Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction

Journal Pre-proof Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction Marcelo Salles Munerato, Claudia Cristina Biguetti, Raquel Barroso Parra da Silva, Ana Claudia Rodrigues da Silva, Ana Carolina Zucon Bacelar, Jordan Lima da Silva, Maira Cristina Rondina Couto, Marco Antônio Húngaro Duarte, Joel Ferreira Santiago-Junior, Paulo Sérgio Bossini, Mariza Akemi Matsumoto PII:

S0928-4931(19)32467-1

DOI:

https://doi.org/10.1016/j.msec.2019.110229

Reference:

MSC 110229

To appear in:

Materials Science & Engineering C

Received Date: 4 July 2019 Revised Date:

19 August 2019

Accepted Date: 18 September 2019

Please cite this article as: M.S. Munerato, C.C. Biguetti, R.B. Parra da Silva, A.C. Rodrigues da Silva, A.C. Zucon Bacelar, J. Lima da Silva, M.C. Rondina Couto, Marco.Antô. Húngaro Duarte, J.F. SantiagoJunior, Paulo.Sé. Bossini, M.A. Matsumoto, Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110229. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Inflammatory

response

and

macrophage

polarization

using

different

physicochemical biomaterials for oral and maxillofacial reconstruction Marcelo Salles Muneratoa, Claudia Cristina Biguettib, Raquel Barroso Parra da Silvab, Ana Claudia Rodrigues da Silvab, Ana Carolina Zucon Bacelarb, Jordan Lima da Silvaa, Maira Cristina Rondina Coutoa, Marco Antônio Húngaro Duartec, Joel Ferreira Santiago-Juniora, Paulo Sérgio Bossinid, Mariza Akemi Matsumotob,* a

Department of Health Sciences, Sagrado Coração University – USC, Rua Irmã

Arminda 10-50, 17011-160, Bauru, SP, Brazil b

Department of Basic Sciences, São Paulo State University (Unesp), School of

Dentistry, Rua José Bonifácio 1193, 16015-050, Araçatuba, SP, Brazil c

Department of Dentistry, Endodontics, and Dental Materials, Bauru School of

Dentistry, University of São Paulo – FOB/USP, Al. Octávio Pinheiro Brisola, 9-75, 17012-901, Bauru, SP, Brazil d

Research and Education Center for Phototherapy in Health Science (Nupen), Rua

Pedro Fernandes Alonso, 766, Jardim Alvorada, 13562-380, São Carlos, SP, Brazil

Corresponding author: [email protected] (M.A. Matsumoto).

Declarations of interest: none.

Abstract Knowledge about the action of immune system in the recognition of biomaterials has been extremely helpful when it comes about understanding host response and biomaterials’ fate in human body. This study aimed to investigate inflammatory response and macrophage polarization during bone healing process of rat’s calvaria critical defects using different bone materials in order to evaluate their influence on bone repair and on the quality of the newly formed bone tissue. Eighty male albinus Wistar rats underwent surgical procedure for the confectioning of a 5-mm diameter bone defect in their right parietal bone, and divided in four groups (n=20 each), according the biomaterial: AG – Control, particulate intramembranous autogenous bone graft, HA/TCP – particulate biphasic calcium phosphate with HA/TCP (60/40), DBB – particulate deproteinized bovine bone, VC – particulate bioactive

vitroceramic. After 3, 7, 21, and 45 days, the specimens were removed and prepared for microcomputed tomography (microCT), light and polarized microscopy, immunohistochemical analysis, and histomorphometry. No significant differences were detected considering percentage of leukocytes among the groups and periods, as well as in relation to immunolabeling for inflammatory (M1) and reparative (M2) macrophages. However, immunolabeling for bone marker indicated a delayed osteoblast differentiation in VC group, resulting in a decrease in mineralized bone matrix parameters in this group, revealed by microCT. In addition, AG and HA/TCP presented a satisfactory bone collagenous content. Despite the distinct origins and physicochemical properties of the tested biomaterials, they presented similar immuneinflammatory responses in the present experimental model, influencing bone-related proteins and bone quality, which must be considered according to their use.

Key words: Biomaterials; bone substitutes; macrophages; osteoimmunology; rats.

1. Introduction The use of biomaterials aiming the recovery and/or reconstruction of bone tissue has become a routine procedure in medical and dentistry fields leading to quite predictable results [1]; [2]; [3]; [4]. The pathway taken for the development of the biomaterials was and still remains involved in a number of difficulties that goes through their characterization, until the understanding of bone biology froma systemic point of view, as part of a complex living organism [5]. In the beginning there was the belief that the lack of tissue reaction to an implanted material was the ideal response, and thus it was considered inert. However, knowledge generated by molecular biology, genomics, and proteomics analysis have been attested the inseparable interaction between host/biomaterial, emphasizing hat all implanted material leads to a recognition process and identification by immunological system, which will orchestrate and determine the initial events of tissue healing [6]. Undeniably, the discovery of titanium integration to bone tissue making possible oral rehabilitation with dental implants using a number of techniques arose interest in the scientific community about the necessity of gaining bone tissue with adequate biological characteristics, in order to fulfill aesthetic and functional rehabilitation [7]; [8]. For this purpose there is a significant amount of available bone substitutes, and amongst them bioceramics deserve attention [9], as the bioactive

glasses, hydroxyapatite, and those based on calcium phosphate [4]; [10]; [11]; [12], which can be found as granules of different sizes, and blocks or scaffolds. Since they are resorbable, they allow the formation of a new bone tissue by osteoconduction, while they are degraded and substituted by the new tissue. A number of studies support their use, both in clinical practice and experimental investigation showing that, although they belong to the same class of biomaterials, they differ from one another,

especially

in

relation

to

rate

resorption,

kind

of

degradation,

osteoconductivity, and bioactivity, that interfere in the quality of the formed bone [12]; [13]; [14]. In this way, their behavior not only reflects the chemical characteristics of the biomaterial, but also their physical aspects as previously cited, which include topography, size and porosity [15]. The tissue damage caused by the insertion of the biomaterial in a living organism causes local release of plasmatic and tissue proteins, as fibronectin and vitronectin, that are adsorbed into its surface according to the physicochemical characteristics. The adsorbed layer becomes the real attractant for the cells [16]. Among the leukocytes that participate of this dynamics, macrophages deserve attention due to their immediate response to any biomaterials that are inserted in an organism [6], and mainly, by their notable morphological and functional plasticity [17]. Macrophages are involved in the recognition, degradation and/or phagocytosis of biomaterials, modulating the inflammatory process and tissue response, and consequently determine inflammation maintenance or repair [6]; [18]. Such abilities come from the polarization capacity of these cells, that can present a proinflammatory (M1) or repair (M2) phenotype, being identified by the molecules that they express and the cytokines they release [19]; [20], already identified both by in vitro [21]; [22] and in vivo studies [17]; [19]. Macrophages can also adapt their phagocytic capacity depending on the size of the material they interact. According to Xia & Triffitt [18], particles until 5-10 µm are recognized and phagocyted by them. However, those particles between 10-100 µm require a fusion of various macrophages, resulting in the so-called foreign body giant cells (FBGC) that adhere to the surface of the biomaterial to perform an extracellular degradation. Interestingly, Vasconcelos et al. [23] studying M2 polarization in response to quitosan revealed that FBGC express M2-like markers in these conditions. Such characteristics of these notable leukocytes may possibly justify the different responses, even minimal, of the various biomaterials available by the host

organism. New findings about macrophages are being revealed. Chang et al. (2008) [24] reported the presence of resident macrophages on endosteal and periosteal surfaces, which were called OsteoMacs (osteal macrophages), and occupy around 1/6 of the local cell population. Among their functions, there is the immunological vigilance, since they are able to respond to antigens and perform phagocytosis [25]; [26]. Strategic location, quantity, and morphology of macrophages urge investigations about their abilities, confirming their influence on osteoblast differentiation [27], and consequently, in bone modeling and remodeling [24]; [28]. In addition, evidences from in vitro studies suggest that macrophages contribute with bone repair by expressing BMP-2 and osteopontin, proteins that are characteristically expressed by osteoblasts [29]. Due to this complex inter-relation between macrophages and biomaterials, this study aimed to identify inflammatory response and macrophage profile focusing on macrophages profile during the repair of bone defects using different bone substitutes.

2. Materials and Methods

2.1 Experimental design This study was approved by the Ethical Committee for Animal Care (protocol 4702160715). All experimental protocols involving animals followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH), as well as Brazilian Society of Laboratory Animal Science (COBEA). Eighty male Wistar rats were used, three months old, mean weight of 450 g, were divided in four groups to be analyzed in four time points (n=5 per group and experimental time point). The animals were kept in cages containing up to four animals, in controlled conditions, under 22±2ºC temperature, 12 hours dark/light cycle, with water and food ad libitum.

2.2 Surgical procedure Surgical procedures for the confectioning of bone defects followed strict aseptic protocol. The animals were first sedated with intramuscular (IM) administration of 1% ketamine (Dopalen®, Agribands Ltda, São Paulo, Brazil) in association with 2% chloridrate of xylazine (Anasedan®, Agribands Ltda, São Paulo, Brazil) using the recommended dose according to individual animal weight. After sedated, trichotomy was performed in the head of the animals, followed by antisepsy

with topic 1% polyvinylpyrrolidone. Local anesthetic with 2% mepivacaine and epinephrine 1:100.000 was used in order to decrease local bleeding and improve analgesia. A linear 2cm dermal incision was performed following the medial suture in order to expose the periosteum, in which the incision was made 3 mm to the left side, for the exposure of the calvaria bone. A round bone defect was made in the right parietal bone using a 5mm-diameter trephine bur with an electrical micromotor (Vector DTCeb /1.500 rpm) under copious refrigeration with sterile 0.9% saline solution, in order to remove bone diploe. Extra care was taken not to damage duramater membrane. The animals were divided in four groups, according to the filling material: AG – particulate autogenous bone graft, 1-2mm; HA/TCP - β-tricalcium phosphate plus hidroxyapatite (Bone Ceramic, 0.5-1mm, Straumann, Basel, Swiss); DBB – deproteinized bovine bone (Bio-Oss, 0.25-1mm,Geistlich-Pharma, Wolhusen, Swiss); and VC – bioactive vitroceramic (Biosilicate, 180212µm,Vitrovita, São Carlos, Brazil). The calvarial bone removed for the confectioning of the defect of C group was particulated and used as graft. All the biomaterials used in the four groups were weighted in ahigh precision scale, and 0.02 g of each one of them was used to fill in the defects, including the bone graft. Before insertion, they were agglutinated with 0.9% saline solution in order to improve handling (Figure 1). Periosteum was repositioned and the skin was sutured with 6-0 nylon. The animals received two doses of 40.000UI benzathine penicillin IM every 48 hours. After 3, 7, 21, and 45 days, the animals were euthanized and the specimens (parietal bones) were removed, immediately fixed in 4% formalin for 48 hours. Then, specimens were first washed in tap water for 24 hours to be processed for microscopic analysis (H&E, Picrosirius-red and immunohistochemistry). Samples from 45 days time point were immersed in alcohol 70o for microCT scanning before to be processed for microscopic analysis.

a

b

c

d

Fig. 1.Biomaterial placed in the parietal defects: a) AG; b) HA/TCP; c) DBB; d) VC

2.3 MicroCT scanning Specimens from 45 days were scanned in a microCT scan (SkyScan, Kontich, Belgium) using the following parameters: X-ray energy level of 50 kVp and 800 µA, 16µm voxel, 1304x1024 pixels and 360o rotation. Images were reconstructed using NRecon v1.6.4.8 software considering the same parameters for all specimens. Data viewer (v1.4.4.0) was used to realign the image projections and CTvox (v2.3) reconstructed the microCT projections in 3D images by volume renderization. The region of interest (ROI) was determined using CTAnalyser (v1.16) with 3.5mm diameter and 3 mm of extension in the center of the defect in order to analyze morphological parameters related to the newly formed bone, such as bone volume (BV, mm3), Trabecular Number (Tb.N, mm), Trabecular Thickness (Tb.Th, 1/mm), and Trabecular Spacing (Tb.Sp, mm) [30].

2.4 Histology and Histomorphometry After scanning, the specimens underwent laboratorial preparation for the confectioning of the histological slices that were stained with hematoxylin and eosin and Goldner trichrome. For histopathological analysis, inflammation, foreign body reaction, bone forming, maturing, and remodeling were analyzed, along with bone viability, and bone/biomaterial interaction. For qualitative assessment of bone repair, six fields of each specimen stained with HE were captured under 40x magnification. Using ImageJ software (National Institutes of Health, Bethesda, USA), a 768-point grid was confectioned and the coincident points that matched with the target cell typewere considered for quantification. Polymorphonuclear neutrophils (PMN),

mononuclear (MN) leukocytes, and multinucleate giant cells (MNGCs) were identified and quantified in a same grid.

2.5 Birefringence analysis Histological slices stained with Picrosirius-red were analyzed under polarized microscopy in order to reveal collagenous matrix. Six fields under 40x magnification were captured from each defect (Eclipse 80i, Nikon, Tokyo, Japan) and analyzed using the software Image ProPlus 5.0 [15].

2.6 Immunohistochemistry and Immunofluorescence For the evaluation of macrophages and bone repair, 3µm sections were prepared as previously described [31] to be incubated with the primary polyclonal antibodies to detect bone proteins Runx-2 (SC#8566), osteocalcin (OC) (SC#18319), and TRAP (SC#30832), macrophages M1, CD80 (SC#9091) and iNOS (SC#649), and macrophages M2, CD206 (SC#34577) and TGF-B (SC#7892, Santa Cruz Biotechnology, Carpinteria, CA, USA).After that, those incubated with anti-rabbit primary antibody were treated with HRP-polymer (Easy Link One, EasyPath, Immunobioscience Corp, USA), and those incubated with anti-goat were treated with Immpress/HRP (Vector Labs, Southfield, USA) for 10-25 minutes. After, they were stained with 3,30-diaminobenzidine tetra hydrochloride (SigmaAldrich, St Louis, MO, USA) and counter-stained with Harris hematoxylin. Primary antibody was omitted for the negative control. Six fields under 40x magnification were captured and registered for the quantification of immunolabeled cells using ImageJ software (National Institutes of Health, Bethesda, USA) using a 391-point grid. For co-localization of M1 and M2 macrophages, CD80 (SC#9091), and CD206 (SC#34577, Santa Cruz Biotechnology, Carpinteria, CA, USA) primary antibodies were used, followed by the incubation with secondary antibodies Alexa Fluor®555 goat anti-rat (Life Technologies, #A2120) with Alexa Fluor®488 donkey anti-rabbit (Life Technologies, #A21206).) After primary antibodies were incubated at 4oC overnight, they reacted with the secondary antibodies for 2 hours. Then, the slices were washed with PBS and incubated with DAPI (D9542-50, Sigma-Aldrich Corp., St. Louis, MO, USA) for 10 minutes. After the slices were washed they were mounted with antifade solution and stored in a dark room until the images were taken

in a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan).

2.7 Statistical assessment Data collected from microCT scanning, and HE and immunohistochemistry morphometry were organized and analyzed in SigmaPlot1 12.0 software for normal distribution (Shapiro-Wilk and Equal Variance Test) and after, One Way Analysis of Variance was chosen when normal distribution was detected, and Kruskal-Wallis when non-normal distribution was detected, under p<0.05. Tukey post-test was performed for the comparison analysis among the groups.

3. Results

3.1 MicroCT and Birefringence Analysis Significant difference was identified considering bone volume (BV mm3) in the comparison among the groups (p≤0.001). Comparative analysis indicated significant differences (p<0.001) between AG (medium: 4.151) vs. VC (medium: 0.851), HA/TCP (medium: 4.408) vs. VC, DBB (medium: 3.939) vs. VC. Also, significant difference was detected in relation to trabecular microarchitecture (p=0.007). Trabecular thickness (Tb.Th mm) was higher in AG (medium: 0.416) in comparison to HA/TCP (medium: 0.165) and VC (medium: 0.151), p<0,05. No significant differences were observed in the comparison with the other groups (p≥0.05). In the same way, differences were detected considering trabecular number (Tb.N mm) p<0,001, since HA/TCP (medium: 0.934) and BO (0.719) presented higher number than AG (0.367) and VC (medium: 0.145), p<0,05. In agreement, trabecular spacing (Tb.Sp mm) was more evident in AG (medium: 1.191) and VC (medium: 1.564) groups than HA/TCP (0.782) p<0,05, and also comparing VC and DBB (0.925), p=0,001 (Figure 2AB). Considering the organic matrix, the analysis of collagen content performed by birefringence comparative analysis among the biomaterials, a higher expression was observed considering red fibers (%) comparing AG (median: 17.3) and HA/TCP (median: 17.6) in comparison to DBB (median: 9.2) and VC (median: 6.7). However, no statistical significant differences were detected among the groups (p = 0.117). Considering the greenish fibers (%), higher expression was identified for VC (median: 93.2) and DBB groups (median: 990.7), and the lower expression for AG

(median: 82.6) and HA/TCP groups (median: 82.32). No statistical significant differences were detected among the groups (p = 0.117). Box plot shows the results, identifying VC group with the higher expression of green fibers, and DBB and HA/TCP groups presenting shorter dispersion (Figure 2 CB).

Fig. 2. AB) MicroCT analysis revealed significant microarchitectural bone differences among the groups indicated by the asterisks. CD) No significant differences were detected among the groups considering the amount of green and red fibers at day 45 (Picrosirius-red, scale bar = 200 µm) 3.2 Histology and Histomorphometry 3 days – Bone defects reconstructed with autogenous graft were filled with non-viable bone fragments, surrounded by a delicate fibrin, red blood cells and a few mononuclear and polymorphonuclear leukocytes. HA/TCP, DBB, and VC groups presented similar histological aspects, with the biomaterial amongst fibrin and red blood cells. No osteoclastic activity or foreign body reaction were noted at this period. 7 days–Autogenous bone grafts presented initial resorption by eventual osteoclasts, amongst a highly vascular granulation tissue. Discrete osteogenesis was noted close to the bone walls. Also, granulation tissue was observed surrounding the HA/TCP particles. Mononuclear leukocytes were concentrated close to the

biomaterial, with a few multinucleated giant cells (MNGCs). Similarly, DBB presented MNGC activity. A highly cell and vascularized granulation tissue surrounded VC particles, with a mild mononuclear infiltrate. Close to the material, larger and vacuolated mononuclear leukocytes were observed. 21 days – Fragments of non-viable bone graft were still seen in AG group, surrounded by maturing newly formed bone, lined by active osteoblasts. Medullar spaces were observed at this period. Eventually, some osteoclasts were seen associated with the graft, amongst loose connective tissue. HA/TCP showed the biomaterial surrounded by numerous thin and elongated MNGCs, in a connective tissue infiltrated by mononuclear leukocytes. Osteogenesis was observed in the periphery of the defects. DBB presented the biomaterial associated to MNGCs in a loose connective tissue. Newly bone formation was predominant in the peripheral areas. VC showed larger and more irregular MNGCs in comparison to the other groups, in a connective tissue infiltrated by mononuclear leukocytes. Discrete osteogenesis areas were in the periphery of the defects. 45 days–Predominantly remodeling bone was filling the defects of AG group, marked by reversal lines and medullar spaces. At this period, some fragments of the bone graft were still seen. HA/TCP group was filled with the biomaterial surrounded by connective tissue, full of fibroblasts, blood vessels and a few leukocytes. In contact with the biomaterial surface, thin and/or irregular, elongated MNGCs were seen. Eventual bone tissue was detected. Differently, DBB group presented the biomaterial surrounded by connective tissue with numerous MNGCs, most of them thin and elongated, or remodeling bone tissue in close contact with it. VC group showed the biomaterial amongst granulation tissue highly infiltrated by cells suggesting macrophages, associated with the MNGCs in the biomaterial surface. Eventually, newly formed bone was observed (Figure 3). Quantitative analysis of PMN and MN leukocytes did not detect significant differences by the statistical tests among the groups and periods. However, significant increase was observed considering MNGCs in HA/TCP group at days 21 and 45, in comparison to AG. Since no MNGCs are present when using autogenous graft, it can be considered that no differences were detected among the groups of the biomaterials (Figure 3).

A

Fig. 3. A) Histological aspect of bone healing using the different filling materials. Higher magnification of the central area of the defects is highlighted by the square of the above correspondent lower magnification. AG healed uneventfully, with bone graft (*) serving as both osteoconductive and osteoinductive material for bone formation, actively remodeling (arrows) at the final periods. The other groups presented particles of the biomaterials (*) until the last period, surrounded by MNGCs (arrows). B-D) Histomorphometric analysis of leukocytes. Significant increase of MNGCs were detected comparing AG and HA/TCP at days 21 and 45 (*indicate statistical significant difference considering p<0.05) (HE; scale bar = 200 µm)

3.3 Immunohistochemistry Runx-2 immunolabeling was significantly higher at day 21 in VC (4.71± 2.21) group when compared to AG (0.66 ±1,30) (p<0.05). On the other side, OC immunolabeling was significantly lower at day 21 in HA/TCP group (0.01±0.02) compared to AG (7.53±3.98) (p<0.05). At day 7, TRAP immunolabeling was higher in AG group in comparison to the other groups (p<0.001). At day 21, significant difference was observed only between AG (median: 11) and HA/TCP (median: 1).

After 45 days, a significant higher number of TRAP positive cells were detected in AG group (median: 2), in comparison to HA/TCP (median: 0) and VC groups (median: 0) (Figure 4). In relation to macrophages, F4/80 positive labeling was observed until day 21 in all groups, whereas at day 45, no labeling was noted. For the identification of M1 macrophages, positive immunolabeling for CD80 was detected in DBB, HA/TCP, and VC groups at day 21. On the other hand, antibodies for M2 detection as CD206 and TGF-B were predominant at days 21 and 45 in all groups (Figure 5).

Fig. 4. Immunolabeling of bone targets. Symbols indicate significant differences in comparison to AG, considering the same period (scale bar = 100 µm)

Fig. 5. Immunolabeling of inflammatory targets. No significant differences were detected among the groups and periods (scale bar = 100 µm)

3.4 Immunofluorescence In general, different cell types were observed during bone healing. Amongst them, our main targets were M1 and M2, which were predominantly in close contact with the biomaterials’ surface or in the granulation/connective tissue surrounding the particles, as shown in Figure 6. However, observing the MNGCs, co-localization positive labeling was detected, especially in DBB and VC. Considering the data obtained from the analysis of conventional immunohistochemistry, the time period of 3 days was omitted.

Fig. 6. Immunofluorescence for M1 and M2 co-localization. MNGCs were also double labeled, indicating polarization of these cells (scale bar = 200 µm) 4. Discussion From the analysis ofthe overall results, interesting outcomes were observed when comparing the different biomaterials used for rats’calvaria reconstruction, including autogenous bone graft. Although for long this specific animal model has been used to evaluate the behavior of a number of bone substitutes [32], it is never enough to emphasize its limitations and site-specificities, such as the lack of supporting bone to adapt the material, which is placed direct on dura mater membrane. In this way, bone formation exclusively depends on the surrounding bone

walls of the defect, and therefore, osteoconductive potential of the biomaterials is put to the test. Considering the distinct origin and physicochemical characteristics of each biomaterial, expectation was that significant differences were found especially related to the inflammatory response and macrophage polarization during tissue repair. DBB is a xenograft from bovine origin, which makes it quite similar to human bone due to its chemical and physical compositions. Its highly interconnected porosity (75-80%) and extremely low degradation rate increase the quality of the newly formed bone [33]. On the other hand, HA/TCP is a purely synthetic porous (90%) biphasic calcium phosphate material which degradation rate can be controlled depending on HA and ßTCP ratio, releasing free ions in the surrounding tissues, especially Ca2+, stimulating cell differentiation and organic matrix mineralization [34]. VC is also synthetic, fully crystallized glass ceramic of the Na2O-CaO-SiO2-P2O5 system, a parent glass of Bioglass 45S5, promising improved bioactive properties promoted by the result of a HCA layer on its surface and by the products of its degradation, as silicon, calcium, sodium and phosphate, which stimulates synthesis activity of the surrounding cells [35]. It can be manufactured in different physical aspects, and it is non-porous in the particulate presentation [36]. It is not uncommon to find mononuclear leukocytes during healing process of an area reconstructed with biomaterials, as reported by Araújo et al. [37], when they carefully described histological aspects of a modified DBB used in a socket repair model in dogs. In previous studies testing Biosilicate® [15]; [38], a population of large cells with clear cytoplasm assembling macrophages associated to the biomaterials particles were observed, different from what was seen in other biomaterials. This intriguing cellular response led to the necessity of investigating their real identity and influence on tissue repair, since immune-inflammatory response is directly affected by any implanted material in a living organism, with special attention to macrophages due to their highly plasticity and polarization capacity, and to be considered decisive cells for this process [39]. As mentioned before, despite the differences among the tested biomaterials, healing process of the bone defects occurred uneventfully in the observed time-points, with all of them presenting mild inflammatory response resulting in no significant differences detected considering the number of MN and PMN. However, significant difference was observed considering MNGCs, which were increased in HA/TCP

group when comparing with AG at late time-periods. Although the affinity of the osteoclastic cells by ß-TCP has been proved, in our study the MNGCs were TRAP negative, but CD206 and CD80 positive, as revealed by immunofluorescence. In this way, it is important to consider the role of MNGCs when it comes to bone substitutes, reminding their origin from phagocytic mononuclear system, the same as macrophages [18]. Although first seen as villains and related to failure and fibrosis, especially when it comes to soft tissue healing, some good news has been recently attributed to MNGCs in bone environment, particularly coming from osseointegration researches, confirming their importance for the maintenance of the implants in a balanced condition [40]; [41]. The role of these cells are so intriguing that recently an attempt to attribute the capacity of MNCGs polarization has been made, classifying them as pro-inflammatory M1-MNCGs and would healing M2-MNGCs [42]. However,

caution

must

be

taken

when

comparing

osseointegration

and

osteoconduction processes considering the biomechanical aspect involved in the first one, and that does not occur during the biological integration of bone materials. However, these evidences add important information about the role of MNCGs in biomaterial field [43]; [44]; 45]; [46]. One important point was brought to light by Jensen et al. [47] when analyzing what they called osteoclast-like cells on DBB and HA/TCP, in mandibles of minipigs. In this model, some morphological differences were found related to MNGCs between the tested biomaterials and a more evident TRAP labeling in HA/TCP. In our study, these evidences were not detected, and no positivity for TRAP was found in this group. This condition may have contributed to M1 and M2 balance and lack of differences among the groups and periods, considering the number of immunolabeled cells, a positive result especially for VC since it is still in experimental phase and not available in the market. In addition, co-localization of CD80 and CD206 antibodies indicating M1 and M2 profiles, respectively, revealed predominance of M2 at days 7 and 21 in AG and DBB, while in HA/TCP it was more evident at the later period. Besides macrophages, MNGCs were also positive labeled for both antibodies clearly identified by immunofluorescence, confirming that inflammatory and reparative profiles can also be attributed to these cells, since they are supposed to derive from macrophages fusion [42].

As mentioned before, although their true role in the process of biomaterial integration in host body is still obscure, there are evidences that they may contribute for healing process, releasing important growth factors such as VEGF [48]. Despite inflammatory and macrophage profiles were found to be quite similar among the groups, Runx-2 bone marker was significantly higher in VC at day 21, indicating a delay in osteoblastic differentiation in the presence of this biomaterial, while OC was lower in HA/TCP at the same time-point. However, when analyzing mineralized bone matrix, the defects reconstructed with DBB and HA/TCP presented improved microarchitecture in relation to VC, probably due to the above mentioned delay in osteoblasts differentiation. At this point, degradation rate of the biomaterial might have exerted an important influence. DBB is known to be highly slow degraded, being detected in human specimens after four to ten years of implantation [49]; [50], and its long-lasting existence would offer conditions for continuing bone formation, maturing and strengthening [51].

The HA/TCP ratio of the biphasic

calcium phosphate used in this study is of 60% and 40%, respectively, which offers the biomaterial a balanced degradation property [52], long enough to permit osteoblasts differentiation and bone maturing. It is known that higher concentrations of HA (above 75%) impede resorption by the cells, while higher concentrations of ßTCP accelerate biomaterial’s degradation [34]. Probably the earlier resorption of VC particles was responsible its results, since some of them were almost totally resorbed at day 45, resulting in a disturbed cell differentiation process and matrix synthesis, since fewer biomaterial surface of the biomaterial was available. Although it is known that particles’ size interfere on osteoconductive capacity and new bone formation [53]; [54], we used the materials as they are available in the market, except by VC which is still in experimental phase. In this way, this is a physical aspect of this new material that can be modified in an attempt to control resorption rate. Therefore, bone tissue quality is not restrained to its mineralized architecture; on the contrary, its collagenous content is as important as, once it represents the scaffold for mineralization in the nano-hierarchy of bone matrix, and is crucial for the maintenance of itsmechanical properties [55]. It can be attested using a simple but efficient stain method with Picrosirius-red, which reveals the molecular order, organization/orientation of collagenous fibers by polarized light microscopy. The more organized are the collagen fibers, the redder they appear. On the other side, if

they are disorganized, the greener they appear [56]. Considering this, it could be observed that despite DBB showed satisfactory mineralized matrix, since its collagenous content was predominantly made of greenish fibers at day 45 in comparison to AG and HA/TCP groups. Nevertheless, it does not mean a poor quality bone, considering that remodeling process is also happening at this time, but more importantly, it contributes for the understanding of the overall picture of the dynamics of bone forming/repairing process in the presence of bone biomaterials. In conclusion, all the tested biomaterials presented similar inflammatory response and macrophage profile despite their distinct origins and physicochemical properties in the present experimental model, which seemed to exert relevant influence on bone markers and quality, again, probably due to the particular degradation rates of each one, which must be considered according to the bone site they will be implanted, and what is expected in terms of function of the reconstructed area.

Funding: This work was supported by São Paulo Research Foundation (FAPESP) [grant number 2016,03762-7].

References 1. Aghaloo TL, Moy PK. Which hard tissue augmentation techniques are the most successful in furnishing bony support for implant placement? Int J Oral Maxillofac Implants. 2007;22 Suppl:49-70. 2. Gruskin E, Doll BA, Futrell FW, Schmitz JP, Hollinger JO. Demineralized bone matrix in bone repair: history and use. Adv Drug Deliv Rev. 2012 Sep;64(12):1063-77. 3. Kolk A, Handschel J, Drescher W, Rothamel D, Kloss F, Blessmann M, Heiland M, Wolff KD, Smeets R. Current trends and future perspectives of bone substitute materials - from space holders to innovative biomaterials. J Craniomaxillofac Surg. 2012 Dec;40(8):706-18. 4. Bellucci D, Sola A, Cannillo V. Hydroxyapatite and Tricalcium Phosphate composites with bioactive glass as second phase: State of the art and current applications. J Biomed Mater Res A. 2015 Dec 8. doi: 10.1002/jbm.a.35619. 5. Huebsch N, Mooney DJ. Inspiration and application in the evolution of biomaterials. Nature. 2009 Nov 26;462(7272):426-32.

6. Franz S, Rammelt S, Scharnweber D, Simon JC. Immune responses to implants - a review of the implications for the design of immunomodulatory biomaterials. Biomaterials. 2011 Oct;32(28):6692-709. 7. Wang RE, Lang NP. Ridge preservation after tooth extraction. Clin Oral Implants Res. 2012 Oct;23 Suppl 6:147-56. 8. Al-Nawas B, Schiegnitz E. Augmentation procedures using bone substitute materials or autogenous bone - a systematic review and meta-analysis. Eur J Oral Implantol. 2014 Summer;7 Suppl 2:S219-34. 9. Baino F, Novajra G, Vitale-Brovarone C. Bioceramics and Scaffolds: A winning combination for tissue engineering. Front Bioeng Biotechnol. 2015 Dec 17;3:202. 10. El-Ghannam A. Bone reconstruction: from bioceramics to tissue engineering. Expert Rev Med Devices. 2005 Jan;2(1):87-101. 11. Jensen SS, Yeo A, Dard M, Hunziker E, Schenk R, Buser D. Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res. 2007 Dec;18(6):752-60. 12. Hannink G, Arts JJ. Bioresorbability, porosity and mechanical strength of bone substitutes: what is optimal for bone regeneration? Injury. 2011 Sep;42 Suppl 2:S22-5. 13. Simunek A, Kopecka D, Somanathan RV, Pilathadka S, Brazda T. Deproteinized bovine bone versus beta-tricalcium phosphate in sinus augmentation surgery: a comparative histologic and histomorphometric study. Int J Oral Maxillofac Implants. 2008 Sep-Oct;23(5):935-42. 14. Cordaro L, Bosshardt DD, Palattella P, Rao W, Serino G, Chiapasco M. Maxillary sinus grafting with Bio-Oss or Straumann Bone Ceramic: histomorphometric results from a randomized controlled multicenter clinical trial. Clin Oral Implants Res. 2008 Aug;19(8):796-803. 15. Vivan RR, Mecca CE, Biguetti CC, Rennó AC, Okamoto R, Cavenago BC, Duarte MH, Matsumoto MA. Experimental maxillary sinus augmentation using a highly bioactive glass ceramic. J Mater Sci Mater Med. 2016 Feb;27(2):41. 16. Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial-cell interactions by adsorbed proteins: a review. Tissue Eng. 2005 Jan-Feb;11(1-2):118. 17. Palmer JA, Abberton KM, Mitchell GM, Morrison WA. Macrophage phenotype in

response to implanted synthetic scaffolds: an immunohistochemical study in the rat. Cells Tissues Organs. 2014;199(2-3):169-83. 18. Xia Z, Triffitt JT. A review on macrophage responses to biomaterials. Biomed Mater. 2006 Mar;1(1):R1-9. 19. Barros MH, Hauck F, Dreyer JH, Kempkes B, Niedobitek G. Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages. PLoS One. 2013 Nov 15;8(11):e80908. 20. Bosca L, Zeini M, Traves P G and Hortelano S 2005 Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate Toxicology 208 249–58. 21. Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Eng Part A. 2008 Nov;14(11):1835-42. 22. Mueller CK, Schultze-Mosgau S. Histomorphometric analysis of the phenotypical differentiation of recruited macrophages following subcutaneous implantation of an allogenous acellular dermal matrix. Int J Oral Maxillofac Surg. 2011 Apr;40(4):401-7. 23. Vasconcelos DP, Costa M, Amaral IF, Barbosa MA, Águas AP, Barbosa JN. Modulation of the inflammatory response to chitosan through M2 macrophage polarization using pro-resolution mediators. Biomaterials. 2015 Jan;37:116-23. 24. Chang MK, Raggatt LJ, Alexander KA, Kuliwaba JS, Fazzalari NL, Schroder K, Maylin ER, Ripoll VM, Hume DA, Pettit AR. Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo. J Immunol. 2008 Jul 15;181(2):1232-44. 25. Reyes-Botella C, Montes MJ, Vallecillo-Capilla MF, Olivares EG, Ruiz C. Expression of molecules involved in antigen presentation and T cell activation (HLA-DR, CD80, CD86, CD44 and CD54) by cultured human osteoblasts. J Periodontol. 2000 Apr;71(4):614-7. 26. Ruiz C, Pérez E, Vallecillo-Capilla M, Reyes-Botella C. Phagocytosis and allogeneic T cell stimulation by cultured human osteoblast-like cells. Cell Physiol Biochem. 2003;13(5):309-14. 27. Raggatt LJ, Wullschleger ME, Alexander KA, Wu AC, Millard SM, Kaur S, Maugham ML, Gregory LS, Steck R, Pettit AR. Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early

endochondral ossification. Am J Pathol. 2014 Dec;184(12):3192-204. 28. Takeshita S, Kaji K, Kudo A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J Bone Miner Res. 2000 Aug;15(8):1477-88. 29. Champagne CM, Takebe J, Offenbacher S, Cooper LF. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone. 2002 Jan;30(1):26-31 30. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 2010 Jul;25(7):1468-86. 31. Matsumoto MA, de Abreu Furquim EM, Gonçalves A, Santiago-Júnior JF, Saraiva PP, Cardoso CL, Munerato MS, Okamoto R. Aged rats under zoledronic acid therapy and oral surgery. J Craniomaxillofac Surg. 2017 May;45(5):781-787. 32. Vajgel A, Mardas N, Farias BC, Petrie A, Cimões R, Donos N. A systematic review on the critical size defect model. Clin Oral Implants Res. 2014 Aug;25(8):879-93. doi: 10.1111/clr.12194. 33. Orsini G, Traini T, Scarano A, Degidi M, Perrotti V, Piccirilli M, Piattelli A. Maxillary sinus augmentation with Bio-Oss particles: a light, scanning, and transmission electron microscopy study in man. J Biomed Mater Res B Appl Biomater. 2005 Jul;74(1):448-57. 34. Yamada S, Heymann D, Bouler JM, Daculsi G. Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/beta-tricalcium phosphate ratios. Biomaterials. 1997 Aug;18(15):1037-41. 35. Hench LL, Polak JM. Third-generation biomedical materials. Science. 2002 Feb 8;295(5557):1014-7. 36. Renno AC, Bossini PS, Crovace MC, Rodrigues AC, Zanotto ED, Parizotto NA. Characterization and in vivo biological performance of biosilicate. Biomed Res Int. 2013;2013:141427. 37. Araújo MG, Liljenberg B, Lindhe J. Dynamics of Bio-Oss Collagen incorporation in fresh extraction wounds: an experimental study in the dog. Clin Oral Implants Res. 2010 Jan;21(1):55-64. 38. Matsumoto MA, Caviquioli G, Biguetti CC, Holgado Lde A, Saraiva PP, Rennó AC, Kawakami RY. A novel bioactive vitroceramic presents similar biological

responses as autogenous bone grafts. J Mater Sci Mater Med. 2012 Jun;23(6):1447-56 . 39. Christo SN, Diener KR, Bachhuka A, Vasilev K, Hayball JD. Innate Immunity and Biomaterials at the Nexus: Friends or Foes. Biomed Res Int. 2015;2015:342304. 40. Albrektsson T, Dahlin C, Jemt T, Sennerby L, Turri A, Wennerberg A. Is marginal bone loss around oral implants the result of a provoked foreign body reaction? Clin Implant Dent Relat Res. 2014 Apr;16(2):155-65. 41. Trindade R, Albrektsson T, Wennerberg A. Current concepts for the biological basis of dental implants: foreign body equilibrium and osseointegration dynamics. Oral Maxillofac Surg Clin North Am. 2015 May;27(2):175-83. 42. Miron RJ, Bosshardt DD. Multinucleated Giant Cells: Good Guys or Bad Guys? Tissue Eng Part B Rev. 2018 Feb;24(1):53-65. 43. Rosner M, Edward DP, Tso MO. Foreign-body giant-cell reaction to the hydroxyapatite orbital implant. Arch Ophthalmol. 1992 Feb;110(2):173-4. 44. Miron RJ, Bosshardt DD. OsteoMacs: Key players around bone biomaterials. Biomaterials. 2016 Mar;82:1-19. 45. Barbeck M, Booms P, Unger R, Hoffmann V, Sader R, Kirkpatrick CJ, Ghanaati S. Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells-New insights into the material-mediated healing process. J Biomed Mater Res A. 2017 Apr;105(4):1105-1111. 46. Miron RJ, Zohdi H, Fujioka-Kobayashi M, Bosshardt DD. Giant cells around bone biomaterials: Osteoclasts or multi-nucleated giant cells? Acta Biomater. 2016 Dec;46:15-28. 47. Jensen SS, Gruber R, Buser D, Bosshardt DD. Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate: light and transmission electron microscopical observations. Clin Oral Implants Res. 2015 Aug;26(8):859-864. 48. Ghanaati S, Barbeck M, Detsch R, Deisinger U, Hilbig U, Rausch V, Sader R, Unger RE, Ziegler G, Kirkpatrick CJ. The chemical composition of synthetic bone substitutes

influences

histomorphometrical

tissue

analysis

reactions of

the

in

cellular

vivo:

histological

inflammatory response

and to

hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate ceramics. Biomed Mater. 2012 Feb;7(1):015005.

49. Piattelli M, Favero GA, Scarano A, Orsini G, Piattelli A. Bone reactions to anorganic bovine bone (Bio-Oss) used in sinus augmentation procedures: a histologic long-term report of 20 cases in humans. Int J Oral Maxillofac Implants. 1999 Nov-Dec;14(6):835-40. 50. Sartori S, Silvestri M, Forni F, Icaro Cornaglia A, Tesei P, Cattaneo V. Ten-year follow-up in a maxillary sinus augmentation using anorganic bovine bone (BioOss). A case report with histomorphometric evaluation. Clin Oral Implants Res. 2003 Jun;14(3):369-72. 51. Artzi Z, Weinreb M, Givol N, Rohrer MD, Nemcovsky CE, Prasad HS, Tal H. Biomaterial resorption rate and healing site morphology of inorganic bovine bone and beta-tricalcium phosphate in the canine: a 24-month longitudinal histologic study and morphometric analysis. Int J Oral Maxillofac Implants. 2004 MayJun;19(3):357-68. 52. Rh Owen G, Dard M, Larjava H. Hydoxyapatite/beta-tricalcium phosphate biphasic ceramics as regenerative material for the repair of complex bone defects. J Biomed Mater Res B Appl Biomater. 2018 Aug;106(6):2493-2512. 53. Klüppel LE, Antonini F, Olate S, Nascimento FF, Albergaria-Barbosa JR, Mazzonetto R. Bone repair is influenced by different particle sizes of anorganic bovine bone matrix: a histologic and radiographic study in vivo. J Craniofac Surg. 2013 Jul; 24(4):1074-7. 54. Carvalho AL, Faria PE, Grisi MF, Souza SL, Taba MJ, Palioto DB, Novaes AB, Fraga AF, Ozyegin LS, Oktar FN, Salata LA. Effects of granule size on the osteoconductivity of bovine and synthetic hydroxyapatite: a histologic and histometric study in dogs. J Oral Implantol. 2007;33(5):267-76. 55. Burr DB. The contribution of the organic matrix to bone's material properties. Bone. 2002 Jul;31(1):8-11. 56. Lattouf R, Younes R, Lutomski D, Naaman N, Godeau G, Senni K, Changotade S. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem. 2014 Oct;62(10):751-8.

Highlights • • •

Different physicochemical properties of tested biomaterials did not lead to significant inflammatory response and macrophage polarization Experimental animal model has to be considered when it comes to biomaterial behavior Bone formation can be influenced by the type of bone substitute

Title: Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction

Declaration of Interest

The authors declare that there is no conflict of interest regarding financial and personal relationships with other people or organization that could inappropriately influence the present work.

Mariza Akemi Matsumoto