GRANULATION TISSUE *

by

Geoffrey Hadfield, M.D., F.R.C.P.

Sir William Collins Professor of Human and Comparative Pathology,
Royal CoUlege of Surgeons of England.

HOWEVER INAPPROPRIATE IT may be, granulation tissue is the only convenient term in common use for the immature and highly fertile mesenchyme which invades and subsequently replaces dead, dying, degenerate,
ill-nourished, time-expired and useless tissue in any situation in the body,
and under a multitude of pathological conditions. Its fundamental
significance in general pathology will be appreciated from the following
summary of its functions:
(1) It fills up all breaches of surface by occupying the space which
remains after removal of the dead material lying between the
edges of incised, lacerated and burnt surfaces, the ends of fractured
bones, torn or incised muscles, tendons and nerve trunks.
(2) It lines and attempts to fill ulcer craters, abscess cavities, fistulae,
sinuses or cavities produced by infection and ischaemia.
(3) It constitutes the peripheral and a variable part of the substance of
all chronic granulomata and encloses " insoluble" foreign bodies.
(4) It replaces inflammatory exudates of all varieties on skin, mucous,
serous, synovial or meningeal surfaces in addition to those formed
in the substance of solid organs.
(5) It invades all tissues devitalised by arterial ischaemia, totally or
partially replacing infarcts,- and forming the line of demarcation
between living and gangrenous or necrotic material: such material
may be necrotic tumour tissue.
(6) It replaces extravasated blood in any situation and all intravascular
thrombi or coagula, including those in the cardiac chambers.
(7) It forms the reaction zone at the edge of a mass of malignant
tumour tissue grafted from one animal into another of the same
species, and it is not unlikely that a similar reaction eventually
provides all spontaneous malignant tumours with the stroma upon
which their continued existence depends.
The essential function of granulation tissue is to replace useless material
by living mesenchyme. This is achieved by a process of vigorous vascular
and cellular invasion just as aggressive and at least as rapid as the infiltration of normal tissue by a malignant tumour. In the rabbit the advancing
front of granulation tissue, largely composed of newly-formed capillary
blood vessels, invades and replaces a mass of exudate at the rate of
0-2 mm. a day in the absence of infection (Clark and Clark, 1939;
Stearns, 1940).
It is beyond question that the newly-formed, active, proliferating
capillary blood vessels of granulation tissue are primarily responsible
for its capacity to invade. Large numbers of proliferating mesenchymal
* A lecture givea on Se?tenber 29th, 1951, at the Sloane Kettering Institute,
Memorial Center for Cancer and Allied Diseases, New York.

397

G. HADFIELD

cells follow in the wake of the capillary front. These cells need oxygen
and soluble metabolites for the energy-consuming process of mitosis, and
unless the dead tissue ahead is vascularised they would not only fail
to grow and differentiate in it but would certainly perish. Capillary penetration is therefore the basic process which makes mesenchymal replacement possible. New capillaries arise from pre-existing capillaries by the
familiar process of " budding." This is accompanied by widespread
mitotic activity affecting not only the endothelial cells of the penetrating
capillaries but also those behind them. A capillary bud is part of the
cytoplasm of an endothelial cell. It projects from the growing end of a
young capillary or from the convexity of a capillary loop. The nucleus
of the endothelial cell may be seen in this cytoplasmic projection and is
not infrequently seen to be in mitosis. It is highly significant that the buds
always grow in the direction of the dead material undergoing replacement.
They elongate as they grow and in so doing develop a thin cytoplasmic
process which also travels in the same direction (Figure 1). The same
"directed " growth was demonstrated by Clark and his colleagues
(Sandison, 1928; Clark et al., 1931), who watched through a transparent
window the penetration by capillaries of the dead material which filled

A~~~~~~~~~~~~~~~~~~~~~~"

Fig. 1. Capillary New Formation.

(New capillaries "directed" to dead material. indicated by

(A) Endothelial cell in mitosis.

(B) Bud formed from its cytoplasm: early canalisation.
(C) and (D) Endothelial cells gliding into capillary " shoot." Bud persists, developing
a process.

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GRANULATION TISSUE

up the central part of a circular hole cut out of the external ear of the
rabbit. The radial arrangement of the capillaries was a clear indication
that they were taking the shortest route to the centre of the circular
lesion. Quite soon after the bud has formed and elongated, the basement
membrane of the capillary bulges into it. Canalisation then proceeds
but the terminal cytoplasmic bud with its " directed" process still persists and pursues its appointed course. Canalisation involves another
process whereby the endothelial cells lining the capillary wall glide into
the newly-formed culs de sac and eventually line the basement membrane
of the young capillary shoot.
The provision of enough endothelium to line the young capillaries
and the cellular activity in the capillary buds quite clearly call for a
considerable degree of mitotic activity, and it must be significant that the
process of new capillary formation is strictly comparable with the normal
development of capillary vessels in the embryo. We know very little about
the mechanism which directs the capillary buds along the shortest route
to the material which they will eventually invade and replace. A similar
and possibly identical mechanism guides the axons of the embryonic neuroblasts to their appropriate endings. Harrison, who cultivated these cells
in tissue culture forty-one years ago, observed that these cytoplasmic
processes travelled unerringly in a direct line to a small mass of clot
placed at some distance from the growing nerve cells, and in doing so
were able successfully to negotiate obstacles in the shape of particulate
matter lying in their path (see Fig. 2A). The progress of the bulb-like
extremity of the growing axon was found to be of the order of 03 mm.
a day. An identical mechanism is seen during the development of the
central nervous system. The axons arising from the groups of nerve
cells destined to become the posterior root ganglia travel towards the
primitive spinal cord, pierce its external limiting membrane and enter
the lateral column. On the other hand, the axons of the cells, which
will eventually become anterior horn cells, travel in the opposite direction
and make their way to their appropriate peripheral end-organ (see Fig. 2B).
A comparable phenomenon of " directed " movement of cytoplasm is
seen in the flowing movement of the clear ectoplasm of Amoeba towards
food particles, and the similar movement of mobile phagocytes in the
tissues. It is quite obvious that this controlled, purposeful movement
of cytoplasm is a general biological phenomenon of wide significance
in normal development, growth and function. We are, however, almost
entirely ignorant of its cause although there are reasons for assuming
that it may be controlled by rising or falling " gradients." In the case
of capillary penetration, for instance, it may be that the capillary bud
moves from a region where some chemical substance is in low concentration towards dead material which contains this substance in high
concentration.
Having penetrated the dead material, what is the nature of the medium
in which the young capillaries are embedded ? We may safely assume
399

G. HADFIELD

A..

.B

Fig. 2.

that the walls of these immature vessels are abnormally permeable and
it is not at all improbable that plasma proteins, together with hormones
and plasma enzymes, are able to pass through them more freely than
normally. The homogeneous and structureless appearance, together with
the physical properties of the medium, suggests that the exuded protein
is chemically altered and possibly " gelled." If these justifiable suppositions are true, the capillaries of granulation tissue lie in a medium which
is almost an exact copy of the plasma clot which has been discovered
to be so suitable for the growth of cells in tissue culture. Furthermore,
the enzymes contained in the exuded plasma are free to act upon the dead
tissue undergoing replacement, and by breaking down some of its constituents and bringing about its partial liquefaction, assist in the process of
capillary penetration. We must also conclude that the immature capillary
walls are freely permeable to hormones circulating in the blood. Recent
contributions to our knowledge of the fundamental parts played by
pituitary and suprarenal cortical hormones in controlling the proliferation
of mesenchyme strongly suggest that the hormones of this group, which
stimulate or retard cell multiplication, may directly or indirectly play a
predominant role in the growth and differentiation of the mesenchymal
400

GRANULATION TISSUE

cells of granulation tissue. This question will be reconsidered later in

the lecture.
We must now enquire into the conditions affecting the rate at which
capillary penetration proceeds. In the first place infection, inflammation
and ischaemia will certainly retard its progress or bring it to a standstill.
Two factors favour vascularisation. The first is liquefaction of the tissue
to be removed. This is brought about by enzymes derived from cells
of the dead tissue itself or contained in the " gelled " plasma in which
the penetrating capillaries lie. By this mechanism fibrin, nuclear material,
cytoplasm and the sarcoplasm of muscle are extensively broken down into
soluble products of relatively low molecular weight. Free haemoglobin,
fatty acids, soaps and the disintegration products of myelin are more
resistant to liquefaction and in common with such material as keratin,
mature collagen, and the matrix of dead bone and cartilage, offer considerable resistance. Material such as this, as well as a great variety of exogenous substances which may find their way into the tissues, will only
yield to the enzyme systems contained in the cytoplasm of tissue phagocytes.
Intracellular digestion of "insoluble" material by phagocytic cells
demands that a sufficient number of immature mesenchymal cells shall
differentiate into mobile histiocytes, and that these cells shall migrate
ahead of the advancing capillary front into the material to be replaced.
This implies that histiocytic phagocytes are the first extravascular
mesenchymal cells to differentiate in granulation tissue.
To summarise the early events in granulation tissue formation we may
say that capillary penetration eventually results in capillary vascularisation
of the material to be removed, establishes the biological conditions under
which mesenchymal cells can proliferate by mitosis, and provides plasma
enzymes to liquefy the dead material and phagocytes which digest it by
their own intracellular enzymes (Fig. 3).
There are several reasons for supposing that capillary penetration and
the mesenchymal proliferation which follows it and then accompanies
it are to a large extent under hormonal control, and the facts which favour
this supposition must now be examined. In the preliminary clinical trials of
A.C.T.H. it was noticed in patients who had been given the hormone for
a few weeks, that operation wounds made just before or during treatment
showed little or no tendency to heal. These observations stimulated
research into the rate of healing of skin wounds in cortisone-treated
animals. Some of the results obtained supported the clinical observations
and others did not. There were very marked differences between species
and the healing of skin wounds tend- to be complicated by infection and
involves epithelial regeneration. A far more satisfactory method is to
study the repair of a simple fracture in the cortisone-treated rabbit. This
has recently been carried out by Sissons and Hadfield (1951a). I am
indebted to these authors for permission to use skiagrams and photomicrographs taken during the course of their experiments. The outcome
401

%',. . X_. _,

G. HADFIELD

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;.

w *

'

i,Ll,V, ,,6

<

:<-.- ._{x

_1q,-q
Fig. 3. Granulation tissue.

of this research was the clear demonstration that capillary penetration of

the fracture haematoma in cortisone-treated animals was reduced to an
insignificant degree and as a direct consequence that bone formation was
greatly inhibited (Fig. 4).
Observations on the rate of epiphyseal endochondral bone growth
were carried out during these experiments and yielded results of wide
general biological significance (Sissons and Hadfield, 1951b). In young
rabbits given 20 mgm. of cortisone per day for 11 days there was a
striking reduction in the thickness of the cartilaginous epiphyseal plate
and an equally striking reduction in thickness of the metaphysis.
Photomicrographs show that capillary penetration of the zones of
c4lcified cartilage has been almost completely arrested and the formation
of cancellous bone behind them seriously inhibited. After 24 days on a
daily dose of 10 mgm. there was total suppression of the whole process

(Fig. 5).

To appreciate the significance of these changes it is necessary to

examine the mechanism of normal endochondral ossification, especially
the cytology of the zone of calcified cartilage in which the cells are
considerably enlarged (" hypertrophic " cartilage). The published
accounts of the changes leading up to this increase in size are those of
profound cell degeneration with an accumulation of large closely-packed
droplets in the cytoplasm. This is associated with nuclear swelling,
402

GRANULATION TISSUE

SIMPLE FRACTURE TIBIA: Young growing Rabbits.

Radiographs
at 19th day

Control
Normal growth
Firm union

10 mgm. Cortisone daily

Cessation of growth
Little repair
No clinical union

FRACTURE CALLUS: 20 days

Cortisone 20 mgm. per day

Degenerate cartilage
Little vascularisation

Control
Well vascularised network
bone
Fig. 4.

403

G.

HADFIELD

ENDOCHONDRAL OSSIFICATION AT EPIPHYSIS

Young Rabbit: Normal Control

Regular invasion of degenerate calcified cartilage by

an advancing front of newly-formed capillaries, behind
v,hich there is ccpious new bone formation.
Young Rabbit given Cortisone: 20 mgm.per day for 11 days.

Striking deficiency in
a. New capillary formation
b. New bone formation

(Many empty cartilage cell capsules in calcified cartilage.)

Young Rabbit given Cortisone :10 mgm. per day for 24 days.

Total suppression of bone growth.

Fig. 5.

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GRANULATION TISSUE

Fig. 6. Diagram of Metaphyseal Endochondral Ossification. Two capillary loops

invading calc:fied cartilage. Immature bone lies between them and osteoblasts in
close relation to capillaries. Degenerated cartilage cells and empty capsules in
calcified zone.

followed by shrinkage and pyknosis, and ends in disappearance of many

of the cells close to the invading capillaries, leaving their empty capsule
behind. I would suggest that the zone of calcified cartilage is in reality
a time-expired and useless tissue and that most of its cells are moribund.
This zone is actively invaded by the young capillaries and proliferating
osteoblasts of the metaphysis where they differentiate into cancellous bone
some distance behind the advancing capillary front (Fig. 6). It is
difficult to find any structural or functional difference between this purely
physiological process and the capillary invasion of dead material and its
replacement by immature mesenchyme in pathological conditions.
Furthermore, an identical process to normal endochondral ossification
governs the transformation of the cartilaginous skeleton of the foetus
into bone, and removes the spongy bone which fills the central part of
immature bones and replaces it by haemopoietic mesenchyme. Normal
pre- and post-natal development and growth of the skeleton are therefore
determined by a cellular mechanism identical with, or closely allied to, the
formation of the granulation tissue of disease. Normal bone growth is
under the control of pituitary somatotrophin and the administration of
this hormone to young animals causes a spectacular increase in thickness
-

-405

G. HADFIELD

of the metaphyseal zone of penetrating capillaries and an equally

spectacular increase in bone growth. This major effect of somatotrophin
is completely inhibited by cortisone, making it highly probable that the
two hormones are mutually antagonistic and part of a balanced
physiological mechanism. The inhibition by cortisone of endochondral
bone growth and of fracture repair would therefore appear to be brought
about by the " neutralisation " of somatotrophin, and the probability
that we are dealing with a balanced hormone mechanism leads to the
conclusion that cortisone has a direct effect on the general process of
mesenchymatous replacement.
The general physiological process of mesenchymatous replacement
would thus appear to have two phases, the first being one of capillary
vascularisation which creates an environment in which cells can multiply.
This is controlled directly or indirectly by somatotrophin. This phase
ceases when vascularisation has been achieved and a sufficient number
of immature mesenchymal cells-fibroblasts, chondroblasts and osteoblasts-are in position. The second phase is characterised by a pro.ess
of capillary devascularisation which is just as spectacular as the vascularisation of the first phase. Cell proliferation is halted and the immature

GRANULATION TISSUE FORMATION

A TWO-PHASE PROCESS
PHASE I

PHASE 11

PHASE 1: MESENCHYMAL PROLIFERATION.

PHASE II: MESENCHYMAL DIFFERENTIATION.
Fig. 7.

cells differentiate. The experiments quoted suggest that the primary

effect of cortisone is to inhibit vascularisation. This hormone may
control early differentiation but full differentiation is so profoundly
influenced by ascorbic acid that the formation of fully differentiated
mesenchyme might be regarded as the cortisone-ascorbic acid phase
(Fig. 7).
406

GRANULATION TISSUE

In conclusion I would again stress that the granulation tissue of disease

has a strict physiological counterpart in the immature mesenchyme
which plays a major role in bone development and growth. A very
similar, and probably identical sequence can be observed in the ovary
during the absorption and replacement of the ovarian follicles by scar.
It has been estimated that approximately 100,000 primary follicles are
produced. The oocyte in the great majority of these never leaves the
ovary and perishes in situ. The replacement of the useless cellular follicle
is carried out by penetration of capillaries derived from the theca folliculi.
The end-result is the corpus atretica. About 400 ova leave the ovary
and the corpora lutea which remain are reduced to scar by the same
mechanism, a somatotrophic phase of vascularisation being followed by
a cortisone-ascorbic phase of devascularisation and differentiation.
Other examples of the formation of " physiological granulation tissue"
during normal growth and development are seen whenever a useless and
time-expired tissue undergoes regression, absorption and mesenchymatous
replacement. The absorption of Meckel's cartilage and of a considerable
part of the foetal suprarenal cortex is probably carried out by this or a
very similar process.
The formation of the granulation tissue of disease can thus be regarded
as nothing more than an adaptation of a basic physiological process
governed by a balanced hormonal mechanism.
REFERENCES
CLARK, E. R. and CLARK, E. L. (1939) Amer. J. Anat. 64, 251.
CLARK, E. R., HITSCHLER, W. J., KIRBY-SMITH, H. T., REX, R. 0. and SMITH, J. H.
(1931) Anat. Rec. 50, 129
HARRISON, Ross (1910) J. Exper. Zool. 9, 787.
SANDISON, J. C. (1928) Amer. J. Anat. 41, 447; 475.
SISSONS, H. A. and HADFIELD, G. J. (1951a) Brit. J. Surg. 39, 172.
(1951b) In the press.
STEARNS, M. L. (1940) Amer. J. Anat. 66, 133.

SAYINGS OF THE GREAT

"In the struggle for power, there is no middle course between the
highest elevation and destruction. (Imperium cupientibus nihil medium
inter summa et praecipitia.) " Tacitus (History, Bk. ii, sec. 74).
" It is easy in the world to live after the world's opinion; it is easy in
solitude after our own; but the great man is he who in the midst of the
crowd keeps with perfect sweetness the independence of solitude."
Emerson, Essays, First Series: Self-reliance.
Knowledge is as food, and needs no less
Her temperance over appetite, to know
In measure what the mind may well contain,
Oppresses else with surfeit, and soon turns
Wisdom to folly, as nourishment to wind."
Milton (Paradise Lost).
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