homeostasis of blood parameters and inflammatory … bone model - rbl...babes; 2 eftimie murgu;...

Romanian Biotechnological Letters Vol. 22, No. 6, 2016 Copyright © 2016 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 22, No. 6, 2016 12018

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; 5Department 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

Homeostasis of blood parameters and inflammatory markers analysis during bone defect healing after scaffolds implantation in mice calvaria defects

Romanian Biotechnological Letters, Vol. 22, No. 6, 2016

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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.

BALTA CORNEL, HILDEGARD HERMAN, MARCEL ROSU, CORALIA COTORACI, ALEXANDRA IVAN, ALEXANDRA FOLK, ROBERT DUKA, SORINA DINESCU, MARIETA COSTACHE, ALEXANDRU PETRE, ANCA HERMENEAN


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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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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 0.5-3 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



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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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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 post-implantation, 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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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



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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. References

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