Increase in periosteal angiogenesis through heat shock conditioning.

RESEARCH Open Access
Increase in periosteal angiogenesis through heat
shock conditioning
Majeed Rana*, Constantin von See, Martin Rücker, Paul Schumann, Harald Essig, Horst Kokemüller,
Daniel Lindhorst and Nils-Claudius Gellrich
Abstract
Objective: It is widely known that stress conditioning can protect microcirculation and induce the release of
vasoactive factors for a period of several hours. Little, however, is known about the long-term effects of stress
conditioning on microcirculation, especially on the microcirculation of the periosteum of the calvaria. For this
reason, we used intravital fluorescence microscopy to investigate the effects of heat shock priming on the
microcirculation of the periosteum over a period of several days.
Methods: Fifty-two Lewis rats were randomized into eight groups. Six groups underwent heat shock priming of
the periosteum of the calvaria at 42.5°C, two of them (n = 8) for 15 minutes, two (n = 8) for 25 minutes and two
(n = 8) for 35 minutes. After 24 hours, a periosteal chamber was implanted into the heads of the animals of one of
each of the two groups mentioned above. Microcirculation and inflammatory responses were studied repeatedly
over a period of 14 days using intravital fluorescence microscopy. The expression of heat shock protein (HSP) 70
was examined by immunohistochemistry in three further groups 24 hours after a 15-minute (n = 5), a 25-minute (n
= 5) or a 35-minute (n = 5) heat shock treatment. Two groups that did not undergo priming were used as
controls. One control group (n = 8) was investigated by intravital microscopy and the other (n = 5) by
immunohistochemistry.
Results: During the entire observation period of 14 days, the periosteal chambers revealed physiological
microcirculation of the periosteum of the calvaria without perfusion failures. A significant (p < 0.05) and
continuous increase in functional capillary density was noted from day 5 to day 14 after 25-minute heat shock
priming. Whereas a 15-minute exposure did not lead to an increase in functional capillary density, 35-minute
priming caused a significant but reversible perfusion failure in capillaries. Non-perfused capillaries in the 35-
minute treatment group were reperfused by day 10. Immunohistochemistry demonstrated an increase in
cytoprotective HSP70 expression in the periosteum after a 15-minute and a 35-minute heat shock pretreatment
when compared with the control group. The level of HSP70 expression that was measured in the periosteum
after 25 minutes of treatment was significantly higher than the levels observed after 15 or 35 minutes of heat
shock exposure.
Conclusion: A few days after heat shock priming over an appropriate period of time, a continuous increase in
functional capillary density is seen in the periosteum of the calvaria. This increase in perfusion appears to be the
result of the induction of angiogenesis.
Keywords: Heat shock, periosteum, animal, intravital microscopy, calvaria, microcirculation
* Correspondence: rana.majeed@mh-hannover.de
Department of Oral and Maxillofacial Surgery, Hannover Medical School,
Hannover, Germany
Rana et al. Head & Face Medicine 2011, 7:22
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HEAD & FACE MEDICINE
© 2011 Rana et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Background
The periosteum is a highly vascularized membrane that
covers bone. It consists of a fibro-elastic layer of tissue
that is firmly attached to the bone surface. Although
the bone cortex is the main beneficiary of the principal
anatomical and physiological functions of the perios-
teal membrane, periosteal activity influences the beha-
viour of the entire bone [1]. Above all, the periosteum
participates in osteogenesis, serves as an attachment
site for muscles and ligaments and contributes to the
blood supply to cortical bone [2,3]. Apart from its
nutritive function, the periosteum has also a mechani-
cal function and plays an important role in tissue
repair. Following the surgical treatment of osseous
defects, the periosteum is believed to be of paramount
importance in the healing process [4,5]. In addition,
the periosteum contributes substantially to bone
growth. Capillary perfusion impairment or failure in
the periosteum is reported to lead to disturbed bone
growth especially in association with bone augmenta-
tion, bone distraction and cleft surgery [6]. A basic
requirement for the preservation of periosteal func-
tions is the presence of adequate blood flow in perios-
teal vessels. Especially in bone augmentation
procedures, which are routinely performed prior to the
insertion of dental implants, the presence of a well-
vascularized recipient bed is essential for a successful
outcome [6,7].
Exposure to a local sublethal heat shock is a possible
method of increasing stress resistance. In response to
heat shock priming, cells are believed to be more resis-
tant against stress such as surgical trauma and reperfu-
sion [8-10]. A heat shock leads to the expression of
cytoprotective heat shock proteins (HSPs), which belong
to a family of proteins that induce immunological cell
activities, thermotolerance, buffering of expression of
mutations and macrophage-mediated wound healing
[11,12]. The upregulation of HSPs, however, induces not
only intracellular but also extracellular processes
[13-15]. In tissues, stress conditioning can reduce inter-
stitial edema formation and improve perfusion as a
result of blood flow upregulation [16]. Moreover, a rela-
tionship between heat shock proteins and angiogenic
factors was detected in acute models [17,18]. Long-term
effects on local microcirculation have not yet been
investigated.
The objective of our study was therefore to study the
effects of local heat shock priming on periosteal vascu-
larization and inflammation using an in-vivo rat model.
A further objective was to analyze the influence of the
duration of exposure to a heat shock and the associated
expression of HSPs using immunohistochemistry.
Material and methods
Animals
The study involved 52 male Lewis rats weighing
between 300 to 330 g. All animals had ad libitum access
to food and water. The rats were housed singly in cages.
They were kept and the experiments were performed in
accordance with the German Animal Protection Act. All
animal procedures (dated 1 January 2007) had been
approved by the Animal Protection Department in the
Office of Consumer Protection and Food Safety of the
State of Lower Saxony in Oldenburg.
Heat shock priming
The periosteum of the calvaria of the anesthetized ani-
mals was exposed to a heat shock. For this purpose, the
foreheads of the rats were shaved. Two copper tubes
were placed on the shaved skin through which water
was delivered. The heating procedure was standardized
by exposing the periosteum of the calvaria to a tempera-
ture of 42.5°C for either 15, 25 or 35 minutes. During
heat shock pretreatment, periosteal and systemic tem-
peratures were measured using a modified thermometer.
Chamber implantation
Intravital microscopy using a periosteal chamber has
been previously described in detail.[19]
Briefly, the animals were anesthetized using an intraperi-
toneal injection of ketamine (Ketavet®, 100 mg per kg
bodyweight, Parke-Davis, Germany) and xylazine (Rom-
pun®, 5 mg per kg bodyweight, Bayer HealthCare, Ger-
many). The periosteum was then exposed. Collagenous
connective tissue was carefully excised using microsurgical
instruments and a 3D microscope until the vascular layer
of the periosteum was exposed. The frame of the chamber
was placed on the periosteum and sutured to adjacent
skin in such a way as to prevent drying (Ethicon Vicryl
sutures 5-0, Johnson & Johnson, Germany). The cover
glass was secured to the frame with a circlip.
Intravital fluorescence microscopy
For intravital microscopy, the rats were again anesthe-
tized with ketamine and xylazine immediately after the
implantation of the chamber as well as on days 3, 5, 10,
and 14 after surgery.
For high-resolution imaging of microcirculation, we
injected fluorescein-isothiocyanate-labeled dextran
(FITC-dextran, 150 000 MW, Sigma Chemicals, United
States) for contrast enhancement by intravascular stain-
ing of blood plasma and rhodamine 6G (MG 476, Sigma
Merck, Germany) for direct visualization of leukocytes.
Immediately before each examination, 0.5 ml of FITC-
dextran (150 mg/ml of 0.9% NaCl solution) and 0.5 ml
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of rhodamine 6G (1 mg/ml of 0.9% NaCl solution) were
injected intravenously. Subsequently, the animals were
immobilized on a special plexiglass table in such a way
that the area to be examined was visible under the
microscope and head movements caused by respiration
were minimized.
Epi-illumination fluorescence microscopy was per-
formed using a modified microscope (Zeiss microscope,
Zeiss Fluoartic, Germany) at 20x magnification. A blue
filter block (450- 490 nm) was used for the detection of
FITC. A green filter block (530-560 nm) was used to
visualize leukocytes labeled in vivo with rhodamine 6G.
The microscopic images were recorded by a charge-
coupled device (CCD) video camera (Pieper, FK-6990
IQ-S, Germany) and were transferred to a DVD recor-
der (LQ-MS 800, Panasonic, Osaka, Japan) for off-line
evaluation. Images were recorded for 30 seconds at four
different regions of interest (0.18 mm²). During micro-
scopy, the body temperature of the animals was main-
tained at +36°C using a heating pad.
Inflammatory parameters
We analyzed leukocyte-endothelial cell interaction in
order to study inflammatory responses. For this purpose,
leukocytes were classified as rolling or adherent cells
depending on their interaction with endothelium.
Adherent leukocytes were defined in each vessel seg-
ment as cells that did not move or detach from the
endothelial lining within a specified observation period
of 20 seconds. Results for adherent leukocytes are
expressed as the number of cells per square millimeter
of endothelial surface, calculated from the diameter and
length of the vessel segment. Cylindrical vessel geometry
was assumed. Rolling leukocytes were defined as cells
that moved with a velocity less than two fifths of center-
line velocity. Results for rolling leukocytes are expressed
as the number of cells per minute that passed a defined
reference point in a microvessel.
Vascular perfusion
Microscopic images were analyzed off-line using image
analysis software (CapImage, Zeintl, Heidelberg, Ger-
many). Vessel diameter (μm) and functional microvessel
density (mm/mm²) were determined for the assessment
of microcirculatory parameters. Functional microvessel
density was defined as the total length of blood cell-per-
fused microvessels per observation area and was
expressed in mm/mm2. For the purpose of our analysis,
we measured microvessel density at five observation
areas at the various time points.
Immunohistochemistry of heat shock protein (HSP) 70
After 24 hours of recovery from a 15-minute, a 25-min-
ute or a 35-minute heat shock, specimens from the
anesthetized animals were prepared for immunohistolo-
gical analysis.
For the immunohistochemical detection of HSP70,
paraffin-embedded specimens were cut into 5-μm-
thick sections, deparaffinized with xylene and rehy-
drated. The sections were exposed to 2% normal goat
serum (Dianova, Hamburg, Germany) diluted in phos-
phate-buffered saline (PBS, Biochrom, Berlin, Ger-
many) to block non-specific binding. They were then
incubated overnight at 4°C with a monoclonal mouse
anti-HSP70 antibody (1:200, Acris, Hiddenhausen, Ger-
many). Negative controls were not exposed to the pri-
mary antibody but to normal goat serum. After
washing with PBS, the sections were incubated with
biotinylated goat anti-mouse antibody (1:200, Dianova,
Hamburg, Germany) and then with streptavidin-horse-
radish peroxidase complex (1:500, Dianova, Hamburg,
Germany). Color was developed with aminoethylcarba-
zole (AEC) substrate (Vector, Burlingame, CA) at
room temperature under microscopic examination.
The sections were then washed with water, counter-
stained with hematoxylin, mounted using an aqueous
mounting medium (Aquatex, Merck, Darmstadt, Ger-
many) and examined by light microscopy (DM4000B
Leica Mikrosysteme, Wetzlar, Germany).
The intensity of immunohistochemical staining for
HSP70 was assessed using image analysis software (Ana-
lysis, Olympus Soft Imaging Solutions, Muenster, Ger-
many). Briefly, digital micrograph data obtained for the
immunohistochemical slides were imported from the
microscope-mounted digital imaging system for the ana-
lysis of staining intensity. Regions of interest were
defined. Staining intensity was measured in four samples
from each animal and expressed as the percentage of
positive pixels to total pixels.
Study protocol
The animals were randomized into eight groups. In four
groups (n = 20), the effects of heat shock exposure were
analyzed after 15-minute (n = 5), 25-minute (n = 5) or
35-minute (n = 5) heat shock priming of the perios-
teum. The fourth group (n = 5) served as controls.
For intravital microscopy, 32 animals were placed into
4 groups (each with 8 animals). Periosteal chambers
were inserted into all animals. Twenty-four hours prior
to chamber implantation, three groups received local
heat shock priming for 15, 25 or 35 minutes. Microcir-
culation and inflammation were studied immediately as
well as 3, 5, 10 and 14 days after heat shock priming
using intravital fluorescence microscopy.
Statistical Analysis
Results are expressed as means ± SEM. Differences
between groups were evaluated with a one-way analysis
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of variance (ANOVA). Differences within groups were
analyzed by one-way repeated measures ANOVA. Stu-
dent-Newman-Keuls or Dunn’s post-hoc tests were used
to isolate specific differences. A p-value <0.05 was con-
sidered significant.
Results
During the entire observation period, the periosteal
chamber enabled us to reliably view and monitor the
periosteum covering the calvaria. No animal had macro-
scopic inflammation at the surgical site.
Inflammatory response
Compared with the control group, the groups of ani-
mals that underwent 15-minute or 25-minute heat
shock priming showed slightly elevated numbers of
rolling leukocytes (Figure 1). By contrast, a 35-minute
heat shock induced a marked inflammatory response
as indicated by a significantly higher number of rolling
leukocytes during the entire observation period. The
control group and the group that received 25-minute
heat shock priming showed constantly low numbers of
adherent leukocytes during the observation period. In
the group of animals that were exposed to a 15-minute
heat shock, the number of adherent leukocytes was
slightly increased until day 9 after surgery and then
declined to levels similar to those of the control group.
When compared with all other groups, the group with
a 35-minute treatment showed a significant increase in
the number of adherent leukocytes during the entire
observation period (Figure 2).
Microcirculatory parameters
Intravital fluorescence microscopy allows us to study the
network of microvessels that run parallel to the tissue
surface over a period of 14 days. Vessels that are
oriented perpendicular to the tissue surface and connect
either to subcutaneous tissue or underlying bone cannot
be identified. An examination of the periosteum
revealed that the majority of capillaries were arranged in
a single layer.
A comparison between the control group and the
group with 15-minute heat shock priming showed no
differences in vessel diameters (Figure 3). By contrast, a
significant increase in vessel diameters was found over a
period of five days in those groups that underwent heat
shock treatment for 25 or 35 minutes. After day 5, all
groups showed similar results.
The control group and the group that received 15-
minute heat shock priming showed similar results for
functional microvessel density (Figure 4). These results
were almost constant over the entire observation period.
In the group with a 25-minute heat shock pretreatment,
functional capillary density continuously increased from
day 5 to the end of the observation period (Figure 4).
After day 5 a significant increase (p < 0.05) in microves-
sel density was observed compared to day 0 after heat
shock conditioning. Both buds and sprouts were identi-
fied morphologically. By contrast, a significantly lower
functional microvessel density was detected until day 5
in the group that underwent 35 minutes of heat shock
priming. Non-perfused microvessels were detected until
day 5 after heat shock exposure. From day 5 onwards,
reperfusion of individual non-perfused capillaries was
Figure 1 Rolling leukocytes (n/min) at day 0, 3, 5 and 10 for
controls (black bar) or after heat shock conditioning for 15
minutes (light grey bar), 25 minutes (dark grey bar) or 35
minutes (white bar), as assessed by intravital fluorescence
microscopy and computer-assisted off-line analysis. There were
significant higher rolling leukocytes detectable after 35 minutes of
heat shock conditioning over the entire observation time (* p <
0.05 vs. control group).
Figure 2 Adherent leukocytes at day 0, 3, 5 and 10 for controls
(black bar) or after heat shock conditioning for 15 minutes
(light grey bar), 25 minutes (dark grey bar) or 35 minutes
(white bar), as assessed by intravital fluorescence microscopy
and computer-assisted off-line analysis. There were significant
higher adherent leukocytes detectable after 35 minutes of heat
shock priming over the entire observation time (* p < 0.05 vs.
control group).
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observed. Reperfusion of all non-perfused capillaries was
completed on day 10. All groups showed similar results
for red blood cell velocity in perfused microvessels.
Histology
Regardless of the duration of heat shock priming, there
were no morphological differences in the structure of
the periosteum between the groups with and without
heat shock exposure. Rather, the periosteum was struc-
turally intact and similar in heat-shocked and control
animals.
An analysis of immunohistological specimens from the
control group (0.06% ± 0.04) revealed a very low level of
HSP70 expression near the detection limit. Twenty-four
hours after heat shock treatment, different levels of
HSP70 expression were induced in the periosteum (Fig-
ure 5). Irrespective of the duration of priming, the levels
of HSP70 expression in the periosteum were signifi-
cantly higher in the groups of heat-shocked animals
than in the control group. It was particularly noteworthy
that the level of HSP70 expression was higher after 25
minutes (6.38% ± 09) of heat shock priming than after
15 (1.42% ± 0.4) or 35 (1,18% ± 0.6) minutes of
exposure.
Discussion
After 15 minutes of heat shock priming, there were only
minor changes in the microcirculatory perfusion of the
periosteum. After 35 minutes of pretreatment, there was
a temporary decrease in perfused capillary density. After
25 minutes of heat shock treatment, however, minor
signs of local inflammation and a constant increase in
functional capillary density were observed until day 14
after the application of a heat shock.
Several methods such as laser Doppler flowmetry or
polarographic oximetry can be used for examining per-
fusion of different tissues in vivo [2,20,21]. These meth-
ods, however, have the disadvantage that they can
visualize blood flow only indirectly. It is therefore
impossible to measure blood perfusion of individual
microvessels using these techniques. By contrast, intravi-
tal microscopy allows the perfusion of microvessels to
be examined over an extended period of time [21,22].
The periosteum of the calvaria is difficult to examine
on account of its anatomical location and physiological
adherence to underlying bone. For this reason, only a
few methods are available for investigating the microcir-
culation of the periosteum of the calvaria in vivo. Pre-
vious studies of the periosteum have therefore been
based on acute examinations especially of histological
specimens [23,24]. The chamber model presented here
allowed us to evaluate the periosteum in vivo repeatedly
over several days [19]. As expected, the control group
did not show any significant changes in microcirculatory
perfusion or local signs of inflammation during the
observation period. Likewise, there were hardly any
inflammatory tissue responses to 15 minutes of heat
shock exposure. The animals that received a 15-minute
heat shock treatment showed results similar to those
obtained for control animals not only in terms of
inflammatory tissue responses but also in terms of vessel
diameter. Fifteen minutes of heat shock priming induced
only minor changes in capillary blood flow and func-
tional capillary density. In addition, this animal group
showed a low level of HSP70 expression near the
Figure 4 Functional microvessel density at day 0, 3, 5 and 10
for controls (black bar) or after heat shock conditioning for 15
minutes (light grey bar), 25 minutes (dark grey bar) or 35
minutes (white bar), as assessed by intravital fluorescence
microscopy and computer-assisted off-line analysis. There was
significant lower functional microvessel density detectable up to
day 5 after 35 minutes of heat shock conditioning.
Figure 3 Microvessel diameter (μm) at day 0, 3, 5 and 10 for
controls (black bar) or after heat shock conditioning for 15
minutes (light grey bar), 25 minutes (dark grey bar) or 35
minutes (white bar), as assessed by intravital fluorescence
microscopy and computer-assisted off-line analysis. There was a
significant higher microvessel diameter up to day 5 detectable after
25 and 35 minutes of heat shock conditioning (* p < 0.05 vs.
control group).
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