Abstract
To overcome the morbidity of autogenous graft removal and limitations of allogeneic and xenogeneic grafts, a great interest exists in the development of biomaterials of synthetic origin. Objective: The aim of this study was to evaluate the biological behavior of a novel bioactive glass (60% SiO2- 36% CaO-4% P2O5) as bone substitute in critical calvaria defects of rats, in comparison to hydroxyapatite. Methods: Sixty male Wistar rats were divided in three groups, according to the treatment: Control Group (C) - blood clot; Hydroxyapatite (HA) - particulate hydroxyapatite (≤0,5 mm); and Bioactive Glass (BG) - particulate bioactive glass (0.04–1 mm). Results: From the intergroup analysis, it was observed that Group C presented a greater newly formed bone area (NBA) when compared to Groups HA and BG. In addition, Group HA showed higher NBA when compared to Group BG at 30 and 60 days (P < 0.05). Immunohistochemistry revealed that groups HA and BG presented high and moderate osteocalcin immunolabeling respectively. Group HA displayed a greater number of TRAP-positive cells compared to Groups C and BG at 30 and 60 days (p < 0.05). Conclusion: From these results, we can conclude that the resorption rate of hydroxyapatite is higher than the novel bioactive glass, which maintained significant higher volume until the last experimental period. Both of the tested biomaterials acted as osteoconductors during bone repair, and their physical characteristics importantly influenced this process.
Keywords: Hydroxyapatite, Bone regeneration, Bioactive glass, Rats
1. Introduction
Congenital or acquired bone defects in the maxillofacial complex still demand challenging therapies.1 Current surgical approaches include the reconstruction of the injured area with autogenous, allogeneic, xenogeneic, and synthetic materials,2 and, amongst them, allogeneic and xenogeneic grafts are the most commonly used for the reconstruction of resorbed alveolar ridge. Bone substitutes are a therapeutic option to correct abnormal intermaxillary relationships, as well as to achieve an adequate bone volume and morphology, since they act as scaffolds,3 and as a mineral reservoir, allowing new bone formation and reestablishing the damaged bone area.
Autogenous bone is still considered the gold standard for surgical corrections of maxillofacial deformities and for reconstructive surgeries4; however, they present disadvantages related to morbidity of the donor site, additional surgical time, pain, hematoma, infection, surgical fracture, and limited availability.5 The frozen allogeneic graft from a human bank tissue would be an alternative to the autogenous graft, but there is the possibility of diseases transmission and immunogenic reactions, and some patients are resistant in accepting the idea of having a transplanted tissue from someone else's body.6
Biomaterials are widely used in medicine, dentistry and biotechnology, and have the ability to contact the biological system and living tissues, aiming to repair and/or substitute damaged tissue and organs, and restore compromised functions by degenerative processes or trauma. Other options of grafting material are the xenografts from animal origin, as the materials from bovine origin, which are often used in oral surgical procedures.7 These materials consist of a highly porous hydroxyapatite, similar to cortical bone, and their organic components are removed chemically or by slow heat.
In a study evaluating patients' preference regarding the origin of the material to be grafted, Fernández et al.2 reported that the highest rate of treatment refusals was for allogeneic and xenogeneic grafts. The most relevant reasons for the refusal of these materials was the fear of possible transmission of diseases and infections, and also by sociocultural reasons, such as the non-acceptance of materials from animal origin or other human beings for their own benefit. In order to overcome the limitations of the autogenous graft removal, as well as to eliminate the risks of immunogenic reactions from the allogeneic and xenogeneic grafts, surgeons can benefit from the use of biomaterials from synthetic origin that, in many cases, reduce or even eliminate the use of materials from biological origin.8
The group of synthetic biomaterials that most resemble bone composition is that of calcium phosphate bioceramics. The main advantage of the calcium phosphate-based graft such as hydroxyapatite is that its ionic content do not interfere with the body's physiology.9 Besides being highly biocompatible, calcium phosphates act as scaffolds and osteoconductors after implantation in the bone. When placed in a stable condition adjacent to the bone, an osteoid matrix is formed directly on the surface of calcium phosphate.10 The incorporation of trace elements such as Na+, Mg2+, Zn2+, Si4+, Sr2+, Mn2+ among others, that perform some role in bone formation or growth, alters the characteristics of bioceramics leading to differences in biological response and in bone apatite conversion rate.11
Another alternative of synthetic biomaterial, which can be used as a bone substitute, is the bioactive glass. The first 45S5 Bioglass was discovered by Hench in 1969, with calcium oxides, sodium, phosphorus and silicon dioxide in its composition. With the addition of magnesium and potassium oxides to the basic composition of the Hench's bioglass, the 13–93® glass was discovered, revealing an improved biomechanical capacity.12 These two bioglasses form a silica gel, which slowly and incompletely converts into hydroxyapatite (Ca10(PO4)6(OH)2) when exposed to body fluid. Crystals that do not convert can remain in the body for an extended period of time.13 While 45S5 Bioglass® and 13–93® demonstrated a slow and incomplete conversion into hydroxyapatite, bioglasses containing boron in its composition have demonstrated a faster and more complete conversion.14 Boron silicate glass (13–93B1) and borate glass (13–93B3), that partially or totally replace silica by boron oxide, respectively, have been raising the conversion rate into hydroxyapatite by 3–4 times, as the levels of boron oxide increases. Nevertheless, due to their rapid resorption and trabecular microstructure, they have been indicated only for bone defects that have well-defined bone walls.15 Sol-gel technique has also been used in the production of bioactive glasses, presenting a greater porosity, and increasing contact surface and conversion to hydroxyapatite. However, they lose in resistance when compared to bioglasses produced by high melting temperatures.16
Thus, the objective of this study was to evaluate the biological behavior of two synthetic biomaterials, HAP-91® Hydroxyapatite and a novel Bioactive Glass compound, as bone substitutes in critical size defects in rat calvaria, considering tissue response, and new bone neoformation after 30 and 60 post-operative days.
2. Material and methods
2.1. Development of the study
The present study was submitted and approved by the Ethics Committee on Animal Use (CEUA) registered under the protocol #673/2015, following the current norms adopted by the College of Animal Experimentation. Sixty male rats (Rattus novergicus albinus, Wistar) were used, weighing between 375 and 450 g, with an average of three months of life. They were kept under standard conditions with water and food ad libitum, under 22±2 °C room temperature, with 12-h light/dark cycles.
2.2. Surgical procedure
The rats were anesthetized with an insulin needle (13 mm × 0.04 mm), associating 0.14 ml/kg of ketamine hydrochloride and 0.06 ml/kg of xylazine hydrochloride. After, trichotomy was performed in the area of interest, followed by asepsis with a product based on polyvinylpyrrolidone (PVPI) in aqueous solution, containing 1% active iodine (Fig. 1, A). Afterwards, the animals were submitted to local anesthesia in both edges (anterior and posterior) of the incision area with 2% Lidocaine + Epinephrine 1:100.00. A semilunar dermoperiostal incision using a 15c carbon steel scalpel blade was then performed in the anterior region of the calvaria, allowing reflection of a full thickness flap toward the posterior direction.17
Fig. 1.
Animal Surgery. A) Trichotomy and asepsis with PVPI; B) Bone defect with the L-shaped marks; C) Defect filled with Bioactive Glass; D) Suture with single stitches.
The bone defect was created with an 8 mm circular trephine drill coupled to a low speed handpiece under constant irrigation with 0.9% sterile saline solution. The bone defect was made in the middle portion of the calvaria measuring 8 mm in diameter and approximately 1.5 mm in depth, corresponding to an area of about 50 mm2. A hollenbach 3S spatula was used not only to check the depth of the perforation during surgery, but also to perform the careful removal of the sectioned bone tissue. Two L-shaped marks were made, one 2 mm anterior and another 2 mm posterior to the margins of the surgical defect using a spherical diamond drill, thus creating two surgical fissures that were filled with amalgam (Fig. 1, B) to allow identification of the central area of the defect during laboratory processing. The marks also served as references to locate the original bone margins of the surgical defect during histomorphometric and immunohistochemical analyses.18
The animals were then randomly divided (by a draw method) into three groups according to the following local treatments: Control Group (C) - bone defect filled with blood clot; Hydroxyapatite Group (HA) - filled with Hydroxyapatite; and Bioactive Glass Group (BG) - filled with novel Bioactive Glass compound (Fig. 1, C). The grafted materials were supplied by a national private company (JHS biomaterials®). In the HA group, a synthetic hydroxyapatite composed of Ca10(PO4)6(OH)2 (HAP-91®), with hexagonal particles 0.5 mm in diameter, formed by long, short or tubular prismatic crystals in needles and porosity of 110.6 A0. In the BG group, a novel Bioactive Glass compound consisting of 60% SiO2- 36% CaO-4% P2O5 was used, with particles ranging from 0.04 to 1 mm in diameter, without sharp edges and with crystallinity close to 100%. In these two groups, the amount of grafted material was standardized with a measuring spoon (approximately 50 mg of each biomaterial), and its accommodation in the defect was standardized with a metal instrument that compacted them in the same way and with the same force. The suture was carefully performed by repositioning the flap over the surgical area, where the tissues were approximated with two tweezers for transfixation of the needle, with the intention that the material would receive the smallest possible movement and remain in the desired location. The suture was made with single stitches with 3–0 silk thread (Fig. 1, D). After the surgical procedure, the animals were medicated with two drops of analgesic (Sodium Dipirone 500 mg/ml).
Ten animals from each experimental group were euthanized using anesthetic overdose of 10 mg/ml lidocaine (0.7 mg/kg body weight) associated with 2.5% sodium thiopental (150 mg/kg body weight). Euthanasia was performed 30 and 60 days after surgery. The calvaria were removed and fixed in 10% buffered formaldehyde for a period of at least 48 h and dissected for the analysis.
2.3. Histological processing
The specimens were demineralized for approximately 45 days in a buffered EDTA solution in the proportion of 250 mg of disodium EDTA salt per 1750 ml of distilled water and neutralized (pH = 7.0) with the addition of approximately 25 g of sodium hydroxide. The material was processed following the protocol of the histology laboratory of the University, the tissue analyzed was paraffin embedded with the part of the center of the bone defect facing downwards, the blocks were divided longitudinally and the sections were made from the center of the defect for histomorphometric and immunohistochemical analyzes. Five serial sections of 4 μm thick were then made with a microtome and captured in a water bath at 40 °C with slides previously prepared with 1 ml solution of Poly-l-Iysine and 10 ml of distilled water. Histological slices were stained with Picrosirius-red. For immunohistochemical analysis, silanized slides were used.
2.4. Histopatological and histomorphometric analysis
Histopathological analysis was performed at 100X and 400X magnification.19 The histomorphometric analysis was performed using a computer image evaluation system, ImageLab 2000 software (Diracon Bio Informática Ltda., Vargem Grande do Sul, SP, Brazil) and by a single examiner, calibrated and blinded for the periods and treatments performed.
Five histological sections were selected from the central area of each specimen's surgical defect. Each section was captured by means of a digital camera coupled to an optical microscope and saved on a computer. In each image, a delimitation of the analyzed area was performed, which corresponded to the region of the calvaria bone where the defect was originally created, called Total Area (TA).20 This area was first determined by the identification of the external and internal surfaces of the original calvaria on the right and left margins of the surgical defect. These surfaces were connected with drawn lines following their respective curvatures. Considering the total length of the histological specimen, 2 mm were measured from the right and left extremities of the specimen, towards the center of the defect, in order to identify the margins of the original surgical defect.
The Newly formed Bone Area (NBA), as well as the areas occupied by the remnants of the implanted materials, called Hydroxyapatite Area (HAA) and Bioactive Glass Area (BGA), were delineated within the limits of TA. The TA value was considered to be 100% of the analyzed area and the values of NBA, HAA and BGA were calculated as a percentage of TA.21 The NBA, HAA and BGA of the respective specimens were evaluated three times by the same examiner and at different days. The three measurements obtained were statistically analyzed and significant level was set at 5% (Kappa test). The mean values were ascertained and statistically compared. Digital images were created with a combination of three smaller images, in view of the impossibility of capturing the entire bone defect in only one image due to the magnification used. The image was created with reference to anatomical structures (such as blood vessels, bone trabeculae) in each of the histological sections.19
2.5. Immunohistochemical analysis
For immunohistochemical reactions, the slides were treated by indirect immunoperoxidase technique employing the primary polyclonal antibodies to Osteocalcin (OC) and Tartrate-Resistant Acid Phosphatase (TRAP) (Goat anti-trap, Goat anti-oc - Santa Cruz Biotechnology, USA). The primary antibodies were diluted in 10 ml of bovine serum albumin (BSA) with 2 ml of diluent (DAKO - Carpinteria, CA, USA) and 120 μL of normal serum (3%, Sigma, CA, USA).
Initially, the histological sections were deparaffinized at 56 °C for 30 min, and a second cycle of deparaffinization started with xylol baths, followed by rehydration in decreasing solutions of alcohols, and finally washed in successive baths in sodium phosphate buffer (SPB). After that, the slides were placed in a solution containing 198 ml of distilled water with 2 ml of 100X citrate buffer at 95 °C for 5 min for antigen retrieval.
The histological sections were treated for blockade of the endogenous peroxidase employing 3% hydrogen peroxide in SPB for 1 h and then washed again with SPB. Endogenous biotin blockade was performed with a solution containing SBP and skimmed milk powder 3% for 1 h. Blocking of non-specific sites was also performed with a solution of bovine serum albumin (BSA) overnight. Thereafter, the sections were incubated with the above-mentioned primary antibodies at room temperature for 18–24 h and washed with SPB. A second incubation was performed using universal biotinylated secondary antibody (Anti-Goat made in Horse, DAKO-Carpinteria, CA, USA) for 2 h at room temperature, followed by a wash with SPB. A third incubation was performed with a solution containing streptavidin conjugated to peroxidase (DAKO - Carpinteria, CA, USA) at room temperature for 2 h.
Immunoperoxidase reaction was performed with buffer (DAB-Substrate, DAKO - Carpinteria, CA, USA) and diaminobenzidine (DAB-Chromogen, DAKO - Carpinteria, CA, USA) for 5 min for OC and 60 s for TRAP, at room temperature. Finally, histological sections were washed several times in SPB and counterstained for 15 s with hematoxylin. All immunoperoxidase reactions were accompanied by a negative control, when primary antibodies were ommited.
Immunoblots located at both margins and at the center of the defect were analyzed at 400X magnification by light microscopy. The expression of OC was measured semiquantitatively and labeled as: absent, mild, moderate and severe.22 TRAP positive cells were counted and the results expressed in units. In order to be considered TRAP-positive cells, mature osteoclasts should contain three or more nuclei.
2.6. Statistical analysis
Statistical analysis of the histometric and immunohistochemical data were performed by BioEstat software (Bioestat Windows 1995 Sonopress, Brazilian Industry, Manaus, AM, Brazil). The hypothesis that there was no statistically significant difference among the different groups and periods was tested. The normality of the data was evaluated by the Shapiro-Wilk test and it was observed that the data were parametric (normal distribution of the data). The parametric analysis of variance ANOVA with Tukey complementation at p < 0.05 was used.
3. Results
3.1. Histopatological and histomorphometric analysis
Regarding the descriptive histological aspects, in group C, in all experimental periods, the surgical defects were not completely filled with bone tissue, presenting only a small amount of newly formed bone within its margins. A thin layer of connective tissue with a chronic inflammatory infiltrate, small number of fibroblasts, and collagen fiber bundles parallel to the wound surface were observed (Fig. 2, Fig. 3).
Fig. 2.
Histological images of the calvaria of groups C, HA and BG. Period of 30 days. A) Group C; B) Group HA; C) Group BG. (Picrosirius-red; 50X magnification).
Fig. 3.
Histological images of the calvaria of one of the animals of groups C, HA and BG. Period of 60 days. A) Group C; B) Group HA; C) Group BG. (Picrosirius-red; 50X magnification).
In groups HA and BG, hydroxyapatite and bioactive glass particles were surrounded by dense connective tissue and a high number of fibroblasts were observed in the majority of the defects. At 30 days, the surrounding connective tissue was disorganized. BG group showed a more orderly orientation of collagen fibers around their particles. In the HA group, cracks of its particles occurred, with invaginations of connective tissue, which was noted in all analyzed periods.
Regarding the newly formed bone area (NBA), the intragroup analysis showed that there was a higher NBA at 60 days compared to 30 days in all experimental groups (p < 0.05). There was a higher percentage of hydroxyapatite (HAA) and bioactive glass (BGA) at 30 days compared to 60 days (p < 0.05).
In the intergroup analysis, Group C presented increased NBA compared to Groups HA and BG at 30 and 60 days postoperatively (p < 0.05). In addition, HA group showed increased NBA compared to BG group at 30 and 60 days postoperatively (p < 0.05) (Table 1).
Table 1.
Mean and standard deviation (M ± SD) of NBA (mm2) according to each group and period.
| Periods Groups | 30 days | 60 days |
|---|---|---|
| C | 25,37 ± 1,05a,b | 32,11 ± 0,78a,b |
| HA | 19,87 ± 1,10a,b | 23,47 ± 1,34a,b |
| BG | 15,61 ± 1.63a,b | 18,76 ± 0,13a,1 |
| N | 30 | 30 |
Statistically significant difference between the periods, same groups (Anova and Tukey with p < 0.05).
Statistically significant difference between groups, same periods (Anova and Tukey with p < 0.05).
Regarding the area occupied by the particles of the biomaterials, it was observed that the group BG presented increased BGA compared to HAA group HA at 30 and 60 days postoperatively (p < 0.05) (Table 2) (Fig. 4).
Table 2.
Mean and standard deviation (M ± SD) of HAA and BGA (mm2) according to each group and period.
| Periods Groups | 30 days | 60 days |
|---|---|---|
| HA | 75,15 ± 0.45a,b | 69,47 ± 1,65a,b |
| BG | 80.25 ± 2,06a,b | 77.11 ± 1,62a,b |
| N | 20 | 20 |
Statistically significant difference between the periods, same groups (Anova and Tukey with p < 0.05).
Statistically significant difference between groups, same periods (Anova and Tukey with p < 0.05).
Fig. 4.
Physical characteristics of the biomaterials Hydroxyapatite and Bioactive Glass. NBA – New bone area, HAA – Hydroxyapatite area, BGA – Bioactive Glass area A) Group HA- 30 days; B) Group HA- 60 days; C) Group BG - 30 days and D) Group BG - 60 days. (Picrosirius-red; 100X magnification).
3.2. Immunohistochemical analysis
Regarding the number of TRAP positive cells, the intragroup analysis showed that the BG group had a higher number of cells at 30 days compared to 60 days (p < 0.05). In the intergroup analysis, the HA group showed a higher number of TRAP positive cells compared to groups C and BG at 30 and 60 days postoperatively (p < 0.05). In addition, group BG presented a higher number of TRAP-positive cells compared to group C at 30 postoperative days (p < 0.05). (Table 3) (Fig. 5).
Table 3.
Mean and standard deviation (M ± SD) of TRAP expression (un) according to each group and period.
| Periods Groups | 30 days | 60 days |
|---|---|---|
| C | 1,00 ± 1,50b | 0,16 ± 0,21b |
| HA | 6,90 ± 0,31b | 6,10 ± 1,45b |
| BG | 2,70 ± 1,63a,b | 0,10 ± 0,31a,b |
| N | 30 | 30 |
Statistically significant difference between the periods, same groups (Anova and Tukey with p < 0.05).
Statistically significant difference between groups, same periods (Anova and Tukey with p < 0.05).
Fig. 5.
anti-TRAP immunohistochemistry (Arrows) in groups C, HA and BG at 30 and 60 days. A) Group C, 30 days; B) Group HA, 30 days; C) Group BG, 30 days; D) Group C, 60 days; E) Group HA, 60 days; F) Group BG, 60 days. (400x magnification).
Considering OC expression, it was observed that group C did not present expression at day 30, while groups HA and BG showed high and moderate expression, respectively (Fig. 6). At 60 days, groups C and BG showed a slight expression of OC and group HA a moderate expression of OC.
Fig. 6.
anti-OC immunohistochemistry (Arrows) in groups HA and BG at 30 days. A) Group HA; B) Group BG. (400x magnification).
4. Discussion
Critical bone defects are those that do not heal spontaneously during the life of the animal or during the time of the experiment, however, new bone formation can be seen.23 In critical defects, bone neoformation will occur from their margins, however, there will never be complete closure by bone tissue. A bone defect of 8 mm in diameter in the skull of the rat is considered critical because it does not close completely, and represents a greater biological challenge than the defects of 5 and 6 mm.24 This was decisive for choosing the experimental model, in order to assess new bone formation capacity of the tested biomaterials. This was demonstrated by the results, when we observed a new bone formation restricted to the margins of the defect in 30 days in Group C, with a thin layer of connective tissue between them. At 60 days, a greater amount of newly formed bone tissue was observed, however, without the complete closure of the defect. The results also demonstrate a significant higher NBA in group C compared to groups HA and BG, corroborating other studies in the literature.25, 26, 27 Cardozo et al.25 found no statistical difference considering new bone formation when comparing blood clot, Biogran® and Perioglas® in 8 mm calvaria defects of rats, at 15, 30 and 60 days. The authors correlated these findings with the limited amount of osteoblastic cells in the calvaria of adult rats, requiring the recruitment of mesenchymal cells from the perivascular tissue of the dura mater for complete bone regeneration. In another study, with 5 mm bone defects in the calvaria of rats, Nagata et al.26 found a greater amount of newly formed bone in the defect filled with blood clot, in comparison with BG with or without the use of membrane, after 4 and 12 weeks. The authors believe that these findings are due to the low rate of BG resorption, by the presence of remaining BG particles. On the other hand, other authors1 demonstrated increased bone formation with synthetic biomaterials compared to blood clot. The differences in the results may be related to the characteristics of the tested materials such as chemical composition, surface size, roughness and crystallinity.
The biomaterials used in the study provided the necessary scaffold for bone repair, with new bone formation occurring in both HA and BG groups, at 30 days, limited to the original margins of the surgical defect. At 60 days, increased bone formation occurred, with the presence of bone tissue in the center of the defect, characterized by “isolated islets” (Fig. 7, a). In some samples, there was limited high cellular woven bone, that could also be seen around the particles of the biomaterial (Fig. 7, b). These results demonstrated the phenomenon of osteoconduction, which refers to the biomaterial's ability to boost the development of new bone tissue through its support matrix.28 However, for this to occur, it is necessary that the biomaterial resorbs, providing space for bone formation,29 and this required a longer evaluation period, as it was done in the present study. These factors may also explain the increased NBA found in group C and the immunohistochemical results that revealed a higher concentration of positive TRAP cells around biomaterials, which may result in space for the new bone. Corroborating with our findings, a study27 using two different degrees of hydroxyapatite crystallinity, did not find increased new bone in groups treated with biomaterials, compared to the control group (blood clot), at 30 and 60 days. Thus, in order that new bone is formed, these biomaterials must be first resorbed, requiring a longer time for bone repair, compared to areas treated with blood clot. If a longer study period was carried out, the results could be different.
Fig. 7.
NBA – New bone area, HAA – Hydroxyapatite area, BGA – Bioactive Glass area. A) “Islets” of bone tissue being formed in the middle of the bone defect at 60 days, Group HA.; B) Formation of bone tissue among the particles of Bioactive Glass at 60 days, Group BG. (Picrosirius-red; 50X magnification).
Regarding the area of particles occupied by the remaining biomaterials’ particles, it was observed that group BG presented higher BGA compared to group HA in all periods. The BG particles also showed a more well-defined morphology and a larger size in relation to those of HA, and this difference in the resorption rate may be related to the larger particle size, higher crystallinity, less roughness and less purity in the composition of the BG.30 The conversion rate of BG to HA13 could also partly explain these findings, as HA exhibits a faster resorption rate compared to BG, leading to faster bone formation; although, BG maintains a greater volume than HA, due to its lower solubility rate.
Biomaterials with characteristics similar to BG, constitutionally based on calcium phosphate, are osteoconductive, biocompatible31 and biodegradable,32 can be indicated for defects in which rapid bone formation is not the predominant factor. This biological process was demonstrated in this study, which showed a higher amount of BGA particles compared to HAA, in all periods. The maintenance of this space is essential due to aesthetic or functional aspects, in cases of filling gaps between the implant and the oral bone, in immediate procedures after extraction or in bone reconstructions.33 However, the size of the particles can influence the results, and synthetic bone substitutes of nanometric particles have shown better properties for bone regeneration and accelerated wound healing. However, they present increased fragility and less ability to resist mechanical forces.28
The body's ability to resorb the tested biomaterials during the repair process was observed through the largest remaining area of hydroxyapatite (HAA) and bioactive glass (BGA) in bone defects at 30 days compared to 60 days. In addition, the higher rate of HA resorption can be explained by the higher number of TRAP-positive cells in this group, demonstrating a more intense cellular response in the body when in contact with the HA Group, and a milder response when in contact with the BG Group. Van Gestel et al.,29 suggested that the higher expression of OC in samples from the HA Group may indicate a more advanced stage in bone remodeling process, and that the silica layer under calcium phosphate in the BG Group prevents osteoclasts from performing its rapid degradation. These phenomena were confirmed through our histometric results, with a greater area of new bone formation in Group HA compared to Group BG, at 30 and 60 days. These findings corroborate with other studies in the literature,17,22,34 which associated greater expression of OC, with improved formation of bone tissue in critical defects in rats' calvaria.
Although there is a considerable physiological, cellular and molecular similarity between animal and human models, there are limitations in the use of the former, and extrapolation of the results should be performed with caution. In addition, the oral cavity represents a unique environment in our body, belonging to the digestive system and which allows access to the airways, providing favorable conditions for the proliferation of various microorganisms.35 This would be another factor to be analyzed before applying our results.
Therefore, the application of such conditions in humans becomes extremely difficult, taking into account their anatomical and physiological differences in organ function, metabolism, drug absorption, genetic mechanisms, among others.36 Therefore, the present study presents limitations in relation to the actual application of these results in dental practice. However, the new methods proposed to replace animal experimentation also have their flaws. The results found in our study demonstrated that low resorption rate of the tested biomaterials requires future long-term research in the literature, in order to better evaluate the characteristics of HAP-91® Hydroxyapatite and the novel Bioactive Glass compound as substitute biomaterials for the treatment of bone defects.
5. Conclusion
From these results, we can conclude that the resorption rate of hydroxyapatite is higher than the novel bioactive glass, which maintained significant higher volume until the last experimental period. Both of the tested biomaterials acted as osteoconductors during bone repair, and their physical characteristics importantly influenced this process.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
None of the authors have any potential conflicts of interest to declare.
Acknowledgment
We thank the company JHS BIOMATERIAIS for kindly ceding the biomaterials used in this research.
Research conducted at the Federal University of Alfenas - Unifal/MG. The experimental part was carried out in the Central Bioterium of the institution. Histological processing in the Department of Structural Biology - Unifal/MG. Immunohistochemical analysis in the Department of Basic Sciences of Paulista State University “Júlio de Mesquita Filho” Unesp- Campus of Araçatuba. Analysis of the results and statistics in the Department of Clinics and Surgery Unifal/MG.
Contributor Information
Eduardo Quintão Manhanini Souza, Email: eduardoquintao@hotmail.com.
Leandro Araújo Fernandes, Email: leandro.fernandes@unifal-mg.edu.br.
References
- 1.Brunello G., Panda S., Schiavon L., Sivolella S., Biasetto L., Del Fabbro M. The impact of bioceramic scaffolds on bone regeneration in preclinical in vivo studies: a systematic review. Materials. 2020;13(7):1500. doi: 10.3390/ma13071500. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Fernández R.F., Bucchi C., Navarro P., Beltrán V., Borie E. Bone grafts utilized in dentistry: an analysis of patients' preferences. BMC Med Ethics. 2015;16(1):71. doi: 10.1186/s12910-015-0044-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Doi K., Kubo T., Makihara Y. Osseointegration aspects of placed implant in bone reconstruction with newly developed block-type interconnected porous calcium hydroxyapatite. J Appl Oral Sci. 2016;24(4):325–331. doi: 10.1590/1678-775720150597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Gao R., Watson M., Callon K.E. Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis. J Tissue Eng Regen Med. 2018;12(1):e620–626. doi: 10.1002/term.2348. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bellucci D., Braccini S., Chiellini F., Balasubramanian P., Boccaccini A.R., Cannillo V. Bioactive glasses and glass‐ceramics versus hydroxyapatite: comparison of angiogenic potential and biological responsiveness. J Biomed Mater Res. 2019;107(12):2601–2609. doi: 10.1002/jbm.a.36766. [DOI] [PubMed] [Google Scholar]
- 6.Sarkar S.K., Lee B.T. Hard tissue regeneration using bone substitutes: an update on innovations in materials. Korean J Intern Med. 2015;30(3):279–293. doi: 10.3904/kjim.2015.30.3.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Schmitt C.M., Doering H., Schmidt T., Lutz R., Neukam F.W., Schlegel K.A. Histological results after maxillary sinus augmentation with Straumann® BoneCeramic, Bio-Oss®, Puros®, and autologous bone. A randomized controlled clinical trial. Clin Oral Implants Res. 2013;24(5):576–585. doi: 10.1111/j.1600-0501.2012.02431.x. [DOI] [PubMed] [Google Scholar]
- 8.Calasans-Maia M.D., de Melo B.R., Alves A.T.N.N. Cytocompatibility and biocompatibility of nanostructured carbonated hidroxyapatite spheres for bone repair. J Appl Oral Sci. 2015;23(6):599–608. doi: 10.1590/1678-775720150122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Salgado P.C., Sathler P.C., Castro H.C. Bone remodeling, biomaterials and technological applications: revisiting basic concepts. J Biomaterials Nanobiotechnol. 2011;2:318–328. [Google Scholar]
- 10.Larsson S. Calcium phosphates: what is the evidence? J Orthop Trauma. 2010;24(3):41–45. doi: 10.1097/BOT.0b013e3181cec472. [DOI] [PubMed] [Google Scholar]
- 11.Dittler M.L., Unalan I., Grünewald A. Bioactive glass (45S5)-based 3D scaffolds coated with magnesium and zinc-loaded hydroxyapatite nanoparticles for tissue engineering applications. Colloids Surf B Biointerfaces. 2019;182 doi: 10.1016/j.colsurfb.2019.110346. [DOI] [PubMed] [Google Scholar]
- 12.Bi L., Jung S., Day D. Evaluation of bone regeneration, angiogenesis, and hydroxyapatite conversion in critical-sized rat calvarial defects implanted with bioactive glass scaffolds. J Biomed Mater Res. 2012;100A(12):3267–3275. doi: 10.1002/jbm.a.34272. [DOI] [PubMed] [Google Scholar]
- 13.Bi L., Rahaman M.N., Day D.E. Effect of bioactive borate glass microstructure on bone regeneration, angiogenesis, and hydroxyapatite conversion in a rat calvarial defect model. Acta Biomater. 2013;9(8):8015–8026. doi: 10.1016/j.actbio.2013.04.043. [DOI] [PubMed] [Google Scholar]
- 14.Deliormanlı A.M., Liu X., Rahaman M.N. Evaluation of borate bioactive glass scaffolds with different pore sizes in a rat subcutaneous implantation model. J Biomater Appl. 2014;28(5):643–653. doi: 10.1177/0885328212470013. [DOI] [PubMed] [Google Scholar]
- 15.Fu Q., Rahaman M.N., Fu H., Liu X. Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. I. Preparation and in vitro degradation. J Biomed Mater Res. 2010;95:164–171. doi: 10.1002/jbm.a.32824. [DOI] [PubMed] [Google Scholar]
- 16.Skallevold H., Rokaya D., Sultan Z., Zafar M.S. Bioactive glass applications in dentistry. Int J Mol Sci. 2019;20(23):5960. doi: 10.3390/ijms20235960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Nagata M., Messora M., Okamoto R. Influence of the proportion of particulate autogenous bone graft/platelet-rich plasma on bone healing in critical-size defects: an immunohistochemical analysis in rat calvaria. Bone. 2009;45(2):339–345. doi: 10.1016/j.bone.2009.04.246. [DOI] [PubMed] [Google Scholar]
- 18.Garcia V.G., Sahyon A.S., Longo M. Effect of LLLT on autogenous bone grafts in the repair of critical size defects in the calvaria of immunosuppressed rats. J Cranio-Maxillo-Fac Surg. 2014;42(7):1196–1202. doi: 10.1016/j.jcms.2014.02.008. [DOI] [PubMed] [Google Scholar]
- 19.Bosco A.F., Faleiros P.L., Carmona L.R. Effects of low-level laser therapy on bone healing of critical-size defects treated with bovine bone graft. J Photochem Photobiol, B. 2016;163:303–310. doi: 10.1016/j.jphotobiol.2016.08.040. [DOI] [PubMed] [Google Scholar]
- 20.Bizenjima T., Takeuchi T., Seshima F., Saito A. Effect of poly (lactide-co-glycolide) (PLGA)-coated beta-tricalcium phosphate on the healing of rat calvarial bone defects: a comparative study with pure-phase beta-tricalcium phosphate. Clin Oral Implants Res. 2016;27(11):1360–1367. doi: 10.1111/clr.12744. [DOI] [PubMed] [Google Scholar]
- 21.Park J.W., Kim J.M., Lee H.J., Jeong S.H., Suh J.Y., Hanawa T. Bone healing with oxytocin-loaded microporous b-TCP bone substitute in ectopic bone formation model and critical-sized osseous defect of rat. J Clin Periodontol. 2014;41(2):181–190. doi: 10.1111/jcpe.12198. [DOI] [PubMed] [Google Scholar]
- 22.Marques L., Holgado L.A., Francischone L.A., Ximenez K.P.B., Okamoto R., Kinoshita A. New LLLT protocol to speed up the bone healing process- histometric and immunohistochemical analysis in rat calvarial bone defect. Laser Med Sci. 2015;30(4):1225–1230. doi: 10.1007/s10103-014-1580-x. [DOI] [PubMed] [Google Scholar]
- 23.Calori G.M., Mazza E., Colombo M., Ripamonti C. The use of bone-graft substitutes in large bone defects: any specific needs? Injury. 2011;42(2):S56–S63. doi: 10.1016/j.injury.2011.06.011. [DOI] [PubMed] [Google Scholar]
- 24.Vajgel A., Mardas N., Farias B.C., Petrie A., Cimões R., Donos N. A systematic review on the critical size defect model. Clin Oral Implants Res. 2014;25(8):879–893. doi: 10.1111/clr.12194. [DOI] [PubMed] [Google Scholar]
- 25.Cardoso A.K., Barbosa Ade A., Jr., Miguel F.B. Histomorphometric analysis of tissue responses to bioactive glass implants in critical defects in rat calvaria. Cells Tissues Organs. 2006;184(3-4):128–137. doi: 10.1159/000099619. [DOI] [PubMed] [Google Scholar]
- 26.Nagata M.J., Furlaneto F.A., Moretti A.J. Bone healing in critical-size defects treated with new bioactive glass/calcium sulfate: a histologic and histometric study in rat calvaria. J Biomed Mater Res B Appl Biomater. 2010;95(2):269–275. doi: 10.1002/jbm.b.31710. [DOI] [PubMed] [Google Scholar]
- 27.Conz M.B., Granjeiro J.M., Soares G.A. Hydroxyapatite crystallinity does not affect the repair of critical size bone defects. J Appl Oral Sci. 2011;19(4):337–342. doi: 10.1590/S1678-77572011005000007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Tao O., Wu D.T., Pham H.M., Pandey N., Tran S.D. Nanomaterials in craniofacial tissue regeneration: a review. Appl Sci. 2019;9(2):317. [Google Scholar]
- 29.Van Gestel N.A.P., Schuiringa G.H., Hennissen J.H.P.H. Resorption of the calcium phosphate layer on S53P4 bioactive glass by osteoclasts. J Mater Sci Mater Med. 2019;30(8):94. doi: 10.1007/s10856-019-6295-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Hoppe A., Güldal N.S., Boccaccini A.R. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials. 2011;32(11):2757–2774. doi: 10.1016/j.biomaterials.2011.01.004. [DOI] [PubMed] [Google Scholar]
- 31.Sawant K., Pawar A.M. Bioactive glass in dentistry: a systematic review. Saudi J Oral Sci. 2020;7(1):3–10. [Google Scholar]
- 32.Nour S., Baheiraei N., Imani R. Bioactive materials: a comprehensive review on interactions with biological microenvironment based on the immune response. J Bionic Eng. 2019;16:563–581. [Google Scholar]
- 33.Takauti C.A.Y., Futema F., Junior R.B.B., Abrahao A.C., Costa C., Queiroz C.S. Assessment of bone healing in rabbit calvaria grafted with three different biomaterials. Braz Dent J. 2014;25(5):379–384. doi: 10.1590/0103-6440201302383. [DOI] [PubMed] [Google Scholar]
- 34.Alpan A.L., Toker H.I., Ozer H. Ozone therapy enhances osseous healing in rats with diabetes with calvarial defects: a morphometric and immunohistochemical study. J Periodontol. 2016;87(8):982–989. doi: 10.1902/jop.2016.160009. [DOI] [PubMed] [Google Scholar]
- 35.Krishnan K., Chen T., Paster B.J. A practical guide to the oral microbiome and its relation to health and disease. Oral Dis. 2017;23(3):276–286. doi: 10.1111/odi.12509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Kitada M., Ogura Y., Koya D. Rodent models of diabetic nephropathy: their utility and limitations. Int J Nephrol Renovascular Dis. 2016;9:279–290. doi: 10.2147/IJNRD.S103784. [DOI] [PMC free article] [PubMed] [Google Scholar]