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Boock Review
- Mitashov, V. I.
Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 117334 Russia
IZVESTIYA RAN. SERIYA BIOLOGICHESKAYA, no. 2, 1997
STOCUM, D.L. "Wound Repair, Regeneration and Artificial Tissues", Springer, Berlin, 1995
The main task of regeneration studies
is to understand the mechanisms underlying restoration.
It follows from the title of this book that it deals with the patterns
and specific features of key stages
of wound healing, tissue regeneration, and
epimorphic regeneration. It should be stressed at once that this
approach to comparative analysis of the mechanisms
underlying regeneration in three different systems proved to be
especially successful and productive. Another distinctive
feature of the book consists in that the restorative
processes are analyzed against the background of comparison
with the normal development for the corresponding structures. Not all also
that all eight chapters are quite clear and ended by a short
summary. All this make reading of the
book very entertaining. Let us first consider the main
concepts presented in this book. Chapter 1 "General Introduction".
In the history of humanity, regeneration was mentioned already in
the rock drawings. It is also known that in the countries
with ancient civilization, such as Egypt, China, and India, diverse
natural products were applied for wound healing.
The famous Greek and Roman physicians Hippocrates,
Celsus, and Galen used various wound treatments for stimulation
of their healing. The fantastic
concepts of regeneration have been most brightly expressed in
the myths of Ancient Greece, such as
legends of Hydra and Prometheus. The researchers who initiated experimental
studies of regeneration are well known: Trembly,
Reaumur, Spallanzani, Bonnet, and Blumenbach. Intensive
studies of regeneration are under way for no more than
a century. In the end of the
last century, regeneration was considered as an postembryonic regulation.
The book by Thomas Morgan was the best review at that time and it did not
lose its importance even now (Morgan, 1901). What are the main features
of regeneration during wound healing, tissue regeneration, and regeneration
of the entire organ? The wound healing is provided mostly
by the fibroblasts and leads to formation of a scar, while the initial
tissue structure is no restored. During tissue
regeneration, the damaged structure is restored, although not
fully and formation of a scar is also possible.
The tissue regeneration is realized as a result
of proliferation of the non-differentiated
precursor cells (epidermis, bone, or skeletal muscle) or dedifferentiation
of the already differentiated cells
(cardiac muscle, stomachal epithelium,
lens, or retina). The epimorphic regeneration
comprises restoration of the limb and tail in adult
urodelean amphibians and in anuran amphibian
larvae, regeneration of planarians and roundworms, restoration
of fins in fish, tail in lizards, limb in crayfish, and mandible
in urodelean amphibians. It is characterized by formation of
the mesenchymal blastema, epithelial- and neural-mesenchymal
interactions, and restoration of several tissue types
leading to a sufficiently complete regeneration
of the entire organ. In Chapters 2 "Wound Repair
in Adult Skin", 3 "Tissue
Regeneration" and 3 "Regeneration Amphibian Limbs", the
author provides a brief and very accurate
characteristic of the structures and analysis
of their regeneration. Five cell types are involved in
healing of the skin wounds: thrombocytes,
macrophages, epidermal cells, fibroblasts, and cells of the blood vessel
epithelium. These cells interact with the extracellular
matrix molecules, which can either be stored in the matrix or be
synthesized. The growth factors, which are low molecular weight
glycoproteins, stimulate or inhibit proliferation, migration, and differentiation
of the cells. They are intercellular
signal molecules that interact with the tyrosine kinase receptors of the
cell membranes and systems of secondary
messengers. The extracellular matrix
consists of proteoglycans (molecular filters)
and fibrous (collagen and elastin) and
adhesive (fibronectin and laminin) proteins.
Integrins are membrane receptors providing
for connection of the cells
with extracellular matrix. The involvement
of integrins in the transmission of signals to
the nucleus via the cytoskeleton cannot be
excluded. The main phases of wound
healing are inflammation, formation of the granular tissue,
and remodeling of collagen. Still little known data about reaction of the
embryonic skin to trauma present a special interest.
Unlike the adult skin, the embryonic skin is regenerated, rather
than forms scars! This is a very good model for comparative analysis of
scar formation by the traumatized adult skin. The
main distinction concerns the extracellular
matrix and presence of growth factors. When the embryonic
skin is damaged, the inflammation phase is absent and, correspondingly,
there is no infiltration of the neutrophils and macrophages, hypervascularization
is absent, chondroitin sulfate is not synthesized, TGF-beta
and bFGF are absent, excess of hyaluronic acid changes the
collagen organization and reduces the possibility of scar formation.
The author proposed the presence of a certain subpopulation of the fibroblasts
in the embryonic skin that enhance regeneration
and prevent scar formation. Possible heterogeneity of
the fibroblasts is of great interest for elucidating
the causes of scar formation. During tissue regeneration, just as
during wound healing, the first reaction
is inflammatory but the granular tissue is not formed
and, correspondingly, scar formation is absent.
The responses of these two systems to trauma is similar only
at the early stages. The late stages of tissue regeneration
markedly differ from the processes of wound healing. The damaged structure
is restored as a result of active proliferation
of reserve, weakly differentiated, or dedifferentiated cells. It
is not known what triggers cell dedifferentiation but it is clear that
removal of the surface antigens that stabilize the differentiated
state and reorganization of the cytoskeleton during interaction
with the extracellular matrix are key events
formation of a cell subpopulation providing for restoration of the removed
or damaged tissue. During limb regeneration, the key structure is
formed - blastema (Chapter 5 "Blastema Formation: Histolysis and Remodeling
of the Extracellular Matrix"). Its formation is preceded by
histolysis of the tissues adjoining the wound epidermis.
The inflammatory reaction leads to formation of a mesenchymal
blastema, rather than is realized by the fibroblastic
pathway of wound healing. The blastema is a phenocopy of the
developing limb bud. The blastema is characterized
by the presence of wound epidermis and interaction with the latter determines
the blastema growth. The inflammatory phase
is very short at the early stages
of regeneration. During limb regeneration, just as during
tissue regeneration, the neutrophils and macrophages do not produce bFGF
and TGF-beta and this makes the regeneration possible,
rather than the wound healing. The neutrophils and macrophages play only
the bactericide and phagocytic role. In adult Xenopus laevis,
which lack complete limb regeneration and in which the
cartilaginous spicule is formed,
the fibroblastic, rather than mesenchymal blastema is formed.
As a result, regeneration proceeds by the type of wound healing
with elements of tissue and epimorphic regeneration.
The epimorphic regeneration comprises formation of the wound epidermis
with an apical epidermal cap and a fibroblastema underneath. Formation
of a cartilaginous spicule in X. laevis is to
be considered, apparently, as a sign of transition from
the ancient epimorphic pathway of regeneration and
from tissue regeneration to restorative
processes inherent in higher animals. This reflects essential evolutionary
aspects of regeneration. Structural variation of the blastema and
resulting intercellular and intermolecular interaction determine the
further course of restoration (Chapter
6 "Tissue Interactions in
Limb Regeneration"). The extracellular matrix molecules are
involved in both positive and negative regulation:
they stimulate cell migration and proliferation and inhibit cell differentiation.
As a result of the matrix decomposition,
the cell-to-substrate interaction undergoes changes, thus leading
to changes in the cell shape and reorganization
of the cytoskeleton mediated by integrins and to changes in protein phosphorylation.
This leads, in turn, to cell dedifferentiation , changes
in the pattern of gene transcription, and production of an
extracellular matrix more similar with the
embryonic extracellular matrix. The specialized cellular
structures are degraded, apparently, by the lysosomes. The
type II collagen gene is switched off
and expression of the fibronectin and tenascin
genes and genes encoding the enzymes of hyaluronate
synthesis is enhanced. The new matrix preserves the cells in a dedifferentiated
state, thus inducing DNA synthesis and cell
divisions. Fibronectin and hyaluronic acid are largely responsible
for maintenance of the blastema cells in a non-
differentiated and proliferative state. Interrelated changes of the cells
shape and reorganization of their cytoskeleton
combined with reversible
protein phosphorylation lead to changes in the pattern of transcription.
A primary consideration in studies of regeneration is elucidation of
the mechanism underlying restoration of specific
tissue organization of the
amputated limb during repeated differentiation
of the blastema (Chapter 7 "Pattern Formation in Developing
and Regenerating Limbs"). The contribution of the
author to experimental solution of this problem is very big
and the results of his studies have already
been included in the contemporary text book (e.g., Gilbert, 1994). Let
us describe in more detail the most important results of his studies.
There are two possible mechanisms underlying restoration of the
three-dimensional limb structure. The first mechanism
implies that the blastema is non-differentiated and the pattern
of its differentiation is determined by the inducing influence of the adjoining
differentiated tissues. According to the second, alternative
mechanism, the blastema is a determined rudiment
inheriting all factors essential for its development
from the limb parental cells. Initially, the results of experiments
on transplantation of the limb fragmented were interpreted
in such a way that the blastema contains pluripotent
cells and the pattern of
their redifferentiation is determined by inducing
influences of the adjacent stump tissues. These experiments
lacked cell markers that would allow following
of the regenerate origin. Later D. Stocum and his coworkers showed
in experiments on the axolotl larvae that
when the blastema was transplanted in the dorsal fin zone, the
capacity for self-organization was expressed
and sufficiently complete limb regenerates developed.
In case of cross transplantations of the blastema between the anterior
and posterior limb stumps, the blastema were not subject to influence of
the recipient limb stump and differentiated as if in situ.
Moreover, when orientation with reference to the proximodistal
and transverse axes was changed to the reverse one, the blastema
retained its initial polarity. Thus, the
limb regeneration blastema as a whole is not pluripotent with respect
to the types of structures which they can give rise to. During limb
regeneration, there is a strict limitation:
the blastema does not reproduce the structures located proximally
to the site of its formation, since otherwise the regenerate
would not be functional. The blastema is
a self-organizing system containing information about the limb
structure and polarity. Autonomous development of the blastema,
specific for the site of its formation, led to formulation of the
concept, according to which the blastema cells inherit
from the initial limb cells the "memory" about the site of its origin
along the proximodistal axis. This "positional
memory" prohibits the blastema from formation of
structures located proximally to the plane
of amputation and, thus, provides for restoration of only amputated
parts. The positional memory is characterized
by a few important features. It is found exclusively
in the non-skeletal connective tissue of the limb. The dedifferentiated
cells alone express the positional memory and the latter is stable. The
positional memory is an important component
of development of the spatial organization during
regeneration. It can be transmitted across the
cell surface. The blastema cells formed at different levels of the
limb have the level-specific capacity for
recognition providing for their preferential association
with the cells located at these levels. The most elegant experiments
of this kind were also carried by D. Stocum and his colleagues. Some
of them already constitute the elementary bases
of almost all studies in this area and suggest that the positional
memory is transmitted across the cell surface.
According to the results of in vivo and in vitro experiments
suggesting the differences along
the limb proximodistal axis, cell recognition
(affinity) is a variable parameter. As a result of cultivation of the blastema
mesenchymal cells, nine possible combinations of the pairs of blastemas
from three levels (carpus-- metatarsus, antebrachium--tibia,
upper arm--femur) were obtained and analyzed. In order to distinguish
between the pair components, one of them was always labelled with 3H-thymidine.
The pairs of blastemas taken from the same level (carpus--carpus,
etc.) fused to form a uniform boundary, while in the pairs
from different levels, the cells of the proximal
blastema always tended to surround the distal blastema. In
in vivo experiments, the blastemas formed at the level of carpus,
antebrachium, or upper arm were grafted onto the dorsal surface
of the regenerating hind limb amputated at the femur middle, onto the site
of junction between the blastema and stump.
It was expected that the differences of the blastema
cells in the type of recognition determined by the level of their formation
should have been expressed in distal displacement
("sorting") of the transplants to the corresponding
level of the recipient regenerate.
This was what indeed happened! The regenerates from the carpus blastema
were displaced to the level of malleolus, those
from the antebrachium blastema to the level of tibia, while those from
the upper arm blastema remained at the
level of grafting, at the middle of femur. Hence, the
cells of blastemas formed at different levels of the limb have the
level- specific capacity for recognition which
provides for their preferential association with the cells located at he
same levels. In order to demonstrate a causal relationship
between the level- specific type of recognition, it
is necessary to prove that the
experimental change of memory, i.e., change of
the regenerate proximal boundary, is accompanied by modification
of the pattern of cell recognition.
This evidence was obtained in the experiments
with retinoids which shift proximally both
the positional memory and level-specific capacity for
recognition, which is essential for restoration of the normal spatial position
of the cells in the regenerate. During the recent years,
genes have been identified, whose expression
is essential also for specification of
the limb polarity, such as Shh, Hox-D, AV-1, Prx-1, Wnt-3, 4,
5a, 6, 7b, Msx-1, 2, Dlx-1, and En-1. However expression
of these genes was studied so far only in the developing limb. It was quite
logical to assume these genes or some of them may be expressed in the regenerating
limb. And this suggestion of the author was
convincingly confirmed by the Japanese scientists (Imokawa
et al., 1996). These genes are molecular markers of a
blastema zone identical to the zone of polarizing activity of the developing
limb. The mechanisms of morphogenesis
during development and regeneration appear to be similar.
Apparently, the author is quite right in assuming that it
is biologically disadvantageous for the nature
to develop different mechanisms for two similar processes. The last
chapter of the book (Chapter 8 "Artificial Tissues and Regeneration:
Can We Restore Structure and Function in Humans?") concerns the
analysis of attempts to produce artificial tissues for substitution of
defects and stimulation of regeneration. For stimulation of
limb regeneration in mammals it is necessary to develop approaches
to stimulation of the blastema formation. The blastema formation
implies remodeling of the intercellular matrix
to a state similar to that of the embryonic
limb bud matrix. Attempts have been repeatedly undertaken,
so far little successful, to stimulate the blastema
formation in anuran amphibians and mammals. It is necessary
to overcome and rearrange two barriers: pattern of
inflammatory reaction and tissue interactions.
The capacity for limb regeneration was reduced
during evolution, apparently, as a result of internal changes of
some or all limb tissues, rather than the cell environment.
The nature of these changes is so far unclear. The book summarizes
vast experimental materials selected very carefully.
Diverse hypotheses, concepts and
theories of regeneration put forward during
the recent years, sometimes mutually excluding, but nevertheless
co-existing were, as a rule, descriptive. The advantage of the book
by D. Stocum consists in an attempt to reveal the common features
in widespread theories of restorative processes
and this provided for predicting value of his generalizations and visualizing
prospects of research in the nearest future. This is
not only an extension of the science of regeneration, but, mostly,
its deepening, construction of the universal basis. I believe that
the book will be very useful for biologists due to systemic
orderliness of the considered experimental
data and universal principles of subordination of cellular
and molecular interactions both during development and regeneration
of the same tissues and organs. The progress in
studies of regeneration is very fast and I believe that the next
edition of this book is very desirable. The book should have been translated
into Russian. Undoubtedly, this book will become
a manual not only for specialists in regeneration,
but also for biologists of wider profile, professors
at the universities and medical schools, and postgraduate and undergraduate
students. REFERENCES Gilbert, S.F., Developmental Biology,
Sinauer, 1994. Imokawa, Y., Sekigichi, K., and Yoshizato, K., Analyses
of Genes Which Are Involved in Newt Limb Regeneration, Internat. Limb Development
and Regeneration Conference, Univ. York, 1996, p. W8. Morgan, T.H. Regeneration.
New York: Macmillan, 1901.
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