Clinical Science (1998) 94, 549-555 (Printed in Great Britain)
549
Skeletal progenitor cells and ageing human populations
Richard 0. C. OREFFO, Stephanie BORD and James T. TRIFFllT
MRC Bone Research Laboratory, Nuffield Department of Orthopoedic Surgery, University of Oxford,
Oxford OX3 7u), U.K.
1. Stem and progenitor cells present within bone marrow give rise to colony forming units-fibroblastic (CFUF) which can differentiate into fibroblastic, osteogenic,
myogenic, adipogenic and reticular cells. The decrease
in skeletal bone formation and rate of fracture repair
observed with ageing and in osteoporosis has been
suggested to be due to a decrease in numbers of these
progenitors, but human studies are limited.
2. We have tested the potential to form CFU-F in a total
of 99 patients undergoing corrective surgery (16 controls, 14-48 years of age) or hip arthroplasty for
osteoarthritis (57 patients, 28-87 years of age) or
osteoporosis (26 patients, 6%97 years of age). Total
colony number, alkaline phosphatase-positive colony
number and colony size were determined.
3. No decrease in colony forming efficiency under the
culture conditions used was observed in all populations
examined irrespective of age, disease or gender, as
determined by the lack of correlation between colony
formation and age.
3. Examination of colony sizes showed a significant
reduction in colony size with age in osteoarthritis and in
control populations indiclting a change in cellular
proliferative potential witt age.
4. Examination of number and percentage of alkaline
phosphatase-positive CFU-F showed a significant decrease in osteoporotic patients compared with controls
and osteoarthritis patients, indicating altered differentiation potential.
5. These results suggest that the reduction in bone mass
with ageing may be due to reduction of the proliferative
capacity of progenitor cells or their responsiveness to
biological factors leading to alteration in subsequent
differentiation. The maintenance of CFU-F number and
alkaline phosphatase activity in these osteoarthritis
patients may, in part, explain the inverse relationship
observed for the preservation of bone mass between
generalized osteoarthritis and primary osteoporosis.
INTRODUCTION
Bone structure is maintained by a delicate balance
between bone resorption and bone formation during
skeletal remodelling in the young individual [ 1-31.
However, in aged individuals this process is altered,
~~
~~
resulting in net bone loss as a consequence of increased
trabecular and cortical bone resorption and decreased
osteoblast number and bone formation [4-91.Stem
cells within bone marrow give rise to colony forming
units-fibroblastic (CFU-F), which can differentiate
into cells of the osteogenic, adipogenic, fibroblastic
and reticular lineages [10-13]. A proportion of these
CFU-F cells have high proliferative and differentiation
capacity, as shown by the formation of bone in
diffusion chambers in rat, mouse, rabbit, porcine and
human marrow populations, which has led to interest
in the use of these stem cells for skeletal repair [ 14-20].
However, there is little known about the roles of the
osteoblast stem cells, osteoblast progenitors or their
changes in number or regulation during the process of
ageing in normal, osteoarthritic or osteoporotic populations. Osteoarthritis (OA) and osteoporosis affect a
majority of the population over 70 years of age [21,22].
However, these two common age-related musculoskeletal diseases rarely co-exist and a number of
clinical studies have suggested an inverse relationship
between OA and osteoporosis with the increased bone
mineral density observed in OA providing protection
against osteoporosis [22,23].
To date, the culture of marrow cells and quantification of osteoprogenitors with ageing has been
examined in predominantly animal models with contradictory results. Bergman et al. [24] reported that in
mouse marrow cells from 4- and 24-month-old mice,
the numbers of osteoblast progenitors were decreased,
although the basal proliferative rate of these cells in
older animals was increased. No differences in alkaline
phosphatase expression were observed at the time
points studied. Tsuji et al. [25], using marrow cultured
from young and old rats, found reduced nodule
formation and reduced alkaline phosphatase-positive
cells in older animals, and, similarly, Quarto et al. [26]
and Egrise et al. [27] reported reduced osteoprogenitor
number and reduced alkaline phosphatase number in
aged rats. In contrast, Roholl et al. [28] in an
ultrastructural analysis study found the numbers of
osteoblasts in ageing rats decreased 10-fold, but the
numbers of pre-osteoblasts were age independent
suggesting that the diminished maturation of osteoprogenitors to osteoblasts is the main factor in bone
loss associated with ageing. In studies looking at
stromal colony formation from mouse marrow, Xu et
~~
Key words: ageing, colony forming units-fibroblastic, marrow fibroblasts, mesenchymal, osteogenesis. osteoprogenitors.
Abbreviations: CFU-F, colony forming units-fibroblastic; MEM, minimum essential medium; OA, osteoarthritis.
Correspondence: D r JamesT. Triffitt.
R. 0. C. Oreffo. S. Bord and J. T. Triffitt
550
al. [29] observed colony formation was 50% higher
in 1415-month-old mice compared with that in
34-month-old mice.
The number of human studies on osteoprogenitor
number and age are few and conflicting. Evans et al.
[30] observed no correlation in cell growth and donor
age from in vitro studies using trabecular bone explant
cultures, and Knight and Gowen [31] found no
relationship between osteoblast function and age.
However, preliminary reports from Nishida et al. [32]
and Endo et al. [33] showed that CFU-F number and
alkaline phosphatase-positive CFU-F number were
decreased with age, while Shigeno and Ashton [34]
found cell proliferation was decreased in populations
under 30 years of age.
We have examined the effects of ageing on human
osteogenic potential in control, osteoarthritic and
osteoporotic populations and tested the hypotheses
that (i) reduced CFU-F numbers are responsible for
reduced osteoprogenitor and osteoblast number and
consequently reduced bone formation observed with
age in control and osteoporotic populations, and (ii)
that maintained or increased CFU-F numbers or
CFU-F activity are responsible for the maintenance of
osteoprogenitor and osteoblast number/activity in
generalized OA and thus explain, in part, the increased
bone mineral density observed in OA.
METHODS
Materials
Cell culture reagents were obtained from Gibco/
BRL (Paisley, Scotland). Alkaline phosphatase kits
and other reagents were from Sigma Chemical Company (Poole, Dorset).
Cell culture
Primary cultures of bone marrow cells obtained
from haematologically normal patients undergoing
routine total hip replacement surgery or corrective
surgery (Table 1) were established as previously
described [35]. Only tissue which would have been
discarded was used with the approval of the local
hospital management and ethical committee. In brief,
marrow cells were harvested using a-minimal essential medium (aMEM) from trabecular bone marrow samples and pelleted by centrifugation at 500 g for
5 min at 4 "C. The cell pellet was resuspended in 10 ml
of aMEM and passaged through nylon mesh (90p
pore size; Lockertex, Warrington, U.K.). Samples of
cell suspension were diluted with 0.5 % (w/v) Trypan
Blue in 0.16 M ammonium chloride and the number
and viability of nucleated cells determined. Cells were
plated out in 25 cm2 plastic tissue culture flasks at
2 x los nucleated cells/flask in aMEM supplemented
with 10% (v/v) foetal calf serum, penicillin (5000
units/100 ml) and streptomycin sulphate ( 5 mg/
100 ml). To avoid problems that can be encountered
with batch to batch serum variation, all studies were
conducted using the same serum batch, known from
laboratory studies to promote stromal cell adhesion
and cell proliferation in human bone marrow cultures.
Cultures were maintained at 37 "C in a gassed
incubator, 5 % CO, in air. The medium was changed
after 6 days and then at day 10 and cultures were
stopped at day 12. At the completion of cell culture,
the medium was removed, the cell layer washed in PBS
and cultures fixed in 95 % (v/v) ethanol. Histological
examination of the human bone marrow cultures was
performed using a Zeiss Axiophot photomicroscope
(Carl Zeiss Ltd, Welwyn Garden City, Herts, U.K.),
and recorded on Kodak tungsten film (Kodak, Heme1
Hempstead, U.K.).
Colony counting and colony size
A range of plating densities (from 5 x lo5 to 5 x lo6
cells/25 cm2) was examined in extensive preliminary
studies to determine the optimal seeding density. A
seeding density of 2 x los cells provided clear distinct
colonies and reproducible stable colony numbers
(results not shown). Total and alkaline phosphatasepositive colonies were counted by eye using an Anderman colony counter (Anderman and Co. Ltd, Kingston-on-Thames, U.K.) as previously reported [36].
All counts were performed blind by an independent
observer. Counts were repeated randomly to confirm
reproducibility of counts obtained. Mean values for
each age point from each patient were derived from
3-6 flasks. For quantitative measurement of colony
sizes, CFU-F colony images were captured with a
video camera, digitized and analysed using OPTIMAS
image analysis software (Datacell Ltd, Maidenhead,
Berks, U.K.). Mean colony sizes (kS.E.M. ; cm2)for
each patient were determined from 10 colonies selected
at random [37], using a grid of the flask area drawn
up using random number tables to determine x-y
coordinates, and placed over the surface of the flask
(3-6 flasks per patient).
Table I Clinical details of patients
Patient no.
(Male+ Female)
Patient no.
(Male)
I2
2
3
I
I
57
26
0
23
4
Patient no.
(Female)
Age
(Yea4
10
2
I
34
22
14-31
2 1 4
47
28-07
69-97
Clinical information
Coztoplasty/Scoliosis
Fracture of femur
Fusion of spine
Coxarthrosir
Osteoporosis-Thompson
Fracture
Human osteoprogenitor number and ageing
Histochemical staininvlkaline phosphatase
activity
Cultures were rinsed three times in PBS, fixed in
95% (v/v) ethanol and stained using an alkaline
phosphatase kit (Sigma kit no. 85) according to the
manufacturer’s instructions. Colonies were determined to be alkaline phosphatase-positive if they
showed any observable staining by light microscopy.
The percentage of alkaline phosphatase-positive
colonies (for each patient) was based on the mean
for each flask.
Statistics
Values are expressed as meansfS.D. or S.E.M.
Linear regression was performed on all data sets and
statistical significance(critical value oft) calculated on
n - 2 degrees of freedom at the two-sided significance
level using the following equation from Altman [38]:
To test under the null hypothesis Ho that there is no
correlation with the parameter in the population, the
population correlation coefficient p =!= 0
where r = correlation coefficient and n = sample number. If 0 - t , < t < t, conclude p = 0. The minimum
number of samples required to provide a valid result at
the correlation observed was calculated using the
following equation [37]:
n = ( 1.96dl
r-)+2
-r2
where n gives the minimum sample size at the corresponding correlation coefficient ( r ) required to give a
significant two-sided result.
RESULTS
Colony forming efficiency
At the completion of culture on day 12, discrete,
individual alkaline phosphatase-positive and negative
colonies were observed by eye and microscopy and
demonstrated a wide variation in colony size. Colonies
were observed to be either completely unstained or to
contain varying numbers of stained cells. Colonies
containing any alkaline phosphatase-positive cells
were counted as alkaline phosphatase-positive CFUF. The mean fibroblast colony forming efficiency from
the whole patient group (99 patients; 14-97 years of
1.45 x
giving
age) was found to be 2.75 x
approximately 1 colony/40000 plated nucleated marrow cells. The colony forming efficiency, as shown in
Table 2, was similar whether analysed as a whole or in
individual control (16 patients, 14-48 years of age),
osteoarthritic (57 patients, 28-87 years of age) and
osteoporotic populations (26 patients, 69-97 years of
age).
55 I
Variation of CFU-F number with age
The variation in colony number in control, osteoarthritic and osteoporotic populations is shown in
Figure la. Examination of colony number as a
function of age showed no correlation of CFU-F
number (regression value, r = -0.16) in vitro with
increasing age in control populations (Table 2). Similarly, examination of CFU-F number with age in the
osteoarthritic ( r = 0.03, 28-87 years of age) and
osteoporotic (r = -0.02, 69-97 years of age) populations showed no significant correlation as a function
of age (Table 2). It should be noted that the significance
level of the correlation coefficient and the sample size
are inversely related and thus a larger sample size
ensures that a smaller observed correlation value is
required to have a significant result. None of the above
correlation coefficients are significant on this basis.
Variation with gender and age
The current data were analysed for variation in
CFU-F number within the female population sampled
to determine whether variation in CFU-F number in
control, osteoarthritic or osteoporotic patients was
gender specific. There was a wide variation in CFU-F
numbers, when examined as a complete group or
subdivided into disease categories; no significant
correlation in CFU-F number with age was observed
whether from control ( r = -0.195), osteoarthritic
( r = 0.01) or osteoporotic populations (r = 0.120).
Expression of alkaline phosphatase-positiveCFU-F
Examination of alkaline phosphatase-positive
CFU-F in control subjects and patients with OA
revealed no significant age-related differences in alkaline phosphatase expression. Alkaline phosphatasepositive CFU-F colonies were detected in both young
and old marrow samples with a high degree of
variation across all age groups, whether expressed as
numbers of total alkaline phosphatase-positive CFUF colonies or the percentage of alkaline phosphatasepositive colonies irrespective of age or gender (Figures
l b and lc). In contrast, in osteoporotic patients,
although there is no change with age in colony number,
the mean number of alkaline phosphatase-positive
CFU-F colonies and the percentage of alkaline phosphatase-positive CFU-F colonies was significantly
reduced (P < 0.001) compared with control subjects
and patients with OA (Figures l b and lc and Table 2),
indicating altered differentiation potential of CFU-F
colonies from osteoporotic populations.
Variation in colony size
Colony size and thus proliferation of putative
osteoprogenitors was examined in controls (n = lo),
osteoarthritic (n = 22) and osteoporotic (n = 22) populations to examine if this parameter was altered
during ageing. Colony size was significantly reduced
(P < 0.001) with increasing age irrespective of whether
the donors were control subjects (r = -0.618) or
R. 0. C. Oreffo, S. Bord and J. T. Triffitt
552
Table 2 Analysis of CFU-F number and colony size as a function of age in normal subjects or patients
with skeletal disease
Fibroblast colonies from normal, osteoarthritic or osteoporotic male and female patients of differing ages were
analysed by counting colony number and measuring colony size using image analysis. Correlationsof the changes with
age within populationsofnormal, osteoarthriticand osteoporoticpatients werecalculated. *P < 0.00I versus control
using Student's t-test: t P < 0.001 versus O A group using Student's t-test, SP < 0.001. significant correlation in colony
size with age.
Age group
(Yean)
Age
correlation
(r value)
Alkaline
phosphatasepositive CFU-F
(mean +S.E.M.)
%Alkaline
phosphatasepositive CFU-F
(mean fS.E.M.)
Colony forming
eficiency
no.
Colony
number
(mean kS.D.)
16
62 f 27
-0.16
37+3
56+4
3.1
57
48f28
+0.03
33+2
62+3
2.4x I O - ~ ~1 0I- ~. ~ ~
26
66+22
-0.02
I2* I*t
18k2*t
3.3x I O - ~ ~ I . II O - ~
10
0.094+0.033
-0.6181
22
0.062f0.035
-0.6451
22
0.073k0.019
-0.096
Patient
Colony no
Normal
(14-48)
OA
(28-87)
Osteoporosis
(69-97)
1 . 4 I~O - ~
Colony size
Normal
(14-48)
OA
(48-83)
Osteoporosis
(69-97)
patients with OA ( r = -0.646) (Table 2). Similarly,
colony size was reduced in osteoporotic patients
compared with controls and thus an effect of age is
apparent as the osteoporotic population has a mean
age (83.6 f7.1 years) greater than the control population (23.8 f9.5 years). However, colony size within
the osteoporotic population was not age dependent
( r = -0.096) over the narrow age range of 69-97 years
(Table 2). Calculation of the minimum number of
samples required to give a significant result, at the
correlation values observed for colony size change
with age for the control subjects and patients with OA
( r = -0.618 to -0.645 respectively), indicates that
only 4 8 patient samples were required.
DISCUSSION
The present studies indicate that no correlation
between age and CFU-F number or colony forming
efficiency was observed in all age groups of the patients
examined irrespective of disease state or gender. Thus
the potential to form CFU-F, examined in this study,
was found to be independent of age in these situations.
This indicates the possibility that the reduction in bone
formation associated with ageing is not a consequence
of reduced numbers of early progenitor cells as shown
by measurement of colony forming efficiency or colony
number. Rather, it appears more probable that it is a
consequence of alteration of osteoprogenitor proliferation and differentiation and possibly altered responsiveness of these cells to biological factors affecting
these processes. In the populations examined, a significant reduction in colony size with age was observed
within control subjects and patients with OA. When
compared with controls this was also seen in osteoporotic patients, indicating a general lowering of
proliferative capacity of CFU-F with development of
the disease. Furthermore, in osteoporotic patients the
number and percentage of alkaline phosphatasepositive CFU-F colonies was reduced compared with
osteoarthritic and control populations. It has been
observed that there is a general and inverse relationship between OA and osteoporosis [22,23], and this
could be attributable, in part, to a maintenance of
CFU-F number and activity in OA. The present
studies, however, are more supportive of the hypothesis that the differences in these two populations
are a result of elevated growth factor content in OA
which may contribute to the altered bone turnover,
increased bone density and decreased incidence of
skeletal fracture seen in this condition [39,40].
To examine variations in CFU-F number in human
populations, the site and reproducibility of marrow
extraction and the culture conditions employed are
crucial. In all OA samples used in this study, marrow
was from the trabecular bone within the femoral head
and neck. For patients undergoing corrective surgery
marrow was from the rib, and for osteoporotic samples
(all classified as Thompson fractures) it was extracted
from the femoral head and neck. Although only
limited comparisons can be drawn between patients
undergoing corrective surgery and patients presenting
with OA or osteoporosis, as these groups have different
patient age ranges, numbers and pathophysiology, no
significant differences were observed in CFU-F number, indicating the site of marrow extraction was not
an issue. In the current study a wide variation in
alkaline phosphatase-positive CFU-F colonies was
observed in patients presenting with OA. Whether this
553
Human osteoprogenitor number and ageing
a
T
140
l6O1
0
b
140
10
20
30
40
50
70
60
AGE (Years)
1
0
90
100
T
10
20
10
20
30
40
50
60
AGE (Years)
70
80
90
100
I
-10 J
0
80
30
40
50
60
70
80
90
100
AGE (Years)
Figure I Variation in (a) colony number, (b) alkaline phosphatasapositive colony number and (c)
percentage alkaline phosphatasc-positive colony number as a function of donor age
Marrow was from male and female control
(m, 14-48 years), osteoarthritic ( 0 ,28-87
years) and osteoporotic
(0.69-97 years) populations. In (a) each point represents mean colony numberfS.D.. in (b) each point represents mean alkaline phosphatase-positivecolony number+S.D., and in (c) each point represents mean percentage
alkaline phosphatase-positivecolony number S.D.
+
reflects differences in the phenotype of the precursor
pool in OA is unclear. Furthermore, in the present
studies no dexamethasone or other additional osteogenic factors were added to the cultures, as, particularly with dexamethasone, almost all human CFUF become alkaline phosphatase-positive [35,41,42].
Thus this parameter appears to be less indicative of
osteogenic capacity in the presence of dexamethasone
in the human system.
The results presented here differ from some of the
data obtained by using mouse and rat marrow stromal
cells from young and aged animals [4,27,36,43] in
which a negative correlation is observed between the
number of osteoblast progenitors and age. However,
other studies in rodent systems are in conflict with
these observations. Xu et al. [29] found an increase in
CFU-F formation in old mice compared with young
mice, and Roholl et al. [28] found that the changes in
554
R. 0.C. Oreffo, S. Bord and J. T. Triffitt
bone mass observed between young and old mice were
a consequence not of altered osteoblast precursor
number, but rather a failure of osteoblast differentiation into mature osteoblasts. They observed no
evidence, from morphometric examination of bone
surfaces, for a reduction in number of osteoblast
precursors in old mice. Shigeno and Ashton [34]
examined cell outgrowths from human trabecular
explants and noted a decrease in proliferation in
trabecular bone cells as a function of age, but only in
the second and third decades. In the present study,
CFU-F number from controls over five decades
showed no significant correlation ( r = -0.16) with age
over the age range 14-48 years.
The difference between these results and those in
abstracts from Nishida et al. [32] and Endo et al. [33],
reporting a correlation in CFU-F and alkaline phosphatase-positive CFU-F in their human marrow populations, is difficult to reconcile. However, their studies
provided no clinical details of the patients studied. The
large number of patients examined in the present study
argues against limited sample size and numbers of
observations as the cause for discrepancy. It is particularly relevant that studies looking at haemopoietic
status and function in young (2-3 months) and old
(2-2.5 years) mice, where total marrow cellularity and
stem cell numbers for granulocytes, fibroblasts and
erythroblasts were examined, found no evidence for a
decrease in numbers of these cells with ageing [43]. The
current results also indicate that in the stromal system,
like the haemopoietic system, ageing has no effect on
the efficiency of colony formation, or colony numbers.
In support of the possibility of altered responsiveness
of progenitor cells and a decrease in osteoprogenitor
proliferation, Marie et al. [44] found a reduction in cell
proliferation capacity in trabecular bone explant
cultures from women with postmenopausal osteoporosis.
The current results emphasize an important point
made by Friedenstein [45], namely that changes in
observed marrow CFU-F quantity or colony forming
efficiency may be dependent on sensitivity to a variety
of growth factors rather than an actual reduction of
stem cell number in vivo. The present results do not
preclude the possibility that changes in CFU-F number may occur within the first few years of development, but in the population examined here the stem
cell-progenitor pool appears to remain constant from
14 to 97 years of age. The lack of evidence to
substantiate a decrease in CFU-F number but the
evidence of a decrease in proliferation of osteoprogenitors with age and in osteoporotic patients,
suggests alternative reasons for the reduced bone mass
associated with ageing. Thus either CFU-F are not
altered as a function of age, but the responses to
growth factors and hormones are decreased and/or
the progeny of the osteogenic stem cells have altered
differentiation potential, or, differentiation into alternative stromal fibroblastic lineages, such as adipocytes, occurs. It is well known that there is an
association between an increase in adipogenesis in
osteoporotic patients and the elderly and a progressive
loss of bone [6,46,47].
Pfeilschifter et al. [48], using trabecular bone explant
cultures, have presented data in support of a decreased
response of osteoblasts to growth factors and hormones in osteoporosis. In an extension of these studies
Bismar et al. [49], and in separate studies Marie et al.
[50], have also shown that the combination of altered
cytokine production and osteoprogenitor responsiveness may play a major role in the pathogenesis of bone
loss with age. In summary, the known reduction in
fracture repair and bone loss with ageing [51] or in
osteoporosis may be due to reduction of the proliferative capacity of progenitor cells and/or a reduction in responsiveness of osteoprogenitor cells to
biological factors resulting in alteration in their subsequent differentiation. Furthermore, our results indicate that the inverse association between OA and
osteoporosis may be partially explained by the maintenance of CFU-F number and activity in osteoarthritic patients with age. These results indicate that
examination of CFU-F proliferation and activity and
identification of the factors and mechanisms that
regulate these processes may provide useful insights
into bone formation in disease states or in ageing
populations.
ACKNOWLEDGMENTS
We acknowledge the support and essential assistance of Professor John Kenwright, Mr Jeremy Fairbank and other consultant orthopaedic surgeons and
surgical staff at the Nuffield Orthopaedic Centre for
provision of bone marrow samples. We thank Dr
M. Cortina-Borja (Statistics Department, University
of Oxford) for statistical assistance and A. Bennett for
expert technical assistance. Support from the Medical
Research Council is gratefully acknowledged.
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