Romanian Biotechnological Letters
Copyright © 2016 University of Bucharest
Vol. 22, No. 6, 2016
Printed in Romania. All rights reserved
ORIGINAL PAPER
Homeostasis of blood parameters and inflammatory markers analysis
during bone defect healing after scaffolds implantation
in mice calvaria defects
Received for publication, January 15th 2016
Accepted, February 23th 2016
BALTA CORNEL1#, HILDEGARD HERMAN1#, MARCEL ROSU1#, CORALIA COTORACI2#,
ALEXANDRA IVAN1,3, ALEXANDRA FOLK4, ROBERT DUKA1, SORINA DINESCU5,
MARIETA COSTACHE5, ALEXANDRU PETRE6, ANCA HERMENEAN1,7*
1
Department of Experimental and Applied Biology, Institute of Life Sciences, Vasile Goldis
Western University of Arad, 86 Rebreanu, 310414 Arad, Romania; 2Department of Hematology,
Faculty of Medicine, Pharmacy and Dentistry, Vasile Goldis Western University of Arad,
Romania; 3Department of Functional Sciences, University of Medicine and Pharmacy Victor
Babes; 2 Eftimie Murgu; 300041; Timisoara, Romania; 4Department of Pathology, Faculty of
Medicine, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romania;
5
Department of Biochemistry and Molecular Biology, University of Bucharest, 91-95 Splaiul
Independentei, 050095 Bucharest, Romania; 6Fixed Prosthodontics and Dental Occlusion,
Department of Prosthodontics, University of Medicine and Pharmacy Carol Davila
Bucharest’7Department of Histology, Faculty of Medicine, Vasile Goldis Western University of
Arad, 86 Rebreanu, 310414 Arad, Romania; # Authors with equal contribution
*Address correspondence to: “Vasile Goldis” Western University of Arad, Department of
Histology, Faculty of Medicine, Pharmacy and Dentistry, 86 Rebreanu St., 310414 Arad,
Romania. Anca Hermenean, e-mail: [email protected]
Abstract
Implantation of a biomaterial can trigger an inflammatory reaction, which influences the differentiation
pattern of cell populations involvedin the regenerative process, determining implant success or rejection.Five
groups of chitosan (CH) – graphene oxide (GO) scaffolds were implanted in mice calvarial defects for different
intervals (72 h, 2 w, 4w, 6w, 8w). The defects were randomly filled with CH or CH 0.5% -3% GO. Heparinized
blood was used for the complete blood count: Red Blood Cell Count (RBC), White Blood Cell Count (WBC),
Platelet Count (PLT), Hemoglobin (HGB), Hematocrit value (HCT). Blood samples were also analyzed for
C-reactive protein (CRP) levels.Histopathological analysis showed that the chitosan-based scaffolds improved
with 0.5-3 wt.% GO were quickly infiltrated upon implantation. Chitosan scaffolds improved with 3% GO
strongly stimulated the production of TNF-α, correlated with an increase in the metabolic activity.Within the first
8 weeks, the chitosan (CH) – graphene oxide (GO) scaffolds were well tolerated by the mice and no abnormal
conditions were observed. The inflammatory response was present at early stages post-implantation and it
decreased after 2 weeks. Chitosan scaffolds improved with 3% GO proved to be the best tolerated biomaterial,
which renders it an important candidate for the studies of bone regeneration.
Keywords: mice calvaria, scaffolds, homeostasis, blood parameters, inflammatory markers, TNF-alpha
1. Introduction
Natural or synthetic polymer based scaffolds have played an increasingly prominent
role in the success of tissue engineering. Advances in our understanding of regenerative
materials and their roles in new bone formation can potentially open a new frontier in the fast
growing field of regenerative medicine (ROSSI & al. [1]). Chitosan is a polysaccharide
derived from partial deacetylation of chitin, which commonly located in the shells of marine
crustaceans and insects. Chitosan and carbon-based nanomaterials are considered appropriate
for biomedical and clinical applications due to their high biocompatibility, biodegradability
and adsorption properties (BASHA & al., DEPAN & al. [2, 3]). The limitations of bone
reconstruction techniques using scaffolds have led to increased interest in bone tissue
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Homeostasis of blood parameters and inflammatory markers analysis during bone defect healing
after scaffolds implantation in mice calvaria defects
engineering. Implantation of a biomaterial can trigger an inflammatory reaction, which
influences the differentiation pattern of cell populations involved in the regenerative process,
determining implant success or rejection (HANKENSON & al. [4]). Macrophages are
prevalent infiltrating cells that respond rapidly to biomaterial implantation and play a crucial
role in regulating the inflammatory response and tissue remodeling, by secreting large
amounts of bioactive mediators that can initiate inflammation, cell migration and
differentiation, tissue remodeling and blood vessel formation HANKENSON & al. [4]).In
biomaterials research, TNF-α, IL-1, IL-6 and other pro-inflammatory cytokines are best
known as mediators of the inflammatory response that can cause both severe tissue damage
and premature failure of implanted materials, but are also key mediators involved in efficient
tissue regeneration and repair ( WANG& al., WANG & al. [5, 6]).Data regarding the
hematological parameters are of interest due to the involvement of blood elements in basic
physiological processes. Based on their role in the transport of respiratory gases (i.e.,
erythrocytes), in inflammatory processes (i.e., leukocytes) and vascular lesions repair (i.e.,
platelets), homeostasis maintenance is necessary after post-implantation and bone defect
healing ( FEI& al., KINI and NADEESH [7, 8]).The objective of the present work is to
analyze the homeostasis of some biochemical and hematological parameters, together with
inflammatory markers in regulating bone regeneration, in order to demonstrate the importance
of integrating rational control of inflammation and normal blood profile into the design of
tissue engineering strategies.
2. Materials and Methods
Biological assessments were performed on freeze-dried chitosan - graphene oxide
porous 3D scaffolds; the obtaining procedures were thoroughly described in previous works
(DINESCU & al., PANDELE & al. [9, 10]).
Animals and surgical procedure
CD1 mice, housed at stable temperature (22 ± 20°C) and a 12 h light/dark cycle, were
used for the experiments. All experimental procedures were approved by the Ethical
Committee of the Vasile Goldis Western University of Arad. The surgical procedures were
performed under general anesthesia, using ketamine hydrochloride (100 mg/kg b.w., Pasteur Romania) and xylazine hydrochloride (10 mg/kg b.w., Bioveta – Czech Republic). 5
mmcalvaria defects were prepared using a drill (Super NP5, Korea) under constant sterile
0,9% saline solution irrigation, in order to prevent overheating of the bone margins, to remove
the bone debris and with special care not to harm the underlying dura mater (Figure 1). Five
groups of chitosan (CH) – graphene oxide (GO) scaffolds were implanted in mice calvarial
defects for different intervals (72 h, 2 w, 4w, 6w, 8w). The defects were randomly filled with
CH (n=50), CH 0.5% GO (n=50), CH 1% GO (n=50), CH 2% GO (n=50), CH 3% GO (n=50)
or untreated (n=50).The periosteum was repositioned by glyconate monofilament fast
absorbable sutures and skin with polyamide monofilament non-absorbable sutures. After
surgery, the animals were housed individually under constant conditions. No lethality was
detected during the surgery or the post-surgical period. The animals were sacrificed under
anesthesia at 72 h, 2 w, 4w, 6w, 8w post-implant (10 animals/time-interval). The calvarial
defect sites with surrounding bone and soft tissue were harvested for subsequent evaluation.
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BALTA CORNEL, HILDEGARD HERMAN, MARCEL ROSU, CORALIA COTORACI, ALEXANDRA IVAN, ALEXANDRA
FOLK, ROBERT DUKA, SORINA DINESCU, MARIETA COSTACHE, ALEXANDRU PETRE, ANCA HERMENEAN
Figure 1: Surgical procedure. A. Skin asepsis in the region of interest; B. Highlighting the osseous region of
interest;C. Preparing the osseous surgical defect;D. Highlighting the osseous defect with a 5mm side; E.
Rectifying the osseous defect with the scaffold;F. Suture in separate points to close the lesion.
Biochemical analysis
Venous blood samples werecollected in Eppendorf tubes. The samples were
centrifuged at 3500 rpm for 10 min. Samples were analyzed for C-reactive protein (CRP)
levels. The reagent kit used for the quantitative determination of CRP was CRP FL
(ChemaDiagnostica, Monsano, Italy) and the measurements were made with a Mindray
BS-120 Chemistry Analyzer (ShenzenMindray Bio-Medical Electronics Co., Ltd., Nanshan,
Shenzhen, China).
Hematological analysis
An automated digital equipment (Urit 2900 VetPlus, China) was used for blood
profiling. Heparinized blood was used for the complete blood count: Red Blood Cell Count
(RBC), White Blood Cell Count (WBC), Platelet Count (PLT), Hemoglobin (HGB),
Hematocrit value (HCT).
Histopathology
Calvaria specimens were fixed in 4% phosphate buffered formalin, embedded in
paraffin and cut into 6 µm thick sections. Sections for histopathological examination were
stained with Hematoxilin & Eosin, analyzed under light microscopy using an Olympus BX43
microscope, and photographed using a digital camera (Olympus XC30).
Immunohistochemistry
Paraffin embedded calvarial sections of 6 µm thickness were previously
deparaffinized and rehydrated using a standard technique. Rabbit polyclonal anti-TNF-α
(1:100 dilution; Santa Cruz, CA, USA) were used as primary antibodies. Immunoreactions
were visualized employing Novocastra (Leica Biosystems, Germany), Peroxidase/DAB kit
according to the manufacturer’s instructions. Negative control slides were processed by
substitution of primary antibodies with irrelevant immunoglobulins, in the same conditions as
primary antibodies. Stained sections were analyzed by light microscopy (Olympus BX43,
Hamburg, Germany).
Statistical Analysis
Statistical analysis was conducted with a one-way ANOVA using Stata 13 software
(StataCorp LP, Texas, USA). A value of p<0.05 was considered to be statistically significant.
3. Results and Discussion
Successful drug delivery and tissue engineering strategies require in most cases
biocompatible three-dimensional scaffolds with morphological and degradation features, well
tolerated by the host animals, matching the targeted clinical application.Our study offered
initial insight into how the immune system responds to, and then affects the in vivo behavior
of these scaffolds and blood profile balance during post-implantation time.
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Homeostasis of blood parameters and inflammatory markers analysis during bone defect healing
after scaffolds implantation in mice calvaria defects
Activity of C-reactive protein (CRP) inflammatory marker, andbloodprofile
The healing of the bone fractures presumes passing through three important stages: the
inflammatory stage, the reparatory stage and the re-modeling one ( PAPE& al. [11]). Thus,
during the first 72h, an activation of the signaling and of pro-inflammatory cells attraction to
the bone defect sitecan be observed, as these cells are needed for healing process initiation.
The C-reactive protein (CRP) is an inflammation marker, which highlights the activation of
this mechanism. Figure 2 shows the effects of chitosan-based biomaterials improved with 0.53 wt.% GO implantation in mice calvaria on the serum level of CRP inflammatory marker. At
72 hours post-implantation, the CRP blood level has elevated for all experimental groups,
followed by a gradually decrease up to 8 weeks. Only CH GO 2% and CH GO 3% proved a
low post-implant inflammatory reaction, the CRP level being significantly lower, as
compared to the control one (p<0.001) at all time intervals. Thus, we can estimate that these
materials are best tolerated by the body, and the inflammation and other potential
complications, which might lead to implant rejection are avoided.
Figure 2: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice
calvaria on the CRP activity at 72 h and 2, 4, 6, 8 weeks post-surgery . All data represent mean values ± SEM
(n = 5). ***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as
compared to control; *statistical significance at p < 0.05 as compared to control
The white blood cells (WBC) level registered at 72h post-implant were significantly
lower for the CH GO 3%compared to the control (p<0.001). The same tendency was observed
for all the other analyzed post-implant timeframes (Figure 3). This scaffold induced a low
blood afflux of white blood cell, in direct relation with the low level of CRP, favoring the
earlier initiation of the bone healing, as compared to the other biomaterials, and, thus making
it a good candidate for bone tissue engineering.
Figure 3: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice
calvaria on the WBC level at 72 h and 2, 4, 6, 8 weeks post-surgery. All data represent mean values ± SEM (n = 5).
***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as compared
to control; *statistical significance at p < 0.05 as compared to control
Romanian Biotechnological Letters, Vol. 22, No. 6, 2016
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BALTA CORNEL, HILDEGARD HERMAN, MARCEL ROSU, CORALIA COTORACI, ALEXANDRA IVAN, ALEXANDRA
FOLK, ROBERT DUKA, SORINA DINESCU, MARIETA COSTACHE, ALEXANDRU PETRE, ANCA HERMENEAN
The red blood cells (RBC), (HGB) and (HCT) had similar profiles, registering
significantly lower values in CH improved with 1-3% GO, as compared for the chitosan
scaffolds (Figs 4-6). During the first 72h, the slightly elevated levels of these parameters in all
experimental groups may be explained also by the body’s attempt to compensate the losses of
the red cells due to the formation of the hematoma, occurring in the evolution of the created
osseous defect (Figs 4-6).
Figure 4: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice
calvaria on the RBC level at 72 h and 2, 4, 6, 8 weeks post-surgery. All data represent mean values ± SEM (n = 5).
***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as compared
to control; *statistical significance at p < 0.05 as compared to control
Figure 5: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice
calvaria on the HGB level at 72 h and 2, 4, 6, 8 weeks post-surgery. All data represent mean values ± SEM (n = 5).
***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as compared
to control; *statistical significance at p < 0.05 as compared to control
Figure 6: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice calvaria
on the HCT level at 72 h and 2, 4, 6, 8 weeks post-surgery. All data represent mean values ± SEM (n = 5).
***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as compared
to control; *statistical significance at p < 0.05 as compared to control
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Homeostasis of blood parameters and inflammatory markers analysis during bone defect healing
after scaffolds implantation in mice calvaria defects
The significant low level of the platelets noticed in the CH, CH GO 0,5% and CH GO
1% groups compared to the control at 72 h post-implantation (Figure 7), may suggest an
activation of the control systems for the platelets activity and of the platelet forming tissue.
These activities are related to the impairment of the vascular integrity and to the
compensation of this lesion as integrating part of the healing process. Two weeks postimplantation, we noticed the reduction of this parameter value in the CH, CH GO 0,5% and
CH GO 1% groups, as compared to control. The reduction was correlated to the hematoma
reorganization stage and to the formation of the granulation tissue in the respective area,
whilst this parameter becomes normal in the experimental groups in the 8th week.
Figure 7: The effects of chitosan-based biomaterials improved with 0.5-3 wt.% GO implantation in mice calvaria
on the PLT level at 72 h and 2, 4, 6, 8 weeks post-surgery. All data represent mean values ± SEM (n = 5).
***statistical significance at p < 0.001 as compared to control; **statistical significance at p < 0.01 as compared
to control; *statistical significance at p < 0.05 as compared to control
Early post-implantation immune response
During inflammation, monocytes and undifferentiated macrophages are recruited from
blood to the implant site, where they can differentiate into macrophages ( FRANZ&al.
[12]).In our study, the chitosan-based scaffolds improved with 0.5-3 wt.% GO were quickly
infiltrated upon implantation. At 72 h post-implantation, chitosan-based biomaterials began to
be filled by invading macrophages, which were present at the implantation sites of the
scaffolds, covering large areas, proportional to the increase of graphene concentration. Most
macrophages detected at the implantation sites of chitosan/GO-derived scaffolds appeared in
clusters.Only chitosan-based scaffolds prepared with 2 and 3% GO showed marked
expression of TNF-α as compared to control (Figure 8). The TNF-α expression levelsin
chitosan and chitosan 0.5% GO scaffolds were significantly lower.Chitosan scaffolds improved
with 3% GO strongly stimulated the production of TNF-α, correlated with an increase in
the macrophage metabolic activity. Several studies showed that monocyte/macrophage
populations on implantation sites are of M1 and M2 phenotypes, which have been correlated
with a pro-inflammatory and a pro-osteogenic function (BROWN & al., OLIVEIRA & al.
[13, 14]). Almeida et al (ALMEIDA & al. [15]) showed that secretion of the cytokines TNF-α,
IL-6, IL-12/23, IL-10 and TGF-β play a role in regulating tissue repair/regeneration.
Macrophage plasticity implies that cells may shift from one profile to the other
(PORCHERAY & al. [16]) or can express both M1 and M2 markers at the same time
(PETTERSEN & al. [17]).Therefore, it is important to understand which functional reactions
will be stimulated by different scaffolds, cytokines being crucial players in cellular responses
activation.
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BALTA CORNEL, HILDEGARD HERMAN, MARCEL ROSU, CORALIA COTORACI, ALEXANDRA IVAN, ALEXANDRA
FOLK, ROBERT DUKA, SORINA DINESCU, MARIETA COSTACHE, ALEXANDRU PETRE, ANCA HERMENEAN
Figure 8: Morphologies ofchitosan-based biomaterials improved with 0.5-3 wt.% GO implanted in mice calvaria
at 72 h post-surgery. Expression and specific distribution of TNF-αat implantation siteat 72 h post-surgery.
Legend: B-bone; IC – inflammatory cells; S - scaffold
4. Conclusions
Within the first 8 weeks, the chitosan (CH) – graphene oxide (GO) scaffolds were well
tolerated by the host mice and no abnormal conditions were observed. The inflammatory
response was present at early stages post-implantation and it decreased after 2 weeks.
Chitosan scaffolds improved with 3% GO proved to be the best tolerated biomaterial, which
renders it an important candidate for the studies of bone regeneration.
5. Acknowledgements
This work was supported the Romanian CNCS-UEFISCDI Research Project (Grant
No. PCCA140/2012) and by the strategic grant POSDRU/159/1.5/S/133391, Project
“Doctoral and Post-doctoral programs of excellence for highly qualified human resources
training for research in thefield of Life sciences, Environment and Earth Science” co-financed
by the European Social Fund within the Sectorial Operational Program Human Resources
Romanian Biotechnological Letters, Vol. 22, No. 6, 2016
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Homeostasis of blood parameters and inflammatory markers analysis during bone defect healing
after scaffolds implantation in mice calvaria defects
Development 2007-2013. This research was also financed by the Institute for Research of the
University of Bucharest (ICUB), through „Scholarships for Excellence in Research for young
researchers, 2015 competition” Project and START Project: BEST – NetWORK 16_PA07-C1.
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