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

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.

Mineral trioxide aggregate (MTA) is a biocompatible material that can help in bone repair because its composition allows the rapid adhesion and proliferation of cells on its structure. There is evidence that MTA promotes a favourable response in the osseous environment, with direct bone apposition. It has been demonstrated that MTA induces bone morphogenetic protein 2 (BMP2) expression and calcification in human periodontal ligament cells, stimulates human gingival fibroblasts to produce BMP2, and is able to promote a favourable formation of mineralized tissue in rat alveolar sockets, this process being characterized by a mild inflammatory response and complete bone healing. MTA surfaces also support osteoblast cell attachment, matrix synthesis, and RunX2 expression, which are essential for osteogenesis.

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. The structure of BCP consists of particles with an average size of 100–500 μm composed of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (β-TCP).

Compounds based on HA exhibit a low or negligible bioabsorption, whereas those based on β-TCP are more soluble and can be degraded or reabsorbed easily. At the same time, β-TCP is unpredictable and thus is not an appropriate scaffold for bone growth. The association between HA and β-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 (β-TCP).

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. 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. As a result, BMPs are involved in the differentiation process wherein osteoprogenitor cells transform into mature osteoblasts.

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. 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, which is involved in the initiation of bone condensation.

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 Saúde Animal Ltda, Paulinia, São Paulo, Brazil) in the presence of 5% ketamine hydrochloride (60 mg/kg of body weight) (Vetanarcol; Laboratory König 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 high-rotation drill. Amalgam was then applied to mark the centre of the surgical wound.

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 Indústria de Produtos Odontológicos S/A, Londrina, Paraná, 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, São 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-μm-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 μm in thickness were deparaffinized in xylol at 60 °C 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 °C in an oven.

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 μg/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 (mm 2 ). 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. 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

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 ( 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 ).

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 (mm 2 )
Mean ± SD
New bone (mm 2 )
Mean ± SD
% immunostaining
Mean ± SD
Control 2 weeks 10 1.92 ± 0.30 a 1.04 ± 0.13 a 10.02 ± 0.70 a
BCP 2 weeks 10 2.34 ± 0.16 b 1.51 ± 0.35 b 23.65 ± 1.85 b,f
MTA 2 weeks 10 3.13 ± 0.14 c 2.17 ± 0.13 c 40.73 ± 2.50 c
BCP + MTA 2 weeks 10 3.23 ± 0.22 c 1.52 ± 0.14 b,d 40.30 ± 1.81 c,g
Control 8 weeks 10 2.75 ± 0.32 c,d 2.10 ± 0.16 c 14.21 ± 0.78 e
BCP 8 weeks 10 4.34 ± 0.20 e 3.25 ± 0.16 e 22.78 ± 0.83 f
MTA 8 weeks 10 6.25 ± 0.30 f 3.98 ± 0.14 f 31.67 ± 2.77 g
BCP + MTA 8 weeks 10 5.67 ± 0.24 g 3.25 ± 0.16 c,e 29.63 ± 2.98 g,h
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).

Fig. 1
Area of osteoid tissue (mm 2 ) for each treatment and period of time; mean (95% confidence interval).

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

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

Histological analysis

MTA

At 2 weeks after the operation ( Fig. 4 A) , 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.

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×.)

At 8 weeks after the operation ( Fig. 4 B), 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

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. 4 C). The body of 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. 4 D), 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.

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. 4 E). At 8 weeks after the operation ( Fig. 4 F), 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)

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. 4 G); 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. 4 H), 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. 5 A) . 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. 5 B), 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.

Jan 17, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Histological and immunohistochemical evaluation of biphasic calcium phosphate and a mineral trioxide aggregate for bone healing in rat calvaria
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