/. Embryol. exp. Morph., Vol. 16, 3, pp. 581-390, December 1966
With 5 plates
Printed in Great Britain
381
Osteogenesis in transplants of bone marrow cells
By A. J. FRIEDENSTEIN 1 ,1. I. PIATETZKY-SHAPIRO 1
& K. V. PETRAKOVA1
From the Laboratory of Immunomorphology, Gamaleya Institute of
Epidemiology and Microbiology, Academy of Medical
Sciences of the U.S.S.R., and Laboratory of Mathematical
Methods in Biology, University of Moscow
After heterotopic (e.g. subcutaneous) transplantation of bone marrow,
haemopoiesis in the graft ceases; reticular tissue develops instead, and later bone
is formed (Denis, 1958). The result can be achieved by grafting either free pieces
of bone marrow or those placed in diffusion chambers (Petrakova, Tolmacheva
& Friedenstein, 1963; Rosin, Freiberg & Sajnek, 1963). In the case of free transplantation the bone formed is later filled with bone marrow. After transplantation in diffusion chambers haemopoiesis does not recur despite the development
of a considerable mass of bone in the chambers (Friedenstein, 1965).
The population of bone marrow cells is very heterogeneous, including
haemopoietic cells, reticular cells and endosteum elements. According to generally
accepted views this population is a mixture of individual cell lines capable of
mutual transformations within certain limits (Maximov, 1927; Burwell, 1964).
After transplantation some of the pathways of differentiation open to bone
marrow tissue (formation of reticular and bone tissues) are stimulated, while
others (haemopoiesis) are arrested. This effect could be ascribed to the death of
haemopoietic stem cells and to proliferation of cells responsible for the development of reticular and bone tissue. It may, however, depend upon transformation
of haemopoietic cells to reticular and osteogenic cells.
An analysis of these possibilities encounters difficulties as there are no reliable
data concerning the potentialities for transformation of different types of bone
marrow cells, and especially of the stem cells. There is no convincing evidence
for the dependence of various histogenetic pathways in bone marrow tissue
(haemopoiesis, osteogenesis or the development of reticular tissue) upon individual differences of the stem cells concerned. All of them could be provided
by one common line of stem cells whose differentiation is controlled by conditions within the population.
Osteogenesis appearing in bone marrow grafts may serve as a model of
1
Authors' address: Gamaleya Institute of Epidemiology and Microbiology, U.S.S.R.
of Medical Sciences, Gamaleya Street 2, Moscow D-182, U.S.S.R.
24
JEEM l6
382
A. J. FRIEDENSTEIN £T AL.
differentiation in a mixed cell population. Irrespective of whether all or some of
the stem cells of bone marrow tissue are able to form bone, the following questions
may be raised. In bone marrow transplants either bone and reticular tissue, or
reticular tissue alone, develops. Does the difference depend upon whether or not
osteogenic stem cells are included in the graft, their behaviour being unaffected
by other members of the cell population? Or is differentiation towards osteogenesis a result of an interaction within a community of cells? These questions
can be answered when bone marrow cell suspensions are transplanted in
diffusion chambers. Under these conditions bone tissue is formed in the chambers.
This technique makes it possible to vary the number of transplanted cells and
the density of their initial population.
MATERIALS AND METHODS
Bone marrow of adult C57BL mice was isotransplanted intraperitoneally to
adult recipients in diffusion chambers made of Millipore HA filters (pore size
0-45 /i, thickness 150 /A) or of AUFS filters (pore size 0-6-0-9 /*, thickness 100 ju).
The chambers were constructed according to the method of Algire (Algire,
1
1
Text-fig. 1. Construction of the diffusion chamber. A, Diffusion chamber type A;
B, diffusion chamber type B; 1, millipore filter; 2, Plexiglass ring; 3, the cells.
Weaver & Prehn, 1957) and were of two sizes: A, chambers with filter diameters
of 14 and 10 mm; and B, with those of 7 and 3-2 mm, respectively (cf. Text-fig. 1).
Chambers were sterilized in 70° alcohol for 15 min, washed in distilled water and
placed into Hanks's solution.
Bone marrow was extracted from femur and chopped into fragments of about
2 mm, which were placed into diffusion chambers, or bone marrow cell suspension was prepared in Hanks's solution. After elimination of cell clumps by
filtration through Capron net the suspension was diluted to 2 x 106 cells per ml.
To prepare lymphocyte suspensions cervical lymph nodes were used. After
filtration the concentration of lymphocytes was adjusted to 107 per ml. To
introduce cells into a chamber the larger filter was put on glass rails over a
hollow-ground slide into which Hanks's solution was poured until it touched
the filter. A given volume of cell suspension was placed on the filter from above,
the liquid passing across the filter and the cells precipitating on it. Filtration was
performed in a Petri dish lined with cotton-wool soaked in saline. Then a filter
Osteogenesis in transplants
383
of smaller diameter (without cells) was placed over the larger one (with cells)
and the chamber was stuck. The number of viable cells in the remaining cell
suspension was counted to determine that of the cells placed into chambers.
The chambers were fixed with 96° alcohol between 1 and 30 days after transplantation. They were freed from the surrounding tissue, the niters were separated
and put into a cooled fixative. In most cases the Gomori reaction for alkaline
phosphatase was performed on filters (Gomori, 1939) to reveal foci of osteogenesis. Filters then were counterstained with haematoxylin, dehydrated with
alcohol, cleared with xylene and mounted in balsam as total preparations. Some
filters were tested for calcium as the control to the Gomori reaction. After
freeing from the plexiglass rings and fixation some chambers were embedded in
paraffin and cut in serial sections that were stained by the Gomori method, for
calcium, with haematoxylin-eosin, for PAS (counterstained with haematoxylin).
The following experiments were performed:
(1) Transplantation of a piece of bone marrow into chambers of types A
and B.
(2) Transplantation of a suspension of 106 bone marrow cells into chambers
of type A.
(3) Transplantation of a suspension of 105 bone marrow cells into chambers
of type A.
(4) Transplantation of a mixture of bone marrow cells and lymphocytes
(1:9), the total number of cells being 107, into chambers of type A.
(5) Transplantation of a suspension of 105 bone marrow cells into chambers
of type B.
(6) Implantation of empty chambers.
In series 2 and 5 the initial density of nucleated cells on the filter was approximately 210 and in series 3 approximately 30 per 0-05 mm2.
EXPERIMENTAL RESULTS
(1) Morphology of transplanted pieces of bone marrow {HA chambers)
When pieces of bone marrow were placed into chambers small blood vessels
and fragments of spongy bone were included with them (Plate 1, fig. 1). Using
the Gomori method both these tissues are clearly stained. The residual bone
marrow tissue of mice showed a negative reaction for alkaline phosphatase.
During the first days after transplantation the trabeculae of the bone necrosed.
They ceased to show reaction for phosphatase, the osteocytes perished and
empty cellular cavities remained in the bone. The surface of bone became denuded losing its covering layer of osteoblasts (Plate 1,fig.2). At the same time a
large number of cells migrated from the graft and a broad zone of haemopoietic
and reticular cells arose in the chamber. The relative amount of the former
rapidly declined, while that of the latter rose.
On the third day the filters were completely covered by cells usually arranged
24-2
384
A. J. FRIEDENSTEIN£rAL.
in several layers in total preparations (Plate 1, fig. 3). The majority of these cells
were fibroblast-like elements, many in mitosis. Large spindle cells with dense
cytoplasm, large nuclei and clear-cut nucleoli could be easily distinguished
among them; in these cells mitoses were seen particularly often (Plate 1, fig. 4).
Within such tissue osteogenic foci appeared on the third day. When tested by the
Gomori reaction these foci looked like a phosphatase-positive network composed
of elongated reticular cells with phosphatase-positive cytoplasm in a small
amount of phosphatase-positive matrix (Plate 1, fig. 5). The osteogenic cells
were bigger than the fibroblast-like cells on the filter. Meshes in the network
were formed by one or, less frequently, two layers of adjacent cells between which
phosphatase-negative fibroblast-like and haemopoietic cells were distributed, i.e.
elements that covered the remaining area of the filter.
Foci of osteogenic tissue were easily distinguished from other phosphatasepositive structures that could occur in these preparations, such as fragments of
marrow blood vessels and the remnants of dead bone. The vessels possessed a
distinct wall, their branching tubes were of regular shape with their contours
clearly outlined. The fragments of the spongy bone that got into the chamber
during implantation lost all signs of viability by the third day. It is significant
that in none of the cases investigated did foci of developing osteogenesis touch
the fragments of old bone. These two were always observed at a distance from
each other; fragments of necrotic bone were located within the initial graft,
while foci of osteogenesis were found in the zone of outgrowth on the filter.
Between the third and the eighth day after transplantation the number of
bone foci did not increase markedly but their structure changed: the amount of
bone matrix rose, trabeculae became thicker and composed of a greater number
of cells (Plate 1, fig. 6). The alveoli between trabeculae narrowed and the entire
structure became more compact. The osteogenic foci increased in size; on the
eleventh day they were quite distinct even when stained with haematoxylin only.
They showed the structure of typical bone trabeculae with osteoblasts embedded
in the bone matrix. In sections this bone tissue has also a peculiar appearance
(Plate 2, fig. 7). By the twenty-fourth day bone occupied most of the chamber
(Plate 2, figs. 8-10).
Table 1 presents the results of transplantation of pieces of bone marrow. At
the time of transplantation such pieces consisted on the average of 8 x 106 cells.
(2) Morphology of bone marrow transplanted inform of suspension of 106
cells into the chamber of type A (HA chambers)
When a suspension of bone marrow cells was placed in a chamber the initial
population consisted of: haemocytoblasts (11 %), myeloids (33 %), leucocytes
(10 %), erythroids (8 %), monocytes (4 %), lymphocytes (34 %), reticular cells
(0-5 %), only nucleated cells being taken into account. The cells were evenly
distributed over the whole surface of the filters; no phosphatase-positive cells
could be found.
J. Embryo!, exp. Morph., Vol. 16, Part 3
PLATE t
Fig. 1. Transplantation of a piece of bone marrow in a chamber with HA filters. Three days.
Alcohol. Gomori. Total preparation, x 200. Blood vessels chamber with bone marrow.
Fig. 2. The same preparation, x 200. Fragment of dead bone in the chamber.
Figs. 3,4. Transplantation of a piece of bone marrow in a chamber with HAfilters.Three days.
Alcohol. Haematoxylin. Total preparation, x 400. Areas of the zone of growth. M, mitosis.
Fig. 5. Transplantation of a piece of bone marrow in a chamber with HA filters. Three days.
Alcohol. Gomori. Total preparation, x 400. A focus of phosphatase activity in the zone of
growth.
Fig. 6. Transplantation of a piece of bone marrow in a chamber with HA filters. Five days.
Alcohol. Gomori. Total preparation, x 400. A focus of phosphatase activity in the zone of
growth.
A. J. FRIEDENSTEIN ET AL.
facing p. 384
/. Embryo!. exp. Morph., Vol. 16, Part 3
ignwur
mi
PLATE 2
^
"*
^^
Fig. 7. Transplantation of a piece of bone marrow in a chamber with HA filters. Eleven days.
Alcohol. PAS-haematoxylin. A section, x 200. A focus of osteogenesis in the chamber under
the filter, a, Filter; b, bone tissue; c, a layer of osteoblasts.
Figs. 8, 9. Transplantation of a piece of bone marrow in a chamber with HA filters. Fifteen
days. Alcohol. PAS-haematoxylin. x 400. Bone tissue adjoining the filter, a, Filter, b, bone
tissue.
Fig. 10. Transplantation of a piece of bone marrow in a chamber with HA filters. Alcohol.
Gomori. Section, x 200. Bone in the chamber, a, Filter, b, bone tissue.
A. J. FRIEDENSTEIN ET AL.
J. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 3
.1
11
14
12
Fig. 11. Transplantation of 106 bone marrow cells in a chamber of type A with HA filters.
Two days. Alcohol. Haematoxylin. Total preparation, x 400.
Fig. 12. The same experiment. Three days. Alcohol. Haematoxylin. Total preparation, x 200.
A focus of 49 myeloid cells with five mitoses.
Fig. 13. The same experiment. Three days, x 200. Large basophilic cells. Mitosis in one.
Figs. 14-16. The same experiment. Five days. Alcohol. Haematoxylin. Total preparations.
x 280. Bands of large polygonal cells.
A. J. FR1EDENSTEIN ET AL.
J. Embryo/, exp. Morph., Vol. 16, Part 3
PLATE 4
Figs. 17, 18. The same experiment. Five days. Alcohol. Gomori. Total preparations. x400.
Fig. 19. The same experiment. Five days. Alcohol. Haematoxylin. Total preparation. x400.
A focus of myeloid cells with mitoses.
Fig. 20. The same experiment. Seven days. Alcohol. Haematoxylin. Total preparation, x 400.
A focus of osteoblasts with mitoses.
A. J. FRIEDENSTEIN ET AL.
facing p. 385
Osteogenesis in transplants
385
On the second and third days (Plate 3, fig. 11) the filters were covered with a
continuous layer of fibroblast-like cells and of haemopoietic elements, the
majority of the latter showing signs of degeneration. Apart from this, solitary
delimited foci consisting entirely of young haemopoietic cells of the myeloblast
type occurred on filters on the third day (Plate 3, fig. 12). Cell counts in four such
foci gave the following values, respectively, 81, 50, 72, 49 (mean = 63), and 4,
0, 2 and 5 mitoses, respectively, were seen in them. Each focus consisted entirely
of cells of the same types. Large isolated cells with basophilic cytoplasm and
large nuclei occurred among fibroblasts (Plate 3, fig. 13). The reaction for
phosphatase remained negative in them all.
Table 1. Bone formation in chambers containing a piece of bone marrow
Days after transplantation
1
2
3
4
5
8
11
18
24
30
Chambers with bone
Total number of chambers
0/3
0/3
3/3
3/3
3/3
2/2
2/2
3/3
15/15
14/15
On the fifth day the composition of the cell population covering the filters
underwent a change. The number of degenerating haemopoietic cells sharply
declined, most of them being spindle-shaped and fibroblast like. Large polygonal
cells with round oval nuclei and compact dense cytoplasm developed among
them. These cells formed fine bands with close packing (Plate 3, fig. 14); mitoses
were common (Plate 3, figs. 15,16) and they were positive for alkaline phosphates
(Plate 4, figs. 17, 18) which distinguished them from the remaining tissue on the
filters. Apart from such phosphatase-positive foci, large foci of myeloid cells
(Plate 4, fig. 19) occurred on the filters on a network of small fibroblast-like
cells connected by fine processes. Later, 7-12 days after transplantation, the
bands of sharply phosphatase-positive cells became of typical, round, osteogenic foci (Plate 5, figs. 21, 22). Bone matrix appeared in their vicinity, the cells
revealing characteristic features of osteoblasts (Plate 4, fig. 20). The rest of the
filters were covered with fibroblasts. No myeloid foci could be revealed at this
time; however, single haemopoietic cells might occur on filters often lying close to
bone trabeculae (Plate 5, fig. 23). In sections bone foci had the structure typical
of bone tissue (Plate 5, fig. 24).
386
A. J. FRIEDENSTEIN £ r ^ L .
(3) The effect of the number of cells and of their packing upon bone formation
Different numbers of cells were placed in chambers of different size and of
each type. The results in terms of the proportion of chambers in which bone
was formed are given in Table 2. The remaining chambers were filled with
reticular tissue.
(4) Implantation of empty chambers
Empty chambers composed of HA filters sterilized for 15 min with 70°
alcohol before intraperitoneal implantation proved to be impermeable to cells:
in none of five empty chambers were cells found 7 days after implantation. No
cells were detected in the filter material itself in sections of the many chambers
composed of HA filters which were studied.
Chambers composed of AUFS filters were permeable to cells The average
numbers of cells found after implantation of an empty chamber with 0 04 mm2
of filter surface were: 5-0 cells after 2 h; 18-5 cells after 6 h; 14-5 cells after 12 h;
24-5 cells after 24 h, and 50-0 cells after 48 h. During the first 5 h leucocytes
predominated among the cells, later lymphocytes and fibroblast-like cells or
histiocytes were predominant. Many cells were always found in the filter
material in sections of chambers composed of AUFS filters (Plate 5, fig. 25).
Table 2. Bone formation in chambers with suspension of bone marrow cells
Chamber
type
Filter
type
No. of cells at time of
transplantation
A
AUFS
106 bone marrow cells
HA
Fixation
Chambers with bone
time
(days) Total number of chambers
119
12
11
AUFS
105 bone marrow cells
f 24
130
HA
AUFS
B
AUFS
HA
106 bone marrow cells
+ 9xl0 6 lymph.
105 bone marrow cells
11
15
11
11
11/12]
5/5 [24/27
8/10J
2/12
2/6
•4/29
0/5
0/6
2/8
2/8
8/11
l 12/17
4/6 J
DISCUSSION
When bone marrow cells are placed into diffusion chambers reticular or
bone tissue is formed instead of haemopoietic elements. This considerable change
of histogenesis occurs after free heterotopic transplantation of bone marrow as
well (Denis, 1958). Therefore, it does not depend upon special cultivation
conditions in diffusion chambers. The cells transplanted in a chamber are in
novel conditions by comparison with those in situ. Their interaction with surrounding tissues (with bone in particular) and their mutual arrangement (tissue
/. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 5
21
25
Fig. 21. The same experiment. Seven days. Alcohol. Gomori. Total preparation. x200.
A focus of osteogenesis.
Fig. 22. The same experiment. Ten days. Alcohol. Gomori. Total preparation, x 200. A focus
of osteogenesis.
Fig. 23. The same experiment. Eleven days. Alcohol. Haematoxylin. Total preparation, x 200.
Bone trabeculae and haemopoietic cells.
Fig. 24. The same experiments. Twelve days. Alcohol. PAS. A section, x 200. Bone in the
chamber, a, Filter, b, bone tissue.
Fig. 25. An empty chamber composed of AUFS chambers. Five days after implantation.
Alcohol. Haematoxylin. Section, x 200. a, Filter; b, chamber contents.
A. J. FRIEDENSTEIN ET AL.
facing p. 386
Osteogenesis in transplants
387
structure) are disturbed. In the case of transplantation of bone marrow fragments this holds true for the zone of cell outgrowth, while the original transplant
itself usually degenerates. These disturbances seem to cause the main alteration
in differentiation of transplanted cells, i.e. the extinction of haemopoiesis. Which
of the causes mentioned is decisive remains unknown.
The fact that osteogenesis occurs in cell suspension shows that the osteogenic
potency of the bone marrow cell population does not disappear after dissociation of cells.
It is evident that not all the transplanted bone marrow cells can act as osteogenic stem-cells. The results of the present work suggest that in a population of
bone marrow cells cultured in diffusion chambers no cells form osteogenic foci
individually. If this were otherwise, bone would form to the same extent in the
chambers of different size after transplantation of the same number of cells
(i.e. containing the same number of such precursor elements). However, the
same number of bone marrow cells, say 105, behaves in a different way in the
chambers of different size. In those of type B this number of cells formed bone,
as a rule, while in the chambers with an area tenfold larger (type A) it did not.
The area of the chamber was determined by the area of the smaller filter.
Bone formation in larger chambers (type A) requires more cells, namely 106, i.e.
ten times more. A tenfold dilution of 106 bone marrow cells placed in type A
chamber (by the addition of 9 x 106 lymphocytes taken from lymph nodes)
prevents osteogenesis. These data cannot be explained by the fact that general
metabolic processes (e.g. those of metabolism, respiration, etc.) could themselves
inhibit or stimulate bone formation in the chambers containing a different
number of cells. When 106—108 bone marrow cells were placed into A-chambers
osteogenesis usually occurred (Friedenstein, 1964). Yet, when 106 bone marrow
cells + 9 x 106 lymphocytes were placed in such chambers bone was rarely formed.
A lesser dilution of the bone marrow cells does not affect the result of transplantation : penetration of a small number of cells from outside into the chamber
(when the chambers consisted of AUFS filters) did not change the frequency of
osteogenesis when compared with the chambers composed of HA filters
impermeable to cells.
The cells responsible for the formation of osteogenic foci seem to have a high
mitotic activity. This is proved by the frequent occurrence of mitosis in the cells
which, judging by morphological characters, are those which give rise to
osteogenic structures in chambers.
It could be expected, therefore, that cells serving as precursors for osteogenic
tissue at transplantation of 105 bone marrow cells in type B chambers can form
considerable cell clones during 30-day cultivation when placed in type A
chambers. Thus, if the population density of 105 cells in type A chambers is
insufficient for osteogenesis, the cells of which bone foci could be formed either
do not multiply or they proliferate, but differentiation of the corresponding
clone does not proceed towards osteogenesis. This latter possibility seems to be
388
A. J. FRIEDENSTEIN^TAL.
most likely. At any rate, the results presented demonstrate that osteogenesis
requires a certain initial density of bone marrow cells. Thus, the development
of bone in a chamber differs from the formation of haemopoietic foci on the
spleen of irradiated mice at transplantation of bone marrow cells (Till, McCulloch & Siminovitch, 1964; Becker, McCulloch & Till, 1963; Lewis &
Trobaugh, 1964). A haemopoietic stem-cell, if in contact with an appropriate
stroma (e.g. in irradiated spleen) is able to create a haemopoietic clone in
isolation (Becker et al. 1963). On the other hand, when bone is formed in a
chamber the differentiation of osteogenic stem-cells depends upon the interaction
of cells at some initial period. It is characteristic that duration does not affect
the results of transplantation, i.e. if the initial packing does not result in bone
formation during eleven days, no osteogenesis develops in 30 days either.
As is well known, bone marrow cells are mobile and are expected to migrate
within the chamber after transplantation. The fact that osteogenesis requires a
certain density of the initial population of bone marrow cells implies that
osteogenesis arises when necessary cells meet to form a corresponding structure.
Apparently it is easily established when the initial density corresponds to 210
bone marrow cells per 0-05 mm2 being achieved but very rarely when the density
equals thirty bone marrow cells per 0-05 mm2.
In the case of transplantation of a piece of marrow, even if it is sufficiently
small to avoid rapid necrosis, bone formation proceeds only in the zone of
outgrowing cells. This implies that the initial structure of marrow is inappropriate for bone formation, and the structure required for osteogenesis is created
anew. Foci of proliferating haemopoietic cells are found on the third to seventh
day in chambers with cell suspension. It seems very likely that these are clones
originated from individual haemopoietic stem-cells similar to haemopoietic foci in
the spleen of irradiated mice (Till et al. 1964). In chambers these foci disappear
on the tenth day probably due to the fact that haemopoiesis proceeds only in the
presence of a certain structure of haemopoietic tissue whose maintenance
requires contact with either bone or spleen stroma (Friedenstein, 1965).
In the presence of bone (or spleen stroma) the stem cells of haemopoietic
tissue differentiate towards haemopoiesis (Till et al. 1964). In the absence of bone,
osteogenic potencies of marrow cell population are realized and bone is formed
which in turn is necessary for the maintenance of haemopoiesis. This creates the
mechanism supporting differentiation of haemopoietic tissue and controlling the
behaviour of stem-cells in the bone marrow cell population. It should be borne
in mind that bone tissue undergoes sustained reconstruction, i.e. it is resorbed in
some places while being formed in others (Hattner, Epker & Frost, 1965). For
this reason its influence upon the differentiation of stem cells can be expected in
any individual area of marrow tissue.
There are grounds for believing that the formation of cells of marrow stroma
and of blood cells is ensured by common stem cells, but no direct evidence is
available. However, haemopoietic and stromal elements might have different
Osteogenesis in transplants
389
stem cells while control over the composition of the whole population of marrow
cells provides selective effects upon each of these categories of stem cells. At the
present time it cannot be ruled out that bone and reticular tissue too have
separate lines of stem cells in bone marrow, although it seems more likely that
they have a common line.
SUMMARY
1. In bone marrow fragments and bone marrow cell suspensions isotransplanted to mice in diffusion chambers, reticular tissue develops and sometimes
osteogenesis also occurs. No haemopoiesis takes place in the grafts.
2. Bone formation in the chambers requires a certain density of the initial
packing of bone marrow cells. This follows from the considerable differences in
the frequency of bone formation in the case of transplantation of the same
number of cells (105) in chambers of different size, and from cases in which
different cell numbers (105 and 106) are transplanted in the chambers of the same
size.
3. The results obtained show that differentiation of the stem cells towards
osteogenesis requires cell interaction within the cell community at a certain
crucial moment.
PE3I0ME
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The authors would like to express their sincere gratitude to Professor G. V. Lopashov for
his valuable advice and interest in this paper.
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(Manuscript received 26 February 1966)