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Enzymehistochemistry
–
tartrat-resistant acid phosphatase (TRAP)
Enzymatic detections of tartrate-resistant acid phosophatase
(TRAP) activity was performed by incubation of the slices in a solution
of naphtol AS-BI-phosphate (7-bromor-3-hydroxy-2naphthoic-oanisi-
dide phosphate, Sigma-Aldrich, Steinheim, Deutschland) and fast red
violet LB salt (5-chloro-4-benzamido-2-methylbenzenediazonium
chloride hemi [zinc chloride] salt, Sigma-Aldrich) in 0.2 M acetate
buffer (pH 5.0) containing 50 mM tartaric acid for 20 min at 37°C. Then
the slices were counterstained with hematoxyline. As negative control
slices were incubated in 0.2 M acetate buffer (pH 5.2) for 20 min.
Immunohistochemistry for collagen type-1, CD68, osteocalcin,
osteopontin, and eNOS
After deparaffination of tissue sections the endogenous peroxid-
ase was blocked by incubation in 3% H
2
O
2
in Tris-NaCl buffer (pH 7.4)
with 0.025% Triton-X-100 (TBS). After rinsing with TBS the samples
were incubated overnight at 4°C with the following primary
antibodies in dilution buffer (Dako, Glostrup, Denmark): (a) CD68
(1:5 diluted; Dako), (b) Osteocalcin (1:50; R&D Systems, Minneapolis,
MN, USA), eNOS (1:500; BD Biosciences, Heidelberg, Deutschland).
After several washing steps with TBS sections were incubated for
30 min at room temperature with a biotinylated anti-mouse secondary
antibody (1:400; Dako), rinsed again and labelled with the ABC
complex/horseradish peroxidise labelled avidin (Dako) for another
30 min. The cromogen Nova Red (Vector laboratories, Burlingame,
California, USA) was used for visualization of the peroxidise activity.
Counterstaining of nuclei was done with hematoxylin (Shandon
Scientific Ltd, Cheshire, UK).
Connexin-43 in-situ hybridization
DIG-labelled cRNA-probes were generated from a 137 bp PCR-
product of the coding region of human Connexin-43 gene that was
cloned in pGEM
®
-T (Promega, Mannheim, Germany) and transformed
in Escherichia coli XL1-Blue (Stratagene, Heidelberg, Germany). After
extraction of the plasmids by column purification (Qiagen, Hilden,
Germany) the vectors were digested with NcoI and NotI (New England
Biolabs, Frankfurt, Germany) for the production of sense- and
antisense-cRNA. cRNA-probes were generated by using 10x RNA-
DIG-labelling-Mix (Boehringer, Mannheim, Germany) and RNA-poly-
merase T7 and SP6 (Promega).
cRNA-probes were used for in-situ hybridization of deparaffinized
sections that were permeabilized in proteinase K (20 µg/ml; Sigma,
Deisenhofen, Germany) for 25 min at 37°C, postfixed in 4% parafor-
maldehyde, exposed to 20% acetic acid and prehybridized in 20%
glycerol. For hybridization the cRNA-probes were diluted in 1:25 in
hybridization-buffer containing 50% deionized formamide, 10%
dextran sulphate, 2x standard saline citrate (SSC), 1x Denhardt
’
s
solution, 10 µg/ml salmon sperm DNA (Sigma), and 10 µg/ml yeast t-
RNA (Sigma). Hybridization was performed overnight at 40°C in a
humidified chamber. After washing sections were incubated overnight
at 4°C with anti-DIG Fab-antibody conjugated to alkaline phosphatase
(Boehringer Mannheim). After development of staining with nitro-
blue-tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (BCIP/NBT;
KPL, Gaithersburg, MD, USA) sections were mounted with glycerine
gelatine (Merck, Darmstadt, Germany). Sense-cRNA probes were used
as negative controls for each test.
Electron microscopy
For ultrastructural examinations, small samples were postfixed
with 4% paraformaldehyde, 2% glutaraldehyde, 0.04% picric acid in
0.1 M phosphate buffer (pH 7.2, PB) for 6 hours (h) at 4°C, carefully
washed with PB and incubated in 1% osmium tetroxide for 2 h. After
repeatedly washing in PB, specimens were dehydrated in a series of
graded ethanol and embedded in Epon (Serva, Heidelberg, Germany).
Polymerizationwas performed at 60°C for 20 h. Semithin and ultrathin
sections were cut with a diamond knife on an Ultracut (Reichert-Jung,
Germany). Ultrathin sections (70
–
90 nm) were counterstained with
uranylacetate and lead citrate (Reichert Ultrastainer, Leica, Germany)
and examined in a Zeiss EM 109 transmission electron microscope.
Results
Induction of osteoporosis and clinical observation
BMD at the left calcaneus (
n
= 3) dropped from 439.8 ± 50.5 mg/
mm
3
to 334.3 ± 40.1 mg/mm
3
, and at the right calcaneus from 441.3 ±
45.0 mg/mm
3
to 335.3 ± 37.3 mg/mm
3
6 months after ovariectomy
indicating an osteoporotic bone status with a loss of 24 ± 2% of the
initial BMD.
Full-weight bearing was achieved in all three cases in the first post-
operative days and all goats survived the entire observation period
without any wound healing disturbance or other problems.
High-resolution peripheral quantitative computed tomography
(HR-pQCT)
In general, it was observed that the bone defects filled with the
injectable nanocrytalline hydroxyapatite with our without collagen
type I showed higher bone volume with increased trabecular number
and decreased trabecular spacing compared to the empty defect
control group (Figure 2). This finding was confirmed by micro-CT
histomorphometric parameters with HA exhibiting the highest BV/TV
ratio (
p
= 0.008) and smallest trabecular spacing
(Tb.Sp) (
p
= 0.005)
compared to the other groups in the region of interest at the interface
with 1 mm distance to the initially created defect (Table 2). The HA/
col-1 yielded the highest connectivity density (Conn.D) (
p
= 0.034) and
the highest number of trabeculae (Tb.N) (
p
= 0.002) compared to the
HA and the control group.
Histomorphometry
Histomorphometric analysis for the core region of the initially
created defect revealed a statistically higher new bone formation in
the HA (
p
= 0.001) and HA/col-1 group (
p
= 0.001) compared to
the empty defect group including all defect sites (Figure 2A). There
were no significant differences between the HA- and the HA/col-1
group (
p
= 0.15). These results were confirmed for site specific analysis
with significant higher new bone formation for the HA group for
vertebral defects compared to the empty defect group (
p
= 0.029)
(Figure 2B). There were no significant differences for new bone
formation at the iliac crest (
p
= 0.119).
For the interface region, no statistically significant differences were
found between the three groups including all defects (
p
= 0.08)
(Figure 2C).
Histology
Defects of the empty control group were found either to be filled
with granulation tissue or with lipid rich bone marrow with an
accumulation of multinuclear cells degrading bone marrow fat cells
(Figure 3). Bone lining cells of the surrounding trabecular bone turned
into active osteoblasts with production of collagen into direction of
the defect.
Defects filled with HA showed a good biocompatibility without
inflammatory reaction and a high fragmentation of the implant that
was related to some site-specific differences (Figure 3). HA implants in
iliac crest defects were more fragmented than in the vertebrae.
Fragmentation was shown to be caused by multinuclear cells with
V. Alt et al. / Injury, Int. J. Care Injured 47S2 (2016) S58
–
S65
S61