YIJOM-3026; No of Pages 8

Int. J. Oral Maxillofac. Surg. 2014; xxx: xxx–xxx http://dx.doi.org/10.1016/j.ijom.2014.10.018, available online at http://www.sciencedirect.com

Clinical Paper Head and Neck Oncology

Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing in rat calvaria

L. G. R. deC.Silva2, S. H. Kim2, S. M. Luczyszyn2, V. Papalexiou2, A. Giovanini1, L. E. Almeida3, V. A. Tramontina2,* 1

School of Dentistry, Positivo University, Curitiba, PR, Brazil; 2School of Health and Biosciences/Dentistry, Pontifical University of Parana´ (PUCPR), Curitiba, PR, Brazil; 3 School of Dentistry, Marquette University, Milwaukee, USA

L. G. R. deC. Silva, S. H. Kim, S. M. Luczyszyn, V. Papalexiou, A. Giovanini, L. E. Almeida, V. A. Tramontina: Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing in rat calvaria. Int. J. Oral Maxillofac. Surg. 2014; xxx: xxx–xxx. # 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Abstract. This work focused on the process of bone repair of defects in standardized calvaria of Wistar rats treated with biphasic calcium phosphate (BCP), mineral trioxide aggregate (MTA), or a combination of the two. Eighty Wistar rats were divided into four treatment groups and were examined at 2 and 8 weeks. A surgical defect was created in the calvaria using a 6-mm diameter trephine drill. The cavity was treated with BCP, MTA, or BCP + MTA; untreated rats with clot formation served as controls. Samples were evaluated histologically and by immunohistochemical staining for areas of new osteoid tissue and new bone tissue, as well as the percentage of labelled cells using anti-bone morphogenetic protein receptor type 1B (anti-BMPR1B) antibodies. Statistically significant differences were found for all dependent variables (area of new osteoid tissue, area of new bone, and percentage immunostaining) by group (P < 0.0001) and time (P < 0.0001), and for the interaction of the two (P < 0.0001). The MTA group at 8 weeks showed the highest amount of osteoid tissue. The same group also exhibited the highest amount of bone tissue formation. The 2-week MTA samples and 2-week BCP + MTA samples exhibited the highest percentages of stained cells. The best results in terms of the area of osteoid and bone tissue formation and the percentage of BMPR1B were observed for the MTA group, confirming that the combination of BCP + MTA does not result in a significant improvement.

0901-5027/000001+08

Key words: bone regeneration; biomaterials; BMPR1B. Accepted for publication 3 October 2014

# 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

2

Silva et al.

Mineral trioxide aggregate (MTA) is a biocompatible material1 that can help in bone repair because its composition allows the rapid adhesion and proliferation of cells on its structure.2,3 There is evidence that MTA promotes a favourable response in the osseous environment, with direct bone apposition.4 It has been demonstrated that MTA induces bone morphogenetic protein 2 (BMP2) expression and calcification in human periodontal ligament cells,5 stimulates human gingival fibroblasts to produce BMP2,6 and is able to promote a favourable formation of mineralized tissue in rat alveolar sockets,7 this process being characterized by a mild inflammatory response and complete bone healing.8 MTA surfaces also support osteoblast cell attachment, matrix synthesis, and RunX2 expression, which are essential for osteogenesis.9 The use of biphasic calcium phosphate (BCP) as an osteoconductive material has proven to be effective in promoting bone healing in orthopaedic and in oral surgery. In addition, BCP is considered to be a safe biomaterial and exhibits high biocompatibility.10–12 The structure of BCP consists of particles with an average size of 100– 500 mm composed of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (b-TCP).10 Compounds based on HA exhibit a low or negligible bioabsorption, whereas those based on b-TCP are more soluble and can be degraded or reabsorbed easily. At the same time, b-TCP is unpredictable and thus is not an appropriate scaffold for bone growth. The association between HA and b-TCP generates the biomaterial called BCP, which is reabsorbable but also contains a more stable segment (HA) that maintains the stability of the scaffold, the graft, and the more soluble component (b-TCP).10,13,14 Immunohistochemical analyses and in situ hybridization experiments have shown that mesenchymal cells, including osteoblasts, express BMPs and their receptors (BMPR) during the formation of skeletal and fracture repair bone tissue.15,16 These glycoproteins are responsible for bringing osteoprogenitor cells to sites of bone formation and repair and thus have important implications in the cascades of cellular events that regulate bone formation and repair. During these processes, mesenchymal cells induce cell proliferation and differentiation, and promote the synthesis of the extracellular matrix.16–18 As a result, BMPs are involved in the differentiation process wherein osteoprogenitor cells transform into mature osteoblasts.19

The cellular response to BMP depends not only on the expression of the type of protein but also on the expression and location of the surface transmembrane receptors BMPR types 1A, 1B, and 2.20 In the calvaria, where it is possible to observe intramembranous ossification, the signal for the repair or formative process is regulated by the BMPR1B receptor,19 which is involved in the initiation of bone condensation.15 As both BCP and the MTA exhibit features that can help in the bone formation process and there are few data on the use of MTA in bone formation (in isolation or in association with BCP), the aim of this study was to focus on the process of bone repair of defects in standardized calvaria of Wistar albino rats treated with BCP, MTA, or a combination of the two. The main analytical techniques used in this study were histology and immunohistochemistry.

Materials and methods

This study was approved by the institutional research ethics committee for animal use. Eighty Wistar albino rats with an average weight of 250 g were used in this study. The rats were divided randomly into four groups; two experiments were performed, using treatment periods of 2 and 8 weeks (n = 20 per treatment group; n = 10 per time period). Each animal was anaesthetized by injection of 2% xylazine hydrochloride intraperitoneally (8 mg/kg of body weight) (Anasedan; Ceva Sau´de Animal Ltda, Paulinia, Sa˜o Paulo, Brazil) in the presence of 5% ketamine hydrochloride (60 mg/kg of body weight) (Vetanarcol; Laboratory Ko¨nig SA, Avellaneda, Argentina). Having confirmed the efficacy of the anaesthetic, a total flap in the form of a ‘U’ was made in the skull of each animal using a number 15 blade, exposing the surface of the calvaria bone. After opening the flap, a circular osteotomy of 6 mm in diameter was made using a trephine drill attached at an angle to a dental micromotor, in the presence of abundant cooling with a saline solution. Prior to the complete removal of the bone fragment, two small osseous incisions in the shape of an ‘L’ were performed in the anteroposterior skull direction so that the larger base of the ‘L’ coincided with the sagittal suture; this was done to identify the location of the circular defect. The incisions were made using a diamond cylindrical highrotation drill. Amalgam was then applied to mark the centre of the surgical wound.21

After carefully removing the incised portion of the calvaria with the aid of a Molt dissector, BCP was added to the wound of 20 animals (Straumann BoneCeramic; Institut Straumann AG, Basel, Switzerland) as per the manufacturer’s instructions, filling the entire calvaria cavity (up to the edge of the defect). In another 20 animals, MTA (MTA Angelus; Angelus Indu´stria de Produtos Odontolo´gicos S/A, Londrina, Parana´, Brazil) was applied in accordance with the manufacturer’s instructions. A further group of 20 animals was treated with a 1:1 combination of BCP and MTA in a similar manner to the other groups. The formation of blood clots in the surgical wound was allowed to occur in the final group, which thus served as the control for the experiment. In all animals, the flap was sutured with isolated knots of Mono-Nylon 4–0 following the surgical procedure. For pain control, sodium dipyrone 500 mg (D-500; Pfizer/Fort Dodge Animal Health Ltda, Campinas, Sa˜o Paulo, Brazil) was given for 3 days after the surgery. Subsequently, the animals were sacrificed with a lethal dose of anaesthetic at either 2 or 8 weeks (10 animals in each treatment group and 10 animals in the control group).

Histology

Following euthanasia, the entire calvaria was carefully cut open and the soft tissue gently removed from the bone. All samples were stored in 10% formalin prior to mounting of the surgical specimens, and then demineralized in 4.13% ethylenediaminetetraacetic acid (EDTA) for a period of 75 days. The material underwent routine histological processing for paraffin embedding; a microtome was used to cut 3-mm-thick sagittal sections using the ‘L’ marking in the amalgam to determine the location of the largest wound diameter. Two slides were made for each piece of material, for a total of 160 slides. The slides were stained with Masson’s trichrome and analysed using an optical microscope at 40 magnification.

Immunohistochemistry processing

For immunostaining, serial sections of 3 mm in thickness were deparaffinized in xylol at 60 8C and hydrated using various concentrations of alcohol (absolute, 95%, and 80%). After hydration, the specimens were submitted to antigenic recovery using a 1% solution of trypsin (pH 7.2) for 45 min at 37 8C in an oven.

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

Evaluation of BCP and MTA for bone healing The slides holding the histological sections were then washed and immersed in 3% hydrogen peroxide for 30 min to inhibit the activity of endogenous peroxidase. After rinsing with distilled water, the specimens were immersed in a buffered saline solution (phosphate-buffered saline (PBS), pH 7.4) to maintain constant chemical properties of the reaction. Next, sections were incubated overnight with the primary antibody anti-BMPR1B (200 mg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using a 1:100 factor dilution in PBS. To detect the primary antibodies, a universal streptavidin–biotin peroxidase kit (Diagnostic BioSystems, Pleasanton, CA, USA) was used, as per the manufacturer’s instructions. The immune reaction was monitored using a solution of tetrachloride diaminobenzidine (Diagnostic BioSystems). Within 2 min of administering the drug, a brown–brownish precipitate confirmed the presence of the antigen. The specimens were counterstained with Harris haematoxylin for 3 min. For the negative control, we used the polyclonal IgG isotope (2 mg/ml; Abcam, Cambridge, UK) for 10 min after incubation at room temperature as the primary antibody for each sample. Three slides were used for the incubation of each antibody. Analysis of the images

All specimen images were captured using a digital camera (Samsung, South Korea) coupled to a light microscope (Zeiss, Germany) using the original 200 magnification of each digital image with a resolution of 600 dpi. A virtual image of 117 cm  80 cm was produced, which made it impossible to catch all aspects of the defect in a single image at the magnification used. Accordingly, a digital framework composed of the entire defect was then constructed by combining two smaller images. The resulting images were

evaluated in terms of bone tissue deposition and blood vessels by consulting histological references. All histomorphometric measurements were made using ImageTool software v. 2.00 (Department of Dental Diagnostic Science at The University of Texas Health Science Center, San Antonio, TX, USA). The image of a 1-mm slide was used to calibrate all measurements. The histomorphometric data were counted manually and expressed as areas (mm2). The perimeters of the deposited histological bone matrix and osteoid tissue areas were carefully traced; all areas were computed into the total histological area. At the same time, the percentage of positive BMPR1B protein was assessed by automated method, following the protocol established by Di Cataldo et al.22 This automated counting allows only the percentage of protein present in the whole defect to be calculated; it is not possible to distinguish the specific immunostaining in cells or bone matrix.

Statistical analysis

The data obtained were analysed using statistical software SPSS 18.0 for Windows (SPSS, Inc., Chicago, IL, USA). Normality tests (Kolmogorov–Smirnov and Shapiro–Wilk) were performed to examine the data distributions. Levene’s test was used to examine the homogeneity of variance, also using two-way analysis of variance (ANOVA) (group  time), followed by the Games–Howell test for multiple comparisons. The level of significance was set at 5% (P < 0.05). Results

3

(Fig. 1). This group also exhibited the best results with respect to the amount of bone tissue formed (Fig. 2). The groups that exhibited the highest percentage of stained cells were the 2-week MTA and 2-week BCP + MTA samples, with no statistically significant difference observed between the two. We also observed a decrease in the percentage of immunostained cells from 2 to 8 weeks for all three groups that were treated with the biomaterials. In the control group, an increase in immunomarker concentration from 2 to 8 weeks was observed (Fig. 3). Histological analysis

MTA

At 2 weeks after the operation (Fig. 4A), it was possible to identify the presence of bone formation concentrated at the edges of the surgical defect in areas contiguous with the original remaining bone. This area of bone matrix formation was surrounded by osteoid tissue as well as areas of a significant amount of connective tissue. Very little mature bone was observed in the central region of the defect. This region was composed predominantly of granulation tissue, with the richly vascularized osteoid formation concentrated in the peripheral areas of the histological sections. At 8 weeks after the operation (Fig. 4B), greater deposition of bone matrix was observed in the areas adjacent to the remaining bone. The body of the defect was filled with a significant formation of osteoid tissue, surrounded by immature medullar tissue. BCP

Histomorphometric analysis

The results of the histomorphometric analysis are given in Table 1. The group that presented the largest amount of osteoid tissue was the 8-week MTA sample

In the bone repair of the surgical defects filled with BCP, discrete bone formation was observed on the margins of the artificially created surgical defect at 2 weeks after the operation (Fig. 4C). The body of

Table 1. Results for the study groups (control, MTA, BCP, and BCP + MTA groups) by treatment period (2 and 8 weeks). Group

Treatment period

Number

Osteoid tissue (mm2) Mean  SD

New bone (mm2) Mean  SD

% immunostaining Mean  SD

Control BCP MTA BCP + MTA Control BCP MTA BCP + MTA

2 2 2 2 8 8 8 8

10 10 10 10 10 10 10 10

1.92  0.30 a 2.34  0.16 b 3.13  0.14 c 3.23  0.22 c 2.75  0.32c,d 4.34  0.20 e 6.25  0.30 f 5.67  0.24 g

1.04  0.13 a 1.51  0.35 b 2.17  0.13 c 1.52  0.14b,d 2.10  0.16 c 3.25  0.16 e 3.98  0.14 f 3.25  0.16c,e

10.02  0.70 a 23.65  1.85b,f 40.73  2.50 c 40.30  1.81c,g 14.21  0.78 e 22.78  0.83 f 31.67  2.77 g 29.63  2.98g,h

weeks weeks weeks weeks weeks weeks weeks weeks

SD, standard deviation; MTA, mineral trioxide aggregate; BCP, biphasic calcium phosphate. Note: The same letters shown within columns indicate no significant difference (Games–Howell multiple comparisons tests; P > 0.05).

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

4

Silva et al. BCP combined with MTA

Two weeks after the operation, a histological picture of healing, represented by a moderate quantity of osteoid tissue composing the major part of the defect, was observed (Fig. 4E). At 8 weeks after the operation (Fig. 4F), the majority of the defect was still composed of osteoid tissue, which was identified by the intense deposition of bone matrix at the margins of the defect in the form of trabeculae that mimic intramembranous ossification. Control (clot)

Fig. 1. Area of osteoid tissue (mm2) for each treatment and period of time; mean (95% confidence interval).

the defect was composed predominantly of dense vascularized connective tissue, resulting in negative images of exogenous material compatible with the particles of the biomaterial. A small number of giant cells and a specific amount of osteoid tissue were also observed around the

biomaterial. Eight weeks after the operation (Fig. 4D), a histological repair process was observed similar to that of the second week process. In this case, however, a greater deposition of bone matrix on the edges of the defect and the osteoid matrix near the biomaterial were evident.

Fig. 2. Area of new bone (mm2) for each treatment and period of time; mean (95% confidence interval).

The control group showed a predominance of granulation tissue, along with a diffuse and moderate chronic inflammatory process. Little deposition of bone matrix was identified at 2 weeks after the operation in this group (Fig. 4G); however, areas of osteoid tissue were observed, concentrated adjacent to the remaining bone tissue or in areas restricted to the body of the defect. At 8 weeks after the operation (Fig. 4H), mild bone formation was identified; however, the foci of bone mineralization were only observed in the thin connective tissue. Immunohistochemical analysis

All groups tested positive for the BMPR1B antibody. The highest BMPR1B content was identified in the 2-week MTA group (Fig. 5A). At this point in time, the BMPR1B-positive immunostaining occupied nearly the entire area of the defect. In contrast, at 8 weeks after the operation (Fig. 5B), the percentage of positive markers decreased as the proportion of osteoid or bone matrix increased. Despite the numerical difference, the overall pattern of immunostaining for the two periods was similar, resulting in a clear identification of the protein both in osteoprogenitor cells and the surrounding extracellular matrix. A different result was observed for samples treated with BCP alone (Fig. 5C) and BCP + MTA (Fig. 5E). In the group treated with BCP, the BMPR1B-positive areas were found in the extracellular fibrous matrix, with an intense deposition of dense and rough fibres mimicking areas of pre-cortical collagen formation of compact bones, involving the grafted biomaterial (Fig. 5D). In the BCP + MTA group, immunostaining of BMPR1B was more intense and a greater percentage

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

Evaluation of BCP and MTA for bone healing

5

of immunostaining was observed in comparison to the group in which only BCP was grafted, at both 2 (Fig. 5E) and 8 weeks (Fig. 5F). The presence of BMPR1B was identified in osteoprogenitor cells and neoformed bone trabeculae in the extracellular matrix. A smaller number of cells and BMPR1B-positive matrix was found in the control group at both 2 weeks (Fig. 5G) and 8 weeks (Fig. 5H). In the control group, the cells were arranged in the area adjacent to the remaining bone tissue or in isolated areas among the connective tissue. Statistical results

Fig. 3. Percentage of immunostaining for each treatment and period of time; mean (95% confidence interval).

Fig. 4. Histological findings for each treatment group at 2 and 8 weeks post-surgery. MTA group at (A) 2 weeks, and (B) 8 weeks, demonstrating the reparative process in the calvaria; granulation tissue (gt) and new bone formation (arrow) are evident. BCP group at (C) 2 weeks, and (D) 8 weeks; granulation tissue (gt) and biphasic calcium phosphate (BCP) particles are seen at both time points, however little new bone formation is evident in these groups. BCP + MTA group at (E) 2 weeks, and (F) 8 weeks, showing regenerative development in this group; intensive bone deposition (arrow) is seen at 8 weeks, in contrast to the group treated only with BCP. Control group at (G) 2 weeks, and (H) 8 weeks; intensive granulation tissue (gt) is seen at both time points analysed; bone tissue deposition (arrow). (All micrographs stained with Masson’s trichrome stain; original magnification 40.)

For the determination of any differences among mean values for the study variables (area of new bone, area of osteoid tissue, and % of immunostaining) according to the study groups (MTA, BCP, BCP + MTA, or control) and period of time (2 or 8 weeks), the normality of data for each treatment (group  time) was initially tested using the Kolmogorov–Smirnov test, as n < 30; this indicated a normal distribution for all variables in all treatments (P > 0.05). Next, the homogeneity of variances was tested among treatments for each variable using Levene’s test, which indicated heterogeneity of variances among treatments for all three variables (P < 0.05). Following that, two-way ANOVA showed statistically significant differences for all dependent variables (area of new osteoid tissue, area of new bone, and percentage immunostaining) by group (P < 0.0001) and time (P < 0.0001), and for the interaction of the two (P < 0.0001), where the observed power was >0.99. The significance of results was finally demonstrated by Games–Howell test for multiple comparisons with a significance level of 5% (P < 0.05). It was possible to observe that the MTA group at 8 weeks showed the highest mean value of osteoid tissue (6.25  0.30 mm2) against the control at 2 weeks, which showed the lowest mean value (1.92  0.30 mm2). When the area of new bone was evaluated, again the MTA group at 8 weeks showed the highest mean value (3.98  0.14 mm2) and the control group at 2 weeks had the lowest mean value (1.04  0.13 mm2). The mean percentage immunostaining was highest for MTA at 2 weeks (40.73  2.50%), with similar results for BCP + MTA at 2 weeks (40.30  1.81%), and the lowest mean value was found in the control

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

6

Silva et al.

Fig. 5. Immunohistochemical positivity for BMPR1B (brownish colour) for each treatment group at 2 and 8 weeks post-surgery. MTA group at (A) 2 weeks, and (B) 8 weeks, showing the intensive presence of positive cells in the granulation tissue at both time-points. BCP group at (C) 2 weeks, and (D) 8 weeks; BMPR1B positivity in cells and fibres that are present in the granulation tissue only surrounding the biphasic calcium phosphate (BCP) particles is evident at both time-points. BCP + MTA group at (E) 2 weeks, and (F) 8 weeks, revealing the intensive presence of BMPR1B at both time-points. Control group at (G) 2 weeks, and (H) 8 weeks; there is scarce positivity for BMPR1B, except at 8 weeks post-surgery, where positivity is restricted to the border of the defect. (Original magnification 40.)

group at 2 weeks (10.02  0.70%) (Table 1; Figs. 1–3). Discussion

Several studies4,10,21,23–25 reported in the literature have described the tissue reactions, in both soft and hard tissues, using the materials employed in this research; however, there are only a few studies that have compared the use of MTA and BCP in isolation or in association for the healing of standardized critical bone defects. This study aimed to investigate whether the association between BCP and MTA could bring some benefits for bone neoformation. The best results in terms of area of osteoid tissue formed (area of bone tissue formed) and the percentage of BMPR1B were observed for the MTA group. This may be due to the excellent biocompatibility of this material26,27 and the ability of osteoblastic cells to adhere and spread on its surface.9

In addition to stimulating adhesion and cell proliferation1 and the expression of alkaline phosphatase by fibroblasts28 and osteocalcin and other interleukins by osteoblasts,29 MTA is able to induce a greater expression of the BMPR1B receptor, which is important in the process of osteogenesis, being directly involved in the process of bone condensation.15 In the analysis of the area of osteoid tissue formed, an increase in this parameter was observed from 2 to 8 weeks in all groups; however, the increase was more pronounced in groups BCP, MTA, and BCP + MTA, providing evidence that the presence of these materials stimulated an increased formation of this tissue in the type of critical defect used in the present study. These results differ from those found by Frota et al.,30 who investigated the use of b-TCP, a component of BCP; they found no induction of osteoid tissue formation in the control group, but only in the groups where the biomaterial was present. This

effect may be due to the short evaluation time (7, 15, and 30 days). Still, if one compares the control group with the other groups, a greater area of osteoid tissue was formed in the groups in which the materials were implanted, providing further evidence of the effectiveness of osteoconductive materials used in these critical defects.30 The groups with the highest formation of osteoid tissue were MTA and BCP + MTA. Of note, there were no significant differences between the 2-week MTA, 2-week BCP + MTA, and 8-week control groups, indicating a similar pattern of osteoid tissue formation in these groups, although at an earlier point in time when MTA or BCP + MTA was used. The MTA group also exhibited more beneficial results than the BCP group during the same period of evaluation. The area of newly formed bone tissue increased in size from 2 to 8 weeks for the BCP, MTA, and BCP + MTA groups, demonstrating that these materials continue to stimulate the formation of tissue over this period of time. Comparing the control group with the other groups, a greater area of bone tissue formation was observed in the biomaterials groups. The material with the best results regarding the formation of bone tissue was MTA. When MTA was used in association with BCP, the amount of tissue formed was lower than with MTA alone, at both 2 weeks and 8 weeks. These results indicate that the combination of these materials does not lead to significant benefits. Perhaps the space occupied by the BCP particles, especially if the particles are slowly reabsorbed, complicates the process of repair. However, if we evaluated bone repair over a longer period of time, perhaps we would observe a higher reabsorption of these particles and perhaps the results would be more similar. The BCP group when compared to the control group showed a greater amount of bone tissue formation, and these results were statistically significant. Our results corroborate the findings of Schwarz et al.12 and Cordaro et al.,10 who reported microscopic evidence of bone formation in the presence of BCP. Some studies have found evidence that the ceramic calcium phosphate may have osteoconductive properties,10,14,31 while others have discovered that the material becomes encapsulated by connective tissue, without osteoconductive properties.23 Do Nascimento et al.32 reported that the deposition of hard tissue on MTA is related to its sealing ability, biocompatibility,

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

Evaluation of BCP and MTA for bone healing and alkaline pH, the presence of calcium and phosphate ions in its formulation, and the ability to attract blast cells and promote a favourable environment for cementum formation. The authors stated that the osteoconductive effect, stimulation of proliferation and cell adhesion, and stimulation of the expression of alkaline phosphatase by fibroblasts and osteocalcin and other interleukins by osteoblasts, are also characteristics related to this material. These findings may help to explain the higher area of the formation of osteoid tissue and bone in the MTA and BCP + MTA groups observed in this work. According to Singhatanadgit and Olsen,20 the endogenous signalling of BMPR1B is required to initiate the differentiation of osteoblasts. Thus, we evaluated the percentage of cells stained for BMPR1B; we observed an increase in stained cells from 10.02  0.70% in the 2-week control group to 14.21  1.81% in the 8-week control group, suggesting that the beginning of the ossification process in the control group occurred later than in the groups where biomaterials were used. For the MTA and BCP + MTA groups, the percentage of stained cells was higher in the 2-week group than in the 8-week group. This finding suggests that there was a decrease in the quantity of osteoprogenitor cells that had free receptors for BMPR1B, indicating that the phase of formation of bone matrix, with greater synthesis activity, occurred during this period, prior to that in the control group. This result is in contrast to that of the work by Maeda et al.5 in which different levels of BMPR were observed between the control group and the MTA group. While the findings of the present study confirm a positive effect of MTA, it is necessary to conduct a larger study to determine the mechanism by which this material leads to bone tissue regeneration; this is also the case for BCP, which is already used in surgeries. In addition, the assessment periods of 2 and 8 weeks were not sufficient to fully consider the possibility of reabsorption of BCP particles. Within the limitations of the present study, it is possible to conclude that the defects treated with MTA exhibited good results in relation to bone formation and that the use of MTA associated with BCP did not provide any additional benefits. BCP alone showed better results than the control, but the role of BCP particles remaining in the treated wounds needs to be evaluated over longer periods of time.

Funding

None. 11.

Competing interests

None declared. 12.

Ethical approval

This study was approved by the Research Ethics Committee for Animal Use of the Pontifical Catholic University of Parana, Brazil (CEUA/PUCPR; protocol number 390/08). References 1. Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Cellular response to mineral trioxide aggregate. J Endod 1998;24:543–7. 2. Camilleri J, Pitt Ford TR. Mineral trioxide aggregate: a review of the constituents and biological properties of the material. Int Endod J 2006;39:747–54. 3. De Deus G, Ximenes R, Gurgel-Filho ED, Plotkowski MC, Coutinho-Filho T. Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cells. Int Endod J 2005;38:604–9. 4. Yaltirik M, Ozbas H, Bilgic B, Issever H. Reactions of connective tissue to mineral trioxide aggregate and amalgam. J Endod 2004;30:95–9. 5. Maeda H, Nakano T, Tomokiyo A, Fujii S, Wada N, Monnouchi S, et al. Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells. J Endod 2010;36:647–52. 6. Guven G, Cehreli ZF, Ural A, Serdar MA, Basak F. Effect of mineral trioxide aggregate cements on transforming growth factor b1 and bone morphogenetic protein production by human fibroblasts in vitro. J Endod 2007;33:447–50. 7. Gomes-Filho JE, Costa MT, Cintra LT, Lodi CS, Duarte PC, Okamoto R, et al. Evaluation of alveolar socket response to Angelus MTA and experimental light-cure MTA. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:e93–7. 8. Gomes-Filho JE, Costa MM, Cintra LT, Duarte PC, Takamiya AS, Lodi CS, et al. Evaluation of rat alveolar bone response to Angelus MTA or experimental light-cured mineral trioxide aggregate using fluorochromes. J Endod 2011;37:250–4. 9. Perinpanayagam H, Al-Rabeah E. Osteoblasts interact with MTA surfaces and express Runx2. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:590–6. 10. 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

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

7

randomized controlled multicenter clinical trial. Clin Oral Implants Res 2008;19: 796–803. Daculsi G, Pilet P, Cottrel M, Guicheux G. Role of fibronectin during biological apatite crystal nucleation: ultrastructural characterization. J Biomed Mater Res 1999;47: 228–33. Schwarz F, Herten M, Ferrari D, Wieland M, Schmitz L, Engelhardt E, et al. Guided bone regeneration at dehiscence-type defects using biphasic hydroxyapatite + beta tricalcium phosphate (Bone Ceramic) or a collagen-coated natural bone mineral (BioOss Collagen): an immunohistochemical study in dogs. Int J Oral Maxillofac Surg 2007;36:1198–206. Schopper C, Moser D, Goriwoda W, ZiyaGhazvini F, Spassova E, Lagogiannis G, et al. The effect of three different calcium phosphate implant coatings on bone deposition and coating resorption: a long-term histological study in sheep. Clin Oral Implants Res 2005;16:357–68. Singhatanadgit W, Mordan N, Salih V, Olsen I. Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2. Int J Biochem Cell Biol 2008;40:2854–64. Ashique AM, Fu K, Richman JM. Signalling via type IA and type IB bone morphogenetic protein receptors (BMPR) regulates intramembranous bone formation, chondrogenesis and feather formation in the chicken embryo. Int J Dev Biol 2002;46:243–53. Ishidou Y, Kitajima I, Obama H, Maruyama I, Murata F, Imamura T, et al. Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation. J Bone Miner Res 1995;10:1651–9. Allori AC, Sailon AM, Warren SM. Biological basis of bone formation, remodeling, and repair—Part I: Biochemical signaling molecules. Tissue Eng B: Rev 2008;14: 259–73. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res 1993;8:565–72. Zhao M, Harris SE, Horn D, Geng Z, Nishimura R, Mundy GR, et al. Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation. J Cell Biol 2002;157:1049–60. Singhatanadgit W, Olsen I. Endogenous BMPR-IB signaling is required for early osteoblast differentiation of human bone cells. In Vitro Cell Dev Biol Anim 2011; 47:251–9. Nagata M, Messora M, Okamoto R, Campos N, Pola N, Esper L, et al. Influence of the ratio of particulate autogenous bone graft/plateletrich plasma on bone healing in critical-size defects: an immunohistochemical analysis in rat calvaria. Bone 2009;45:339–45. Di Cataldo S, Ficarra E, Acquaviva A, Macii E. Automated segmentation of tissue images

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

YIJOM-3026; No of Pages 8

8

Silva et al.

23.

24.

25.

26.

for computerized IHC analysis. Comput Methods Programs Biomed 2010;100:1–15. Fleckenstein KB, Cuenin MF, Peacock ME, Billman MA, Swiec GD, Buxton TB, et al. Effect of a hydroxyapatite tricalcium phosphate alloplast on osseous repair in the rat calvarium. J Periodontol 2006;77:39–45. Lu JX, Gallur A, Flautre B, Anselme K, Descamps M, Thierry B, et al. Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical, and medullar bone sites in rabbits. J Biomed Mater Res 1998;42:357–67. Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 2000;21:393–411. Mitchell PJ, Pittford TR, Torabinejad M, McDonald F. Osteoblast biocompatibility of mineral trioxide aggregate. Biomaterials 1999;20:167–73.

27. Torabinejad M, Ford TR, Abedi HR, Kariyawasam SP, Tang HM. Tissue reaction to implanted root-end filling materials in the tibia and mandible of guinea pigs. J Endod 1998;24:468–71. 28. Bonson S, Jeansonne BG, Lallier TE. Rootend filling materials alter fibroblast differentiation. J Dent Res 2004;83:408–13. 29. Moretton TR, Brown Jr CE, Legan JJ, Kafrawy AH. Tissue reactions after subcutaneous and intraosseous implantation of mineral trioxide aggregate and ethoxybenzoic acid cement. J Biomed Mater Res 2000; 52:528–33. 30. Frota R, Da Silva-Ju´nior VA, Teixeira M, Sobral AP, Emanuel-Dias-De Oliveira e Silva. Da Silveira MM, et al. Histological evaluation of bone repair using b-tricalcium phosphate. Med Oral Patol Oral Cir Bucal 2011;16:190–4. 31. Bowers GM, Vargo JW, Levy B, Emerson JR, Bergquist JJ. Histologic observations

following the placement of tricalcium phosphate implants in human intrabony defects. J Periodontol 1986;57:286–7. 32. Do Nascimento C, Issa JP, Iyomasa MM, Regalo SC, Sie´ssere S, Pitol DL, et al. Bone repair using mineral trioxide aggregate combined to a material carrier, associated or not with calcium hydroxide in bone defects. Micron 2008;39:868–74.

Address: Vinı´cius Augusto Tramontina School of Health and Biosciences/Dentistry Pontifical University of Parana´ (PUCPR) Rua Imaculada Conceic¸a˜o 1155 Curitiba PR CEP 80215-901 Brazil Tel: +55 41 3271 1637 Fax: +55 41 3271 1637 E-mail: [email protected]

Please cite this article in press as: Silva LGR, et al. Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing. . ., Int J Oral Maxillofac Surg (2014), http://dx.doi.org/10.1016/j.ijom.2014.10.018

Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing in rat calvaria.

This work focused on the process of bone repair of defects in standardized calvaria of Wistar rats treated with biphasic calcium phosphate (BCP), mine...
2MB Sizes 0 Downloads 6 Views