WOUND HEALING AND COLLAGEN FORMATION IV. Distortion of Ribosomal Patterns of ...

25 Pages·2003·3.09 MB·English

WOUND HEALING AND COLLAGEN FORMATION
IV. Distortion of Ribosomal
Patterns of Fibroblasts in Scurvy
RUSSELL ROSS, Ph.D., and EARL P. BENDITT, M.D.
From the Departnmnt of Pathology, School of Medicine and the Department fo Oral Pathology,
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School of Dentistry, University of Washington, Seattle ow
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ABSTRACT from
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The changes in scorbutic wounds following the administration of ascorbic acid have been ttp
investigated using the techniques of electron microscopy, histochemistry, and autoradiog- ://rup
gorfa phy. the fibroblasts Particular and attention to the idhaes ntity been of paid the to extracellular the changes filamentosueesn in the endmoapltaesrmiiacl characteristic reticulum ress.org
of scorbutic wounds. Seven-day-old wounds in scorbutic guinea pigs were examined prior /jcb
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to and from one to 27 hours following the administration of vitamin C. Fibroblasts from rticle
wounds of normal animals demonstrate a characteristic configuration of the ribosomes of -p
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the endoplasmic reticulum which is suggested to be analogous to polyribosomes described f/2
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in cells synthesizing protein such as the reticulocyte. Tangential views of the membranes of /2/3
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the ergastoplasm show the ribosomes to be grouped in paired rows which take both straight 5/1
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and curved paths. This configuration is lost in scurvy and can be seen to begin to reappear 81
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as early as 4 hours after giving ascorbic acid. With increasing time, the morphology of the 98
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ribosomal aggregates approximates that seen in normal cells, so that by 24 hours their 65
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reorientation is complete. It is suggested that one of the disturbances in scurvy may relate df b
to an alteration either in messenger RNA, in the ability of the ribosomes to relate to the y g
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mhesysdenrgeorx, yamino or in acids the membranneecesss ary for of the completing ergastoplasm. collagen In additiosynnt,he sis the may lack be of related formation to the of est on
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architecture of the ribosomal aggregates. Extracellular collagen fibrils appear concomitant 0 M
with the restoration of ribosomal and ergastoplasmic morphology as early as 21 hours after arch
administration of ascorbic acid, with complete disappearance of the scorbutic extracellular 2
0
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material within 24 hours. Observations of this scorbutic material do not support the concept 3
that it is a collagen precursor.
INTRODUCTION
Ascorbic acid has a critical role in both the forma- years in man (1, 2). The studies of Aschoff and
tion and maintenance of collagen in healing Koch (3), H6jer (4), and Wolbach and Howe
wounds of man and guinea pigs, as well as in the (5, 6) describing the changes seen in this disease
prevention of hemorrhage from the vascular in a variety of connective tissues are now classical.
components of connective tissue (4-6, 20-22). The latter authors carefully described the lack of
Scurvy has been known for over two hundred collagen formation in dentin, bone, and healing
563
wounds, during the ascorbic acid-deficient state. label into normal cells with its subsequent
They described the appearance of a substance, reease, and appearance over the extra-
lacking the ordinary staining characteristics of cellular collagen fibrils. Several differences
collagen, in the interstitial spaces of these tissues. were noted in the scorbutic wounds. The
After the administration of ascorbic acid, they uptake of proline was somewhat slower than
observed the prompt appearance of argyrophilic in normal cells, possibly due to the alteration
fibers, followed shortly by fibers having the typical in the vasculature of these wounds. In ad-
staining characteristics of collagen (5, 6, 25). dition, the scorbutic fibroblasts took up the
The studies with the light microscope left the same relative amount of label as the normal
following questions unanswered: cells, but lost it at a slower rate. The appear-
.1 What is the nature of the cellular alteration ance of the label in the extracellular fibrillar
resulting from scurvy? material in wounds of scorbutic animals al-
2. What is the nature of the material appearing so occurred more slowly, and continued to
in the extracellular spaces and adjacent to increase in amount to the end of the experi-
the abnormal fibroblasts during their pro- ments. In contrast to this, the label in the
liferation in scorbutic wounds? Where does collagen of normal animals leveled off after
this material come from?; is it elaborated by 24 hours, and began to decrease, presumably D
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the fibroblasts, or is it produced elsewhere? due to dilution, after the 3rd day. This con- n
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3. Do the argyrophilic fibers and the collagen, tinuing increase of label in the scorbutic ad
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found very soon after ascorbic acid adminis- material could possibly represent an influx fro
tration, come from material newly elaborated of labeled protein from the blood, although m h
by the fibroblasts, or are these formed from the reasons for this rise are not yet clear. ttp
material pre-existing in the interstitial space? The present investigation was designed to ://rup
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In our previous investigations (7-9), the ob- examine the wounds following the administration ss.o
servations of the light microscopist were extended, of ascorbic acid and the restoration of a normal rg
and certain aspects of the cellular alterations were diet. Of particular interest is the advent of col- /jcb
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presented. The aim of the present studies is to lagen formation and its association with the rticle
further characterize the deviations seen in the changes in appearance of the various cellular -p
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scorbutic state from those observed in the normal organelles associated with protein synthesis. In f/2
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state during the process of wound healing, and to addition, it was hoped that further examination /2/3
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answer, if possible, some of the remaining ques- by autoradiography and enzyme histochemistry of 5/1
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tions. The principal previous findings (7-9) these wounds would shed light on the identity of 81
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established : the filamentous extracellular material in the 98
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.1 Several aberrations in the appearance of the scorbutic wounds. 65
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fibroblasts in the scorbutic state. These df b
consisted of (a) an altered endoplasmic MATERIALS AND METHODS y g
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rreotuincdueldu m structures,w hich appeared rather tihna n the long form con- of Tissue Preparation for Light Microscopy est on
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tinuous canaliculi, and (b) large accumula- Female guinea pigs weighing approximately 300 0 M
tions of intracytoplasmic lipid. grams were placed on a scorbutigcnic diet (Nutri- arch
2. The appearance in the extracellular spaces of tional Biochcmicals, Cleveland) for 14 days. All of 20
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a fine, fibrillar material in scurvy which the operative procedures wcrc performed under ether 3
lacks the characteristic banding of collagen. anesthesia. Circular wounds werc created in the dorsal
The material was found by ni situ histo- skin, as described previously (7), and allowed to heal
for 01 days. A wound was removed from each animal
chemical methods to be low in tryptophan,
on the 10th day and ascorbic acid (100 mg) was ad-
and was intermixed with strands of fibrin
ministered intraperitoneally. The animals were then
present in these wounds. It was concluded
placed on a diet containing adequate ascorbic acid.
that the morphologic findings in scurvy
The remaining wounds were removed at 24-hour
were consistent with an alteration in protein intervals for 5 days following vitamin C adminis-
synthesis by the scorbutic fibroblasts. tration.
3. The autoradiographic studies with tritiated The wounds were fixed in neutral buffered formalin
proline demonstrated a rapid passage of the and embedded in paraffin. Sections were stained
366 THE LANRUOJ FO CELL YGOLOIB • EMULOV ~, 1964
with hematoxylin and eosin, the Van Gieson stain, micrographs taken were of the wound contents. At
Mallory's connective tissue stain, Wilder's reticulin least 50 sections were examined of wounds from hcae
stain, phosphotungstic acid-hematoxylin, and Wei- of the 5 time periods.
gert's stain for fibrin. In an effort to resolve the problem of sampling, a
large number of micrographs (ranging from 20 to 50)
Histochemical Techniques
were taken randomly of each section. Each micro-
Cross-sectional blocks of scorbutic wounds were graph contained from 1 to 5 cells, with an average of
removed and quick frozen in liquid nitrogen-cooled 2 cells; consequently, no less than 2,000 cells, and
isopentane. Six-micron sections were cut from this often many more, were examined from each of the five
material in a Pearce cryostat. The sections were time periods. The sections were stained with either
treated in the following way: uranyl acetate, lead hydroxide (12), or a combina-
.1 Sections from each animal were examined for the tion of uranyl acetate followed by lead hydroxide.
presence of tryptophan by the Adams' indole The tissues were examined with an RCA-EMU 3E
method (31). or 3G electron microscope. The description of fibro-
2. A group of sections from each animal were incu- blasts from non-deficient animals represents a much
bated in collagenase (Worthington Biochemi- larger number of observations from previous and
cal Corp., Freehold, New Jersey) (pH 7.0) for present experiments (7-9, 51).
30 minutes, and for 1, 2, and 4 hours.
Techniques Used for Autoradiography
3. Another group of sections were incubated in Do
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crystalline trypsin (Armour Pharmaceuticals, A third group of four female guinea pigs was n
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Kankakee, Illinois) (pH 8.0) for 30 minutes, placed on a scorbutigenic diet for 14 days. Each ani- ad
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and for ,1 2, and 4 hours. mal was wounded under anesthesia by making seven d fro
Several groups of blocks of wound tissue were fixed linear incisions in the dorsal skin. The wounds were m
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in cold acetone, or alcohol (for 12 hours), or 70 per allowed to heal for 7 days, and each animal was then ttp
cent methanol for 3 hours. These were then embedded given proline-3,4-H 3 (New England Nuclear Corp., ://ru
in paraffin, sectioned, and exposed to either col- Boston) (12 #c/gin body weight). A wound was re- pre
lagenase or trypsin. moved from each animal at 6 days after proline-H 8 ss.o
eosin, All Vasenct ions Gieson, were or stainbeyd the Awdiatmhs ' hematoxylini ndole method. and agdumiinneias tration. pig was givAefnt er 1r00 emoval mg of of ascorbic this wound, acid intra-e ach rg/jcb/a
bated Control in the sections appropriate for each buffer, of the in abovea fashion were similar incu- pweerrei toneally then removed and placed at 4, o12n, 24a , normal 36, 48, diet. and 27Wounds hours rticle-p
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to that of the enzyme incubations, and subsequently following vitamin C administration. These tissues f/2
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stained. were fixed and embedded as for electron microscopy, /2/3
sectioned at 1-micron, and prepared for autoradiog- 65
Tissue Preparation for Electron Microscopy /1
raphy using NTB-3 nuclear track emulsion (East- 38
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With the animal under ether anesthesia (8), linear man) as previously described (9). 69
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incisions, approximately 1 cm long, were made in the The autoradiographs were developed after 6 /36
dorsal skin of a separate group of 12 female guinea weeks' exposure, and random photomicrographs were 5.p
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pigs after these animals had been fed for 14 days on taken of sections from three different blocks of tissue f b
the scorbutigenic diet. A wound was removed, as from each wound. The micrographs were enlarged y gu
previously described (7), prior to ascorbic acid admin- and the numbers of grains were counted per unit est o
istration. Each animal was then given 100 mg of area of cells, collagen, filamentous material, erythro- n 2
ascorbic acid intraperitoneally, placed on a normal cytes, and debris, as previously described (9). These 0 M
diet, and wounds were removed at 4-hour intervals counts were then corrected for background. arch
for the first 24 hours and at 24-hour intervals for the Nutritional Behavior of the Animals 20
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next 3 days. 3
The wounds were cut into transverse slices 1 to 2 Each animal was weighed every other day. Earlier
mm thick, as described previously (7), and were then experiments (8, 9, 35, 54) demonstrated that regular
fixed. Sections from each of 4 to 5 blocks were ex- examination of weight, prior to the period of extreme
amined from the central portion of each wound. weight loss from inanition, is a reliable indication of
These tissues were fixed in 2 per cent osmium tetroxide food intake in these animals. In addition, previous
buffered with s-collidine (10), fixed again in neutral pair-feeding experiments (unpublished data) demon-
buffered formalin, and embedded in Epon 812 (11). strated that the weights of pair-fed animals are similar
Adjacent 1-micron sections were stained with Azure to those of the scorbutic animals during the experi-
II, Methylene Blue (30) and used for orientation and mental period. Gross also observed this and noted, in
specimen trimming so that the thin sections repre- addition, that pair-feeding indicates that the weight
sented the center of each wound. Hence, all of the loss in the latter stages of scurvy is not due to inanition
R. Ross DNA E. P. BENDITT Wound gnilaeH dna negalloC .noitamroF IV 367
alone (35). As in the previous experiments in scurvy trois, scorbutic and recovery wounds examined
(8, 9), the animals gained weight on the seorbutigenic after the various time intervals.
diet for the first 12 days. Their weights subsequently In addition, a comprehensive review was made
leveled off and remained stable for approximately 4 to of electron micrographic data from previous
6 days, after which time they began to decrease. The
wound-healing experiments from normal and
weight curves are very similar to those observed by
scorbutic animals (7-9, 51). The observations of
Gross (35) in his studies of scurvy following ascorbic
the morphology of the cells in the present study
acid administration. After being given ascorbic acid,
are in agreement with those from these earlier
all of the animals returned to a normal weight-gain
pattern. Due to the trauma of wounding, each animal investigations.
characteristically loses weight for a day and then pro- One group of animals, not on a deficient diet,
ceeds to gain weight. was pair-fed to a group of scorbutic animals. The
At the end of the experiments, each animal was pattern of ribosomes in fibroblasts from the
sacrificed and the molars and incisors were examined wounds of the pair-fed animals were identical in
for evidence of scorbutic change in areas in the odon- appearance to those from wounds of animals fed a
toblasts of the pulp. Changes characteristic of scurvy normal diet da .mutibil
followed by healing were observed.
Wounds Prior to the Administration of
LIGHT MICROSCOPE OBSERVATIONS Ascorbic Acid Dow
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The sequence of events observed in the light lo
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microscope is as follows: Prior to ascorbic acid Many of the features of wounds from scorbutic ded
administration, the extracellular spaces contain animals have been described previously (8, 38). fro
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wmmiaattthee rriiaall either doews hich the noVt an is stain Gipeoosrolny for or strruecttiucrueldi n Mallory or connec(8t).i ve collagen This Wtceiho annsg es should which in like the are to fibrobleaa mstpsh asize general in thfreeateu re scurvy. specific of Thesea ll observa- of are: the http://rupre
tissue stains. This configuration appears essentially (~) The rounded up, somewhat dilated, irregular ss.o
utrnacthiaonng ed of ascourpbi c to 24 acidh;o urs howevfeorl,lo wing by the 24 adminis- hours dceicsrteearsneade of amountt he endoplasmic of rough-surfaced reticulum, endo- and rg/jcb/a
some argyrophylic fibers appear. These fibers are plasmic reticulum. rticle
(b) A disappearance, in scurvy, of the character- -p
n48a rrow hours. at Mosfitrs t and of themb ecome appear coarse to and lie in wavy planes by istic orientation of the ribosomes attached df/22/2
parallel to the borders of adjacent cells. Twenty- to the cisternal membranes of the endo- /36
four hours after ascorbic acid administration, there plasmic reticulum seen in normal fibro- 5/13
is material present between the cells that is faintly blasts. 816
(c) An increase in the number of free cytoplasmic 98
positive with the Van Gieson stain. Within 48 /3
ribosomes in scorbutic fibroblasts. 65
hours, however, strongly Van Gieson-positive .p
fibers appear. The same sequence is observed The first of these, the rounded, separated pro- df b
with the Mallory connective tissue stain. These files of the endoplasmic reticulum, was noted y gu
findings parallel those already described by earlier (8, 38). The characteristic appearance of est o
the cells can be seen in Fig. .1 It should be noted n
Wolbach and Howe (5). They re-emphasize the 2
fact that prior to 24 hours after ascorbic acid that most of the membranes of the cisternae of the 0 M
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administration it is difficult to find evidence for endoplasmic reticulum contain a full comple- rch
collagen formation using these techniques. ment of ribosomes and there are few empty sites 202
apparent on the membranes. 3
ELECTRON MICROSCOPE The second change in scurvy is apparent in the
configuration of the attached particles lining the
OBSERVATIONS
rounded-up cisternae. The orientation of the ribo-
The electron microscopic observations cited somes on the endoplasmic reticulum can be seen
below are the result of sampling more than 250 in tangential sections of the cisternal membranes
tissue blocks from all of the animals, followed by that provide an ne face view. In guinea pigs fed
examination of randomly taken electron micro- adequate amounts of ascorbic acid, the ribosomes
graphs representing more than 2,000 cells from of the wound fibroblasts present a characteristic
each time period. The observations listed are and possibly specific configuration. They appear
representative for all of the tissucs from the con- to be grouped in rows of pairs, which often curve
368 THE LANRUOJ FO CELL YGOLOIB • EMULOV ~, 1964
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FIGURE 1 An electron micrograph from a portion of a 7-day-old scorbutic wound. The cisternae of endo-
plasmic reticulum (er) appear as rounded, separate profiles which contain somewhat dense, amorphous
material. The surface of these rounded cisternae often appear ruffled and irregular. Lipid deposits )1( are
evident as well as large mitochondria (m). Numerous free ribosomes (arrows) and peripheral
cytoplasmic, filamentous aggregates (a) can also be seen. The extracellular spaces contain the non-banded
filamentous material (f) characteristic of scorbutic wounds. )< 21,000.
R. Ross D~A E. P. BENDITI' Wound Healing dna negalloC .noitamroF IV 369
and present spiral or rosette forms (Figs. 2 a and 4 Hours after Ascorbic Acid
2 b). In normal cells there are very few free ribo-
Four hours after ascorbic acid administration,
somes and most of the attached particles can be
the wounds are similar to those seen in scurvy.
seen to have this orientation whenever the
The fibroblasts contain numerous profiles of
membranes are tangentially sectioned. Such
ne face views are most commonly seen in regions rounded cisternae of endoplasmic reticulum
which do not appear to be interconnected, at
where the endoplasmic reticulum approximates
least within the plane of section. However, in a
the Golgi zone. A marked change is apparent in
few regions, paired rows of ribosomes in curved
this configuration in scorbutic fibroblasts. In-
shapes can already be seen on the cisternae of the
stead of being regularly arranged in rows of two,
endoplasmic reticulum (Fig. 3, arrows). Lipid
the ribosomes present a different configuration and
deposits previously described are also present in
appear to be randomly distributed on the surface
these cells. The filamentous aggregates located in
of the cisternae (Fig. 2c).
the periphery of the cytoplasm are present in these
Thirdly, numerous unattached ribosomes can
cells just as they appear in the fibroblasts of both
be seen to be distributed throughout the cyto-
normal and scorbutic animals.
plasmic matrix of the scorbutic fibroblasts (Fig.
The intercellular spaces contain large, broad,
1). Some of them appear to be grouped in clusters, D
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interlacing bundles of material composed of what w
although the largest proportion are randomly n
dispersed in the cytoplasm. Fewer free cytoplasmic appear to be fine filaments. No banding can be load
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observed in either the bundles or the filaments d
frrioboms omes normal appear animals. to be present in the fibroblasts after staining with either uranyl acetate or lead from
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Therefore, in scurvy, the ribosomes are altered hydroxide, except in those few areas in which ttp
not only in that numerous free particles are present fibrin can again be identified. Extravasated ://rup
iinn tthhaet cytoplthaossem that of are scorbutic attached fibroblasts, to the membranes but also ceerlyl throcytes debris are and intermsemaslhle d bits of witmhaitner ial this resembling fibrillar ress.org
of the endoplasmic reticulum have lost the pattern material. /jcb
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so characteristic of non-deficient fibroblasts. 8 Hours after Ascorbic Acid rticle
Intracytoplasmic lipid deposits and the peri- -p
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pheral filamentous aggregates in the scorbutic By 8 hours, many of the cisternae of the endo- f/2
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cells have been reported previously (8). plasmic reticulum appear dilated (Fig. 4) and /2/3
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The extracellular material present in the somewhat more interconnected than at 4 hours. 5/1
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scorbutic wounds is generally homogeneous, con- Many free ribosomes are still present in the cyto- 81
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sisting of broad interlacing bundles of fine fila- plasm of these cells, and numerous intracyto- 98
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ments closely packed together. Occasional bundles plasmic lipid deposits are present. At this time, 65
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of fibrin, recognizable by its characteristic perio- the reorientation of the ribosomes attached to the df b
dicity, can be seen intermeshed within this material, endoplasmic reticulum of the fibroblasts appears y g
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as Anw ell additioans al occasional finding bits of of cell interest debris. in wounds of m Thebe more admviatnoccehdo ndrtihaa n are typical earlier of (Fig. those 4). seen in est on
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scorbutic animals is that the rough endoplasmic fibroblasts, and peripheral filamentous aggregates 0 M
reticulum of the macrophages does not appear to are present in these cells. In addition, the Golgi arch
be altered in contrast to that of the fibroblasts. complex appears to be larger and more extensive 2
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ERUGIF ~ b This represents part of a fibroblast from a 7-day wound in a non-deficient
animal. The nucleus (N), mitochondria (m), and Golgi complex )G( of this cell are
apparent. A characteristic feature of this cell is the extensive, well developed rough endo-
plasmic reticulum (er) that is arranged in the form of numerous flat sacs often inter-
connecting with each other. When the membranes of the ergastoplasm are sectioned tan-
gentially, the ribosomes attached to their surface appear to be arranged in characteristic
patterns (arrows), not visible when the membranes are sectioned normally. A higher mag-
nification of such a region is presented in Fig. 2 .b X ~0,000.
370 THE LANRUOJ FO CELL YGOLOIB • EMULOV ~, 1964
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R. Ross DNA E. P. BENDITT Wound Healing dna negalloC .noitamroF IV 371
than that seen in scorbutic cells prior to treat- 24 Hours after Ascorblc Acid
ment.
By 24 hours after the administration of ascorbic
The appearance of the material in the extra-
acid, the wounds appear very similar to those seen
cellular spaces varies from area to area. In most
in normal animals. The extraccllular spaces are
regions the characteristic scorbutic, non-banded
filled with collagen fibrils. The non-banded
fibrils can be seen (Fig. 4). However, there are
filamentous material seen in scurvy has completely
regions in which extracellular condensations of
disappeared (Fig. 7). The fibroblasts now appear
material can be seen adjacent to cell surfaces. In
very much like those seen in non-deficient animals
addition, a fine filamentous material of lower
(7). They contain an abundant endoplasmic
density can be seen to be intermixed with the
reticulum, large mitochondria, and a prominent
material typical of scorbutic wounds.
Golgi region. The attached ribosomes demonstrate
12 Hours after Ascorbic Acid an arrangement similar to that seen in the non-
scorbutic ceils (Fig. 8). Tangential sections show
At this time period, changes can be seen in
rows of pairs of ribosomes lying upon the
both the cells and the extracellular regions. The
membranes of the endoplasmic reticulum in both
numbers of free cytoplasmic ribosomes has de-
straight and curved profiles (Fig. 8). A relatively
creased, and in some parts of many of the cells D
small number of free ribosomes, grouped in ow
tangential views of the cisternal membranes n
clusters or rosettes, remain in the cytoplasm of lo
further demonstrate a tendency to form rows of ad
these cells. Many of the recovering fibroblasts ed
paired ribosomes (Figs. 5, 6). Much less intra- contain numerous peripheral vesicles as well. fro
lcToynhtgeoepr ,l asmic profiles narrower, lipid of einsd opalnadsm ic present more in interconnemcratenetdyi culum of the appear cellts. han ltaor ge A the cnoummmobne r endoplasmic of additiomniaclr ographs reticulum observation was of notedt he cellms adein relation of in the a m http://rupre
previously (Figs. 5, 6). The Golgi regions of the 24-hour ascorbic acid-treatod animals. Whenever ss.o
fibroblasts are prominent and their vesicles and rg
cisternae appear to be dilated. Numerous vesicles the membranes of the cisternae approach the /jcb
of varying size can be seen to line some cell surfaces rpibloassommaes . membrane Numerous they appearpr ofiles to obfe devoid cisternal of /article
at this time. -p
The area between the cells now consists of membranes have been observed so close to the df/2
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fibrils, some of which are clearly collagen, as plasma membrane that they appear to be in /2/3
evidenced by a 700-A periodic banding, inter- contact (Fig. 9). Direct continuity of the cisternal 65/1
mixed with a much larger mass of material con- cavity with the extracellular milieu has, however, 381
6
sisting of fine filaments wh;ch display no banding never been demonstrated in any of this material. 98
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(Figs. 5, 6). Numerous vesicles can also be seen between the 65
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ERUGIF £ b This micrograph displays part of the fibroblast from a 7-day wound seen in 20
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a normal animal in Fig. ~ a. The region of the cell depicted shows part of a mito- a
chondrion, as well as several profiles of rough endoplasmic reticulum (er). Where the rch
2
cisternae are sectioned normally, a single layer of ribosomes may be seen attached to the 02
3
membranes. However, there are many regions (arrows) where the ergastoplasmic
membranes have been grazed tangentially, presenting an en face view of the orientation
of the ribosomes attached to the membranes. Here the membrane appears as an increased
area of density around the ribosomes. The ribosomes are arranged in characteristic curved
and spiral patterns, some of which are in double rows. In some regions (arrows) a fine
thread appears to connect these ribosomes. X 5~,000.
I~GURE 2 C A micrograph from parts of two cells from a 7-day-old scorbutic wound. The
rounded cisternae of endoplasmic reticulum are irregular in shape, and tangential sec-
tions of the membranes provide a view of ribosomal orientation. Here the ribosomes have
no characteristic configuration (arrows), but appear to be randomly dispersed on the
membranes. Lipid droplets and free cytoplasmic ribosomes are also apparent. X 84,000.
372 TIlE JOURNAL OF CELL BIOLOGY • VOLUME ~, 1964
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R. Ross DNA E, P. BENDITT Wound Healing and Formation. Collagen IV 373
endoplasmic reticulum and the plasma membrane stained positively for fibrin with phosphotungstic
of the fibroblasts. acid-hematoxylin and the Weigert's stainl The
bulk of the extracellular material gave none of
Extracellular Changes
these reactions.
Observations of the extracellular, filamentous, Incubation of both frozen and chemically fixed
non-banded material present in scorbutic wounds sections, with collagenase, trypsin, or buffer,
demonstrate its relatively rapid disappearance demonstrated that collagenase removed both fine
following ascorbic acid administration. Within 8 and coarse collagen fibers from the adjacent wound
hours, non-banded filaments of lower density are margins. Trypsin acted on the collagen fibers in
present between the filaments of the scorbutic the wound edges to a lesser extent but in the same
material (Fig. 4). Within 12 hours, collagen manner as did collagenase. Trypsin was also
fibrils are present both adjacent to cells and inter- found to remove the sharply eosinophilic, trypto-
mixed with the scorbutic material (Fig. 5, 6). By phan-negative extracellular material present in
24 hours, the material present during scurvy is no the scorbutic wounds, whereas collagenase did
longer observable, and it has been replaced by not.
mature collagen fibrils (Fig. 7). No evidence of The buffer alone had no effect on the collagen
phagocytosis of the scorbutic material was ob- or the scorbutic extracellular material in any of Do
w
served at any time during the experimental ob- the tissues examined. nlo
a
servations, although phagocytosis of erythrocytes, Autoradiographic Observations ded
and ferritin-containing vesicles, are clearly present fro
m
in the macrophages during this interval. The results of grain counts on the autoradio- h
48 Hours after Ascorbic Acid graphs showed no demonstrable change in the ttp://ru
amount of label in ceils or in extracellular material p
re
As the wounds grow older, they take on the at any of the time periods examined. Limitations ss.o
wcohuanradcst eristics (7) in normparle viously animals. described Few free for ribomsaomteus ring opfr eclude the autoradiogkrnaopwhiincg whether method, any shift as in used isotope here, is rg/jcb/a
are present in the cells, and those lining the endo- occurring either from the cells to the extracellular rticle
plasmic reticulum again have their characteristic material or from the serum to these. -pd
configuration. The cisternae of the endoplasmic f/22
reticulum appear as elongated, interconnected DISCUSSION /2/3
6
5
profiles, and the lipid deposits have completely Ascorbic acid deficiency presents a classical /13
disappeared from the cells. By this time, most of pathologic alteration of the connective tissues with 816
9
the extracellular regions are filled with collagen a concomitant depression in the formation of a 8/3
6
fibrils (Fig. 10). specific protein, collagen (5, 6, 17, 18, 20, 21, 23, 5.p
d
Histochemical Observations 2c4,h aracterize 35, 54). the The fine present structure observations alterations further in f by gu
e
Examination for the presence of tryptophan in fibroblasts, during scurvy, and the reconstitution st o
n
these wounds, prior to and following the adminis- of the normal ultrastructure of the fibroblast 2
0
tration of ascorbic acid, demonstrated a positive following administration of vitamin C. The re- M
a
reaction in rare, isolated, small foci. These loci appearance of the non-scorbutic configuration of rch
2
0
2
3
ERUGIF 3 This electron micrograph contains parts of two fibroblasts from a wound 4
hours after ascorbic acid administration. The extracellular filamentous material (f)
appears similar to that seen prior to ascorbic acid treatment. An occasional body (b),
possibly cell debris, can be seen intermixed in this material. The fibroblast has the ap-
pearance of a typical scorbutic cell, except that a few areas display a tendency for the
ribosomes of the endoplasmic reticulum )re( to aggregate in a fashion similar to that seen
in ceils from normal animals (arrows). X ~4,000.
374 TuE LANRUOJ FO CELL YGOLOIB " EMULOV ~, 1964