Structural and cellular differences between metaphyseal and diaphyseal periosteum in different aged rats.

Abstract

In both physiological and pathological processes, periosteum plays a determinant role in bone formation and fracture healing. However, no specific report is available so far focusing on the detailed structural and major cellular differences between the periostea covering different bone surface in relation to ageing. The aim of this study is to compare the structural and cellular differences in diaphyseal and metaphyseal periostea in different aged rats using histological and immunohistochemical methods. Four female Lewis rats from each group of juvenile (7 weeks old), mature (7 months old) and aged groups (2 years old) were sacrificed and the right femur of each rat was retrieved, fixed, decalcified and embedded. Five-micrometer thick serial sagittal sections were cut and stained with Hematoxylin and Eosin, Stro-1 (stem cell marker), F4/80 (macrophage marker), TRAP (osteoclast marker) and vWF (endothelial cell marker). One-millimeter lengths of middle diaphyseal and metaphyseal periosteum were selected for observation. The thickness, total cell number and positive cell number for each antibody were measured and compared in each periosteal area and different aged groups. The results were subjected to two-way ANOVA and SNK tests. The results showed that the thickness and cell number in diaphyseal periosteum decreased with age (p<0.001). In comparison with diaphyseal area, the thickness and cell number in metaphyseal periosteum were much higher (p<0.001). There were no significant differences between the juvenile and aged groups in the thickness and cell number in the cambial layer of metaphyseal periosteum (p>0.05). However, the juvenile rats had more Stro1(+), F4/80(+) cells and blood vessels and fewer TRAP(+) cells in different periosteal areas compared with other groups (p<0.001). The aged rats showed much fewer Stro1(+) cells, but more F4/80(+), TRAP(+) cells and blood vessels in the cambial layer of metaphyseal periosteum (p<0.001). In conclusion, structure and cell population of periosteum appear to be both age-related and site-specific. The metaphyseal periosteum of aged rats seems more destructive than diaphyseal part and other age groups. Macrophages in the periosteum may play a dual important role in osteogenesis and osteoclastogenesis.

Full text

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This is the author-manuscript version of this work - accessed from
http://eprints.qut.edu.au

Fan, Wei and Crawford, Ross W. and Xiao, Yin (2008) Structural
and Cellular Differences between Metaphyseal and Diaphyseal
Periosteum in Different-aged Rats. Bone 42(1):pp. 81-89.


Copyright 2008 Elsevier

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Structural and Cellular Differences between Metaphyseal
and Diaphyseal Periosteum in Different-aged Rats

Wei Fan1, Ross Crawford1, Yin Xiao1

1Bone Tissue Engineering Lab, Institute of Health and Biomedical Innovation,
Queensland University of Technology, Brisbane, Australia


Running title: Periosteum and ageing

Key words: periosteum, ageing, macrophages, osteoblasts, Stro-1

Corresponding author
Dr. Yin Xiao
Associate Professor
Bone Tissue Engineering Lab, Institute of Health and Biomedical Innovation,
Queensland University of Technology, Kelvin Grove Campus, Brisbane, Qld 4059
Australia
Tel: +61 7 3138 6240
Fax: +61 7 3138 6030
Email: yin.xiao@qut.edu.au

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Abstract
In both physiological and pathological processes, periosteum plays a determinant role
in bone formation and fracture healing. However, no specific report is available so far
focusing on the detailed structural and major cellular differences between the
periostea covering different bone surface in relation to ageing. The aim of this study is
to compare the structural and cellular differences in diaphyseal and metaphyseal
periostea in different-aged rats using histological and immunohistochemical methods.
Four female Lewis rats from each group of juvenile (7-week old), mature (7-month
old) and aged groups (2-year old) were sacrificed and the right femur of each rat was
retrieved, fixed, decalcified and embedded. 5 µm thick serial sagittal sections were cut
and stained with Hematoxylin and Eosin, Stro-1 (stem cell marker), F4/80
(macrophage marker), TRAP (osteoclast marker) and vWF (endothelial cell marker).
1mm lengths of middle diaphyseal and metaphyseal periosteum were selected for
observation. The thickness, total cell number and positive cell number for each
antibody were measured and compared in each periosteal area and different-aged
groups. The results were subjected to two-way ANOVA and SNK tests.
The results showed that the thickness and cell number in diaphyseal periosteum
decreased with age (p<0.001). In comparison with diaphyseal area, the thickness and
cell number in metaphyseal periosteum were much higher (p<0.001). There were no
significant differences between the juvenile and aged groups in the thickness and cell
number in the cambial layer of metaphyseal periosteum (p>0.05). However, the
juvenile rats had more Stro1+, F4/80+ cells and blood vessels and fewer TRAP+ cells
in different periosteal areas compared with other groups (p<0.001). The aged rats
showed much fewer Stro1+ cells, but more F4/80+ ,TRAP+ cells and blood vessels in
the cambial layer of metaphyseal periosteum (p<0.001).
In conclusion, structure and cell population of periosteum appear to be both
age-related and site-specific. The metaphyseal periosteum of aged rats seems more
destructive than diaphyseal part and other age groups. Macrophages in the periosteum
may play a dual important role in osteogenesis and osteoclastogenesis.

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Introduction
The periosteum is a type of connective tissue developed from mesodermal cells
during the embryonic development. It is tightly attached to various bone surfaces
through Sharpy’s fibres, forming a thin but very tough membrane. In both
physiological and pathological processes, periosteum plays a determinant role in both
bone formation and fracture healing in addition to the involvement of other important
factors such as growth factors and mechanical loading [1-3]. The periosteum contains
progenitor cells both osteogenic and chondrogenic in nature, as well as other related
bioactive factors, and is highly vascularised [1, 4, 5]. Transplantation of autogenous
and allogenous periosteum have been applied successfully to repair various-sized
bone or cartilage defects, in particular to large bone defects [6-10]. However, the
availability of periosteum, morbidity associated with harvest and immunological
concerns are still barriers against large scale in vivo application of periosteum
transplantation. Interestingly, the progenitor cells in the bone marrow and periosteum
have been successfully applied in bone tissue engineering with various scaffold
materials [11-17]. Some researchers have attempted to make artificial periosteum by
seeding progenitor cells onto a membrane and achieved positive results in repairing
bone defects in vivo [18, 19]. However, current artificial periosteum, which is based
on single type of cell structure, is still far from clinical application, primarily due to
the questionable cell viability, stability and long-term function. One of the major
challenges in tissue-engineered periosteum is to form the cellular structures similar to
native periosteum, which can represent the stage of active bone formation. Therefore,
it is imperative to understand the cellular structures of periosteum and their
relationship during bone development and ageing. To date, however, no clear
histological basis is available for the selection of donor-site or age-related periosteal
grafts.
Previous studies have revealed that periosteum consists of two different layers [20].
The outer fibrous layer is composed of fibroblasts, collagen, elastin fibrers, nerve, and
microvascular network. The inner cambial layer is highly cellular containing
mesenchymal stem cells, fibroblasts, osteogenic progenitors and osteoblasts. However,
no study has documented the detailed structural and specific cellular differences
between the periosteum on different bone surface, such as the difference between
metaphyseal and diaphyseal periosteum.
Age is another important factor affecting the structure and function of periosteum.
Some age-related changes in periosteum have been reported including decreased
periosteal fibroblast number, fibrous layer thickness, osteoblast number, collagen
formation, osteoid zones and vessel density throughout the periosteum [21-24].
Depending on the locations of bone formation or resorption periosteum shows
corresponding structural changes with ageing [21, 25]. However, the detailed
information about the age-related structural and cellular changes in different
periosteal areas is still unclear. We hypothesize that age-related changes in periosteum
are also site-specific. Therefore, the distribution of mesenchymal stem cells,
macrophages, osteoclasts, blood vessels and structural changes in metaphyseal and

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diaphyseal periostea from the femurs of different aged rats were investigated in this
study.
Materials and Methods
Animal samples and slices
This study was carried out according to the guideline of the Animal Ethics Committee
of the Queensland University of Technology. Three different aged groups of female
Lewis rats were utilized with four rats in each group. The juvenile, mature, and aged
groups were 7-week old, 7-month old, and two-year old rats respectively. The right
femur of each rat was retrieved after the animals were sacrificed. The tissue samples
were fixed with 4% paraformaldehyde for 12 hours at room temperature, then
decalcified in 10% EDTA and embedded in paraffin. Serial sections of 5 µm thick
sagittal slices were cut from the paraffin blocks using a microtome (Leica
Microsystems GmbH Wetzlar, Germany). The slices near the central sagittal plane
were used for subsequent experiments.
Definition and selection of observed tissue areas
The diaphyseal and metaphyseal periostea were selected for the observation. The
diaphyseal periosteum was selected from 1 mm length of periosteum in the middle of
diaphyseal area and the metaphyseal periosteum was selected from 1 mm length of
periosteum from metaphyseal area starting from the mesial border of growth plate on
upper metaphysis.
Structural observation
Four slices from each rat sample were stained with Hematoxylin and Eosin (HD
Scientific supplies Pty Ltd, Kings Park, NSW, Australia). The images were captured
under ×200 magnification. The thickness of fibrous and cambial layers on the middle
line perpendicular to the periosteum in each microscopic field and cell number of
each layer throughout each periosteal area were measured using Axion software (Carl
Zeiss Microimaging GmbH, Göttingen, Germany) under a microscope (Carl Zeiss
Microimaging GmbH, Göttingen, Germany). The data from the four slices were
averaged and recorded for subsequent analysis.

Immunohistochemistry
Four specific cell markers, Stro-1 (mouse anti-human, Chemicom International Inc.,
Temecula, CA, USA), F4/80 (Rat anti-mouse, ABR-Affinity BioReagents Inc.,
Golden, CO, USA), TRAP (tartrate-resistant acid phosphatase)(mouse anti-human,
Lab Vision Co., Fremont, CA, USA) and vWF (mouse anti-human, Chemicom
International Inc., Temecula, CA, USA), were utilized to identify mesenchymal
stromal cells, monocytes/macrophages, osteoclasts and blood vessels in each
periosteal sample of different-aged groups. To validate the results, each experiment
was repeated at least three times.

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Prior to immunoperoxidase staining, endogenous peroxidase activity was quenched by
incubating the tissue sections with 3% H2O2 for 20 minutes. All sections were
blocked with 10% swine serum for 1h. The enzymatic treatment was used to expose
epitopes by incubating the slices with proteinase K (ready-to-use, DakoCytomation,
CA, USA,) for 10 minutes at room temperature. Sections were then incubated with
optimal dilution of primary antibody Stro-1 (1:100), F4/80 (1:50), TRAP (1:20) and
vWF (1:100) overnight at 4 0C. Sections were then incubated with a biotinylated
swine-anti-mouse, rabbit, goat antibody (DAKO Multilink, CA, USA) for 15 minutes,
and then incubated with horseradish perioxidase-conjugated avidin-biotin complex
(ABC) for 15 minutes. Antibody complexes were visualized after the addition of a
buffered diaminobenzidine (DAB) substrate for 4 minutes. The reaction was
stopped by immersion and rinsing of sections in PBS. Sections were then lightly
counterstained with Mayer’s haematoxylin and Scott’s Blue for 40 seconds each, in
between 3 minute rinses with running tap water. Following this, the sections were
dehydrated with ascending concentrations of ethanol solutions, cleared with xylene
and mounted with a cover slip using DePeX mounting medium (BDH Laboratory
Supplies, England).
Controls for the immunohistological staining procedures included conditions where
the primary antibody was omitted. In addition, an irrelevant antibody (IgG), which
was not present in the test sections, was used as a control.
Under ×400 magnification, the positive cell number from each cell population in
each periosteal area was counted using a microscope (Carl Zeiss Microimaging
GmbH, Göttingen, Germany) and AxioVision software (Carl Zeiss Microimaging
GmbH, Göttingen, Germany). Each measurement included three different slices and
the average was recorded for subsequent analysis. To eliminate the effect of the
difference in total cell number and periosteum thickness in different groups on the
positive cell number and blood vessel counting, the Strol-1+, F4/80+, and TRAP+ cell
numbers were normalized to the cell number per 100 total cells in each specific group.
The blood vessel number was normalized to the vessel number in 0.03mm2 periosteal
area.
Statistical analysis
All the data were analyzed according to the age of rats and the periosteal sites by
two-way ANOVA using the General Linear Model and post-hoc testing performed
using Student-Neuman-Keuls comparison (SNK) for group homogeneity. The
significance level was set at p ≤ 0.05. Analysis was performed using SPSS software
(SPSS Inc, Chicago Il).

Results
Structural differences in different periosteal areas and aged groups
The two-way analysis of variance model indicated that the age of rats and the

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periosteal sites both influenced, independently and interactively, the thickness and cell
number in the periosteum (p<0.001). In diaphyseal periosteum, both thickness and
cell numbers decreased with age in both cambial and fibrous layers (Fig.1). However,
no significant differences were found in the thickness of the fibrous layer between
juvenile and mature groups (p=0.39). There was no difference in cell numbers in the
fibrous layer between mature and aged rats (p= 0.07) (Fig.1). Both the thickness and
cell numbers in the cambial layer decreased significantly with age (p<0.001) (Fig.1).
Compared to the diaphyseal area, the thickness and cell number in fibrous and
cambial layers were significantly higher in metaphyseal periosteum (p<0.001) (Fig.1).
In general，the metaphyseal periosteum of juvenile and aged animals was thicker and
more cellular when compared with the mature group. Notably, there were no
significant differences between the juvenile and aged groups in the thickness and cell
numbers in the cambial layer of metaphyseal periosteum (p=0.07 for thickness and
p=0.14 for cell number) (Fig.1). The mature rats had the thinnest and least cellular
cambial layer in the metaphyseal area. The thickness of the fibrous layer in the
metaphyseal periosteum from all three age groups was similar (p=0.09), while both
mature and aged groups were much less cellular in this layer than the juvenile group
(p<0.001) (Fig.1).

Stro-1 expression in periosteum
The normalized Stro-1+ cell number was used in this analysis. Site (p=0.04), age
(p<0.001) and the interactions between site/age were also significant (p<0.001) in the
model for Stro1 expression. In diaphyseal periosteum, Stro-1 was broadly expressed
in the cambial and fibrous layers in juvenile rats (Fig. 2). Very few Stro-1 positive
cells were found in either cambial or fibrous layers from mature and aged rats
compared to the juvenile group (p<0.001) (Fig. 2).
In the metaphyseal area, the juvenile rats had significantly more Stro-1+ cells in both
the cambial and fibrous layers compared with the mature and aged rats (p<0.001) (Fig.
2). In the fibrous layer of aged rats there were more Stro-1+ cells compared with the
cambial layer and the fibrous layer of mature group (p<0.05). The mature and aged
groups had similar number of Stro-1+ cells in the cambial layer. (Fig. 2).


F4/80 expression in periosteum
The normalized F4/80+ cell number was used for this analysis. Periosteal sites and age
were both significant (p<0.001) factors in the model as well as their interactions. The
juvenile rats had significantly more F4/80+ cells in both layers of the diaphyseal
periosteum compared with the mature and aged groups (p<0.001) (Fig. 3). Nearly no
F4/80+ cells were detected in the diaphyseal periosteum in the mature and aged
groups (Fig. 3).

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In the metaphyseal area, the F4/80+ cells were found mostly in the cambial and
fibrous layers of aged periosteum as well as in the cambial layer of juvenile
periosteum. Significantly fewer positive cells were identified in the mature
periosteum (p<0.001) (Fig. 3).

TRAP expression in periosteum
The normalized TRAP+ cell number was used for this analysis. Site and age (both
p<0.001) were significant factors in the model as well as the interaction between
site/age (p<0.001). TRAP+ cells were mostly detected in the both layers of the
metaphyseal area in aged rats compared with the juvenile and mature rats (p<0.001)
(Fig.4). Very few TRAP+ cells were identified in diaphyseal periostum, although the
cambial layer in the diaphyseal periosteum of aged rats had more positive cells than
both the juvenile and mature aged groups (p=0.004) (Fig. 4). No difference was
observed in the fibrous layer of diaphyseal periosteum in three age groups (p=0.147)
(Fig. 4).
vWF expression in periosteum
The normalized vessel number was used in the analysis. Site and age (both p<0.001)
were significant factors for vWF expression with the interaction of site/age also being
significant (p<0.001). Blood vessels identified by the vWF staining in diaphyseal
periosteum revealed that juvenile rats had a higher degree of vascularization in the
cambial layer than the older age groups (p<0.001) with the exception of the fibrous
layer (p=0.77)(Fig.5)..
In metaphyseal areas the juvenile and aged rats had the higher degree of
vascularization in the cambial layer compared to the mature group (p<0.001) (Fig.5)..
The degree of vascularization in the cambial layer of aged rats was also higher than
the fibrous layer (p<0.001). The vessel number in the fibrous layer of mature rats was
similar to that of juvenile rats (p=0.154), but greater than that of aged rats
(p<0.05)(Fig.5).

Discussion
The indispensable role of periosteum in both bone formation and fracture healing is
well documented. When the periosteum is stripped off, the healing of bone defects
and surrounding soft tissues is seriously compromised [2, 26]. This phenomenon has
inspired many studies to focus on the application of periosteal grafts in bone and
cartilage regeneration. It is known that periosteum is a thin membrane-like connective
tissue covering the external surfaces of most bones and several studies have revealed
the general histological and ultrastructural structures of periosteum from various
bones [22, 27]. However, the detailed cellular structure of periosteum and its site
specificity in relation to ageing are not well understood. In this study, the age-related
degeneration (shown as decrease in thickness and cell number) was observed in the
diaphyseal periosteum, especially the decrease in cell numbers in both cambial and

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fibrous layers in the aged group. However, in the metaphyseal areas, it is worth noting
that the aged rats had thicker and more cellular cambial layer than the rats from the
mature group although they were similar in the fibrous layer. There was also a
noticeable change in cell populations within these areas, in particular an increased
fractional number of osteoclasts. These observations would indicate aged-associated
resorptive activity of cortical bone in metaphyseal areas, which in turn may have
clinical relevance with more fractures reported in metaphyseal areas compared with
diaphyseal areas in the elderly. In a recent study by Bliziotes M. et al.,[28] an
increase in osteoclast numbers and eroded cortical bone surfaces was found more
obvious at the femoral neck of nonhuman primates than at the shaft, especially in the
castrated female animals. The nature of the regulation of periosteal bone turnover
activity is not clear, but mechanical force and sex hormones are important modulators
of periosteal activity. It has been found that low sex steroid levels in female animals
are associated with an increase in periosteal resorptive activity [29] and the
application of focal mechanical force results in an increase in local bone formation
[30].
Stro-1 is thought to be a pluripotent cell marker found highly expressed on various
stromal cells [31-33]. Stro-1 positive cells are capable of differentiating into multiple
mesenchymal cell lineages including adipocytes, osteoblasts and chondrocytes, as
well as hematopoiesis-supportive stromal cells, [32, 33]. In this experiment, a higher
percentage of Stro-1 positive cells were found in diaphyseal and metaphyseal
periosteum in juvenile rats indicating the high osteogenic/chondrogenic nature of
periosteum in this stage. In mature and aged groups, Stro-1 positive cells were
significantly decreased and the intensity of Stro-1 staining was weaker in all
periosteal areas compared with the juvenile group. In juvenile rats, the expression of
Stro-1 was found in periosteal cells, pre-osteoblasts, osteoblasts and early osteocytes
embedded in osteoid. Only mature osteocytes were negative for Stro-1. The broad
expression of Stro-1 in both layers of juvenile periosteum suggests that both cambial
and fibrous layers may be involved in the new bone formation.
To our knowledge no other study has documented the age-related distribution of
monocytes/macrophages or osteoclasts in periosteum even though periosteal bone
turnover activity and osteoclast distribution have recently been documented in the
femoral neck in a series of adult rhesus and Japanese macaques [28]. In our study
periosteal monocytes/macrophages and osteoclasts were identified using F4/80 and
TRAP as specific cell markers. F4/80 (or EMR1) is a specific monocyte/macrophage
marker, while TRAP is mainly expressed by mature osteoclasts. Although both
monocytes/macrophages and osteoclasts derive from the haemopoietic precursors and
both can be multinucleate, there are still substantial differences existing between these
two cell types [34]. The differentiation into the osteoclast lineage from haemopoietic
precursors is found prior to macrophage commitment [35]. It has been reported that
some growth factors produced by macrophages, such as BMP2 or TGF-β, could
promote the osteogenesis and proliferation of osteoblasts and chondrocytes in vitro
[36]. Macrophages can develop into osteoclasts only when certain stimuli exist, such
as receptor activator of nuclear factor Kappa B ligand (RANKL),

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Macrophage-Colony Stimulating Factor (M-CSF) or inflammation factors [37]. In this
study numerous macrophages, but limited number of osteoclasts, were found in the
diaphyseal and metaphyseal periosteum especially in the cambial layer of juvenile rats.
In aged rats both macrophages and osteoclasts were increased in the metaphyseal area.
Few macrophages and osteoclasts were found in mature rats and the diaphyseal
periosteum of aged rats.
Blood vessels, identified by vWF staining, showed that both cambial and fibrous
layers in different periosteal areas in juvenile rats were well vascularized while
mature rats had blood vessels predominantly in the fibrous layer. In the juvenile rats
more active periosteal osteogenic activity was found in both periosteal areas, which
was demonstrated by increased cell numbers of mesenchymal stem cells and
macrophages as well as increased thickness of the cambial layer. The high degree of
vascularization in the periosteum of juvenile rats suggests a role in nutrient and
osteoprogenitor cell supply. In diaphyseal periosteum, blood vessel numbers in the
cambial layer decreased with age. However, in the metaphyseal region, the aged rats
had more blood vessels in the cambial layer when compared to the juvenile and
mature groups. The increase of periosteal vascularisation in aged rats could be related
to increased bone resorptive activity. The increased number of blood vessels in the
areas of osteoclastic bone resorption is also reported in bone metastasis [38] and
ectopic bone resorption [39]. In bone development angiogenesis and bone resorption
are closely associated with each other. Vascular endothelial growth factor (VEGF), the
most critical growth factor for angiogenesis, has been found to stimulate osteoclast
activity [40]. Therefore, osteoclast activity and angiogenesis can be regulated by the
common mediators such as VEGF.
Based on the results obtained in this study, it could be concluded that the age-related
periosteal structure and cell populations are site-specific. The diaphyseal periosteum
showed age-related degeneration, whereas, the metaphyseal periosteum is more
destructive in older age. Macrophages in the periosteum may play a dual, age
dependent role in bone metabolism with osteogenesis in young rats and
osteoclastogenesis in aged rats.

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Acknowledgements
We would like to thank Ms Sarah Whitehouse, a statistician in our institute for
assisting the statistic analysis of the data.

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Legends
Figure 1. Diagrams and pictures (×200) of H&E observation of diaphyseal and
metaphyseal periostea from different-aged groups. The thickness and cell number of
the cambium layer in diaphyseal periosteum (Dia-c) decreased with age. No
significant differences were found in the thickness of the diaphyseal fibrous layer
(Dia-f) in juvenile and mature groups. There were no differences in cell numbers in
the fibrous layer in mature and aged rats. There were no significant differences
between the juvenile and aged groups in the thickness and cell number in cambial
layer of metaphyseal periosteum (Meta-c). The thickness of metaphyseal fibrous layer
(Meta-f) from three aged groups was similar to one another, while both mature and
aged groups were much less cellular in this layer than in the juvenile group. *: p<0.05.
J: Juvenile; M: Mature; A: Aged..
A: diaphyseal periosteum from the juvenile group; B: diaphyseal periosteum from the
mature group; C: diaphyseal periosteum from the aged group; D: metaphyseal
periosteum from the juvenile group; E: metaphyseal periosteum from the mature
group; D: metaphyseal periosteum from the aged group; (In pictures, B: bone tissue; P:
periosteum; C: cambial layer; F: fibrous layer)

Figure 2. Diagram and pictures (×400) of Stro-1+ cell distribution in different
periosteal areas and age groups. Stro-1 was broadly expressed in both the cambial and
fibrous layers of diaphyseal periosteum (Dia-c and Dia-f) from juvenile rats. Very few
Stro-1+cells were found in diaphyseal cambial and fibrous layers from mature and
aged rats in comparison with the juvenile group. No significant difference was found
in the Stro-1+ cell numbers in metaphyseal periosteum (Meta-c and Meta-f) of the
juvenile and mature rats when compared with the diaphyseal area, while both mature
and aged groups had much fewer positive cells than the juvenile group in this area. *:
p<0.05. J: Juvenile; M: Mature; A: Aged.
A: diaphyseal periosteum from the juvenile group; B: diaphyseal periosteum from the
mature group; C: diaphyseal periosteum from the aged group; D: metaphyseal
periosteum from the juvenile group; E: metaphyseal periosteum from the mature
group; D: metaphyseal periosteum from the aged group; (In pictures, B: bone tissue; P:
periosteum)

Figure 3. Diagram and pictures (×400) of F4/80+ cell distribution in different
periosteal areas and age groups. The juvenile rats had much more F4/80+ cells in
cambial layer of diaphyseal periosteum (Dia-c) compared with fibrous layer (Dia-f)
and the other two groups. In metaphyseal area, the F4/80+ cell number in both layers
(Meta-c and Meta-f) from aged rats were more than other aged groups. *: p<0.05. J:

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Juvenile; M: Mature; A: Aged.
A: diaphyseal periosteum from the juvenile group; B: diaphyseal periosteum from the
mature group; C: diaphyseal periosteum from the aged group; D: metaphyseal
periosteum from the juvenile group; E: metaphyseal periosteum from the mature
group; D: metaphyseal periosteum from the aged group; (In pictures, B: bone tissue; P:
periosteum)
Fig.4 Diagram and pictures (×400) of TRAP+ cell distribution in different periosteal
areas and age groups. Few TRAP+ cells were found in the diaphyseal periostum
(Dia-c and Dia-f) in any of the three groups. In the metaphyseal area, the aged rats
had much more TRAP+ cells in the cambial layer (Meta-c) than the fibrous layer
(Meta-f) and the other two groups. *: p<0.05. J: Juvenile; M: Mature; A: Aged.
A: diaphyseal periosteum from the juvenile group; B: diaphyseal periosteum from the
mature group; C: diaphyseal periosteum from the aged group; D: metaphyseal
periosteum from the juvenile group; E: metaphyseal periosteum from the mature
group; D: metaphyseal periosteum from the aged group; (In pictures, B: bone tissue; P:
periosteum)

Fig.5 Diagram and pictures (×400) of vWF+ blood vessel distribution in different
periosteal areas and age groups. Juvenile rats had higher degree of vascularization in
the cambial layer of diaphyseal periosteum (Dia-c) than the other two groups except
in the fibrous layer (Dia-f). In metaphyseal area, the rats from aged group had highest
degree of vascularization in cambial layer (Meta-c) among all three groups. The
degree of vascularization in the cambial layer of aged rats was higher than the fibrous
layer (Meta-f). The vessel number in the fibrous layer of mature rats was similar to
that of juvenile rats, but more than that of aged rats. *: p<0.05. J: Juvenile; M:
Mature; A: Aged.
A: diaphyseal periosteum from the juvenile group; B: diaphyseal periosteum from the
mature group; C: diaphyseal periosteum from the aged group; D: metaphyseal
periosteum from the juvenile group; E: metaphyseal periosteum from the mature
group; D: metaphyseal periosteum from the aged group; (In pictures, B: bone tissue; P:
periosteum)

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