After the tissues
being cut, stained with three chrome Hematoxylin and Eosin, Orsein and
Statistical works: On electron microscopic images, surfaces of
different cellular organelles were measured in square millimeter with
transparent millimeter paper. Different surfaces measured in this study
were as fallows the whole cells, cytoplasm, nucleus, lipid beads, endoplasmic
reticulum, light and dark vacuoles, lysosomes and mitochondria. Measurements
of different surfaces were made in 4, 5, 6, 7, 11 and 24 days after regeneration.
So, Table 1 was drawn in which different cellular and
organelle surfaces were compared with each other in different days after
Microscopical results: On the basis of TEM images of punched localities
in 4, 5, 6, 7, 11 and 24 days after regeneration, 3 different cellular
types were observed. Cellular classification is brought in Table
1. In the fifth day the images show tissues` destruction and autolysis.
Figure 11 shows a cell during autolysis.
In the day 7 some cells with big and light colored nuclei can be seen,
in which few organelles, light colored nuclei and the absence of condense
chromatin in them are a sign of activity and probable beginning of the
In light microscope images of the connective tissue under the epithelium,
derm fibrocytes propagation can be identified (Fig. 1-3,
7). In electron microscope images fibrocytes can also
be viewed dispersedly after the second week (Fig. 4-6,
||Different cell types in wound regeneration locality
4-24 days after punching on the basis of cellular type morphology
||Comparison of organelles surfaces mean in 4-5-6-7-11-24
days after regeneration (mm2)
||Angiogenesis and chondrogenesis processes (Magnification
4x10, P.A.S. staining, Light Microscope)
||Derm fibroblasts and collagen fibers propagation (Magnification
4x10, three chrome staining, Light Microscope)
||Angiogenesis in wound regeneration locality (Magnification
4x10, Orsein staining, Light Microscope)
||Cell autolysis 5 days after regeneration. Chromatin
condensation and nuclear degradation are of the reasons of cellular death (Magnification
||Fibrocyte and collagen propagation in the days 11 and
24 of regeneration. A collection of collagen fibers can be observed
around cells (Magnification 7500, TEM)
Before complete closure of rabbit ear holes, presence of stratified squamous
epithelium (epidermis) around the hole is obvious. Figure
7 shows the cells in this phase. Blood vein increase can be observed
in light microscope images, nearly 30 days after the holes closure (Fig.
3). Appearance and increase of endothelial cells at the edges of blood
veins and blood cells can be observed in electron microscope images. Figures
9 and 6 certify angiogenesis process. Chondrocytes presence can be
observed in the week 3. Figure 11 represents chondrogenesis
process which is also certified by light microscopy studies (Fig.
Statistical results: Table 2 indicates comparisons
between different cells and organelles surfaces in several days after
||Blood vessels 11 days after regeneration (Magnification
||Before complete closure of rabbit ear holes extended
epithelium around the hole is obvious (Magnification 4x10, Hematoxylin-Eosin
staining, Light Microscope)
||First row of Table 2 compares cytoplasm
surface amounts in different days after regeneration, the maximum
amount is shown in the 24th day, which shows more volume increase
of the cytoplasm indicative of most active time of the cell. The least
amount belongs to the 6th day which shows the least cellular activity.
||In comparison of nuclear surface amounts in the days after regeneration,
the maximal amount is in the 24th day, which shows strong activity
of the nuclei and more cellular variation in this day. The minimal
amount is in the 6th day which shows the least activity and cell numbers.
||Endoplasmic Reticulum (ER) surface in several days after regeneration
shows the maximal amount in the 24th day, which is indicative of lots
of cellular activity in this day, since the ER is expanded and substance
synthesis and exchange is much in the cell and the minimal amounts
belong to 5, 6 and 7th days, in which there are little activities
in the cells. In the 4th day, there is very little activity, which
with the occurrence of cell death process in days 5 and 6, decreases
to none, just like the 7th day in which only cells with big nuclei
can be observed and there`s not any organelle, but after cellular
gradual differentiation, to different cell types in days 11 and 24,
cellular activity increases and ER reaches to maximal amount.
||Probably a blastema cell 7 days after regeneration.
Organelles are not completed yet (Magnification 3300, TEM)
||A blood vessel longitudinal section 11 days after regeneration
(Magnification 3000, TEM)
||Fibrocytes in regeneration locality (Magnification 2000,
||Chondrogenesis (Magnification: 2500, TEM). A differentiated
cell for chondrocyte formation with vacuoles containing polysaccharides
||Strong activity of cellular secretion in 24 days after
regeneration, with vacuoles containing polysaccharides (Magnification
||Mitochondrial surfaces is compared in different days
after regeneration. The maximal amounts can be seen in the day 24,
which is again indicative of strong cellular activity from the point
of view of energy production and the need to this organelle. Because
of the absence of much activity in the days 4 and 7 and the cell death
in 5th and 6th days, there is not any mitochondrial surfaces in these
||Surface of Golgi apparatus is compared in different days, in which
the maximal amount can again be observed in the day 24, which is indicative
of lots of cellular secretions and substances synthesis and exchanges
and there is none in days 4 to 7.
||Lysosomal surfaces unlike most
of the other organelles reach their maximal amount in 5th and
6th days indicative of cellular death and destruction. Lysosomal surface
increase probably shows the presence of macrophages and cellular phagocytosis
during this period.
||Lipid bead surfaces are compared with each other, in different days
after regeneration, which shows the maximal lipid amount in the day
24 and none in the days 5, 6 and 7.
||In the last row in Table 2 bright colored vacuole
surfaces are compared, in which the maximum is again in the day 24
and the minimum in 6th and 7th days. This fact shows such vacuoles
contain polysaccharides probably and these morphologic findings certify
chondrogenesis which was also shown by light microscopy (Fig.
Understanding blastema tissue cells` ultra structure in mammals is important
and for studying the relationship between form and function in blastema
tissue cells, cellular behaviors and processes should be comprehended.
Considering the fact that such a research has not probably been reported
so far, this study was done on the basis of morphologic studies and with
the emphasis on cellular variation and cells ultra structures in regeneration
locality, by the help of electron and light microscopy in several specific
days after hole making in the rabbit pinna by punching technique. So,
different cell types were observed in different stages of tissues reconstruction.
The observed cells are brought in Table 1 on the basis
of morphological characteristics, cellular behaviors and tissue`s classification.
As Table 1 shows, cellular type A, was observed in
all the days after regeneration. In the 4th day, cells with small and
dark vacuoles were abundant, which probably contain lysosomes which will
lead to cellular destruction.
In the 5th day of regeneration, some cells with dark nuclei although
in destruction, can be observed in which there are dark vacuoles.
In the day 6, few cells with large nuclei were only observed, which these
very few cell numbers indicate complete cellular destruction in the day
before. In the 7th day, the cells seemed with large nuclei, but light
colored, which probably show undifferentiation of this type of cells.
In the days 11 and 24, nearly all A type cells were present which are
indicative of cellular variation in these days. B type cells were named
on the basis of cellular behaviors, in the days 7, 11 and 24. In 7 days
after regeneration, the cellular behavior observed was low stainability
of nuclei which probably shows undifferentiated blastema cells and blastema
appearance must have nearly begun from this day, since there`s much cellular
variation after the 7th day.
In the days 11 and 24, specific cellular behavior such as exocytosis
and intercellular conjunctions between epithelial cells are obvious. Exocytoses
show ER and Golgi apparatus strong activity.
C type cells on the basis of tissue cell types can be found in the days
11 and 24. In day 11th fibrocytes and epithelial cells are observed which
indicate cellular variation and in the day 24 blood cells and chondrocytes
can be seen. So, chondrogenesis probably begins from this day. It`s reported
from Raghe about regeneration which vasculature in pre blastema of pre
organs depends on presence of primitive steps of blastema bud in adult
newts (Regeh, 2002; Giampaoli et al., 2003).
In this study, vasculature needs the presence of blastema cells too.
After 11th day, blood veins and blood cells appear. Revasculature and
cellular redifferentiation in mammals begin in the second week of regeneration.
Presence of endothelial cells of venules walls, certify this fact. In
a study by Paul, from the disturbed locality of limbs, the blastema of
regenerated organs in a population of larva with different ages, was grafted
to the dorsal fin, together with anterior parts of spinal cord. 30 days
after the experiments of blastema grafts, skeletal muscle fibers appeared
in all age groups (Paul, 1962).
In an other study by Poss et al. the experiments on zebra fish
fin cutting was explained, in which blastema formation is a definite step
of facilitating the next regeneration (Poss et al., 2000; Murciano
et al., 2007; Nechiporuk and Keating, 2002).
Fibroblasts and collagen fibers are probably derived from the cells which
are called blastema in the 7th day of regeneration and aren`t differentiated
and will be differentiated to fibrocytes in the 11 and 24 days. In this
study the cells with blastema characteristics, which are probably blastema
were also observed in nearly the first week of regeneration (Fig.
8). It probably seems that undifferentiated blastema cells appearance
in mammals occurs earlier comparing with the amphibians and redifferentiation
is more quickly than amphibians. In a report from Buduin, Lassalle and
Boilly, effects of newts`limb blastema were studied on growth of the axon
from the spinal cord (Bauduin et al., 2000; Tsonis, 2000).
Redifferentiation in amphibians occurs between the days 22 to 40, so
the redifferentiating cells can be identified in these days (Santos et
al., 2002; Endo et al., 2004; Schwartz et al., 1985).
DNA in eukaryote cells are not free and together with proteins enter
in a structure called chromatin.
The chromatin fibers with big diameters show high density of chromatin
which is indicative of inactive chromatin (Wei et al., 2002). In
seven days after regeneration, the cells have large and light nuclei which
indicate low density chromatin and strong cellular activities. In Table
2 nuclei numbers were compared with each other in different days of
regeneration and the maximum could be seen in the day 24, so it seems
that the maximum cell numbers belong to this day.
As it was shown in the Table 2, ER surfaces reach to
maximum in the day 24 which is indicative of strong cellular metabolic
activity, but in the days 5-7 the cellular activities are the least.
Microscopic observations guide us to conclude that after rabbit pinna
punching, new cells and tissues are gradually appeared in the wound locality
and after about one week some big nucleated cells can be observed which
are probably blastema cells and must be the origination of all the other
cells. In 11 and 24 days strong cellular activities can also be seen.
Nuclear division, many propagating fibroblasts, exocytosis in some cells,
many light and dark vacuoles and expanded ER, all are signs of cellular
activities and probably differentiated cells formation.
This research was supported by the grant of the research office of Mashhad
University and special helps of Mrs. Pesian from the centeral lab of Ferdowsi University
of Mashhad, Iran.
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