Comparison of Particulate, Block and Putty Forms of β-tricalcium Phosphate-Based Synthetic Bone Grafts on Rat Calvarium Model

Abstract

Purpose

Bone augmentation is a necessity for atrophied alveolar ridge prior to dental implant placement. Various bone graft types and forms with different characteristics are available in the market for alveolar augmentation. Beta tricalcium phosphate (β-TCP) is a synthetic biomaterial known as the oldest type of calcium phosphate. Studies comparing particulate, block or putty grafts are very limited. The aim of this study was to compare the particulate, block and putty forms of the same β-TCP bone graft and analyze the efficiency in critical size calvarium defects.

Material and Methods

Twenty male Wistar-Albino rats were employed for the study. Four bicortical bone defects with 5 mm diameter were created on each rat calvarium, and three defects were filled with particulate, block or putty β-TCP graft and one defect was left empty. The animals were killed after 8 weeks. New bone formation, residual graft, loose connective tissue, condensed mesenchyme, alkaline phosphatase, proliferating cell nuclear antigen, osteocalcin were measured on the specimens.

Results

Compared to block and putty forms, significantly higher new bone formation and least residual graft were observed in the particulate graft group. The residual graft was significantly higher in the block graft group than both the particulate and the putty groups. The cellular immunoreactivity of the samples in the particulate graft group was significantly higher. There was no significant difference between putty and block graft groups.

Conclusion

Bone regeneration is significantly affected by the form of β-TCP bone graft, and the particulate form was the most successful in our study.

Introduction

Various factors such as bone quality and volume, aesthetics and occlusion should be taken into consideration for a successful implant-supported restoration. Correct implant placement is especially important for ideal prosthetic restoration and oral hygiene [1]. The well-healed alveolar bone after tooth extraction provides mostly favorable conditions for dental implant placement in the correct position and sizes [2]. Severe bone loss may occur after tooth loss especially caused by chronic periodontitis and in the edentulous sites not rehabilitated for a long time. Therefore, bone augmentation is frequently required prior to implant surgery [3].

To achieve a successful augmentation of deficient alveolar ridges for future implant placement, grafting applications should be based on such biological principles: biological acceptability, primary wound closure, absence of infection, angiogenesis, space creation and maintenance, graft immobilization [4].

Autografts are considered as the gold standard because of their osteogenic potential. However, autografts have disadvantages such as donor site morbidity, high resorption rates, limited amount and increased surgical time [5]. Allografts are commonly used materials with osteoinductive features and without donor site morbidity. The procedures applied to reduce the infection transmission risks of allografts change their structural integrity and reduce biological activity. In addition, their standardization is problematic in experimental and clinical studies because they are not uniform [6]. Osteogenic capacities of xenografts obtained from various origins, mostly bovine, are eliminated due to the aggressive methods applied to reduce the foreign body reaction [7]. The disadvantages of autografts such as limited availability and the disease transmission risk of allografts and xenografts have encouraged the production of synthetic graft materials reproducible of their chemical composition and porous architecture [8].

Beta tricalcium phosphate (β-TCP) is the oldest type of calcium phosphate known as a bone graft. In 1920, Albee and Morrison reported the bone ratio increase when injected into the segmental bone defects [9]. The porous structure resembling trabecular bone allows the migration of osteogenic cells, nutrients, growth factors, capillaries, phagocytic cells and vascularization [10]. β-TCP substitutes are nontoxic, noncarcinogenic, immunologically inert materials resorb and replaced by remodeled bone in 13–20 weeks [10, 11]. It can be prepared in a wide variety of forms and sizes, in solid or porous structure [8].

Particulate bone grafts are suitable for filling the extraction sockets and three-wall infrabony defects with ease of use; however, they are difficult to use in defects with fewer bone walls and the graft can spread out of the defect site. Due to their low mechanical stability, complex techniques are often required for the graft immobilization and space maintenance using tools such as pin fixed membranes, tenting screws and titanium mesh [12]. Providing high mechanical stability, block grafts are another alternative for bone augmentation requiring contact surface adaptation and reliable fixation to the receiving area [13]. They are often used with particulate grafts to avoid dead space [14].

In order to provide minimally invasive bone regeneration, calcium phosphate-based injectable bone preparations have been developed. These moldable and in situ hardening products have been shown to act as a solid skeleton for bone formation. In a lateral sinus floor elevation animal model, injectable grafts have been shown to provide sufficient stability for covering the sinus membrane ruptures without the need of placing a barrier membrane [15]. However, materials such as bioceramics, large polymers and chitosan can be associated with various problems such as poor plasticity, leakage to surrounding tissues, low mechanical strength and poor degradability [16].

Various bone graft types and forms with different characteristics are available in the market for alveolar augmentation. Bone grafts in different forms create various ease-of-use advantages and disadvantages for different defect types and augmentation needs. However, the bone regeneration efficiencies of different forms of β-TCP bone grafts with proven osteoinductive and osteoconductive effects have not been compared before.

The aim of this study was to compare the particulate, block and putty forms of the same β-TCP bone graft and analyze the efficiency in critical size rat calvarium defects.

Materials and Methods

This study was carried out with the approval of local ethics committee in Ege University Laboratory Animals Application and Research Center (No:2018–046) in accordance with European Directive 2010/63/EU for conducting animal experiments. A total of 20 male Wistar-Albino rats of 12-week weighing 400–450 gr were determined by veterinary control and employed for the experimental procedure. Animals housed in wire cages in groups of 2–3 per cage with regular rat pellet and water provided ad libitum at an environment with 22 ± 2 ºC, 30–45% relative humidity and 12-h artificial day-night cycle.

The critical size defect filling material was divided into four groups, as follows: group 1, control; group 2, β-TCP particulate graft; group 3, β-TCP block graft; group 4 β-TCP putty graft.

Under general anesthesia with ketamine (60 mg/kg) and xylazine (7 mg/kg), a full-thickness flap was elevated via an incision approximately 1.5 cm down to periosteum over the scalp to reach the calvarium. Four bicortical bone defects with 5 mm diameter were created by using trephine burs on each rat calvarium. Three defects were filled with particulate (Powerbone® Granule 1–2 mm—1 cc), block (Powerbone® Stick 4 × 20 mm 2,60 cc) or putty graft (Powerbone® Putty—1 cc) produced by Bonegraft Biological Materials®, and one defect was left with no grafting application as control. Three defects and one defect (control) were left with no grafting application (Fig. 1). Subjects were given enrofloxacin and meloxicam for 5 days to control pain and infection. The animals were killed 8 weeks after the operation. Histopathological examinations were performed at the Department of Histology and Embryology at Izmir Katip Çelebi University.

Fig. 1.

A Defects with 5 mm diameter are created with trephine bur under saline irrigation. B Defects are shown prior to augmentation. C Three defects are filled with bone graft and one left empty as control. D Calvarium is exposed after 8-week healing

All samples were fixed in 10% buffered formalin for 24 h and then decalcified in 10% formic acid for 6 days and processed for embedding in paraffin using routine protocol. Sections 5 µm thick were cut on a rotary microtome (Leica RM2245) and stained with hematoxylin and eosin (H&E). New bone formation, residual graft, loose connective tissue, condensed mesenchyme were measured on the specimens. Also additional sections were used for immunohistochemical staining for alkaline phosphatase (ALP), osteocalcin (OCN) and proliferating cell nuclear antigen (PCNA).

Tissue samples were stored at 60 °C overnight and then were deparaffinized by xylene for 30 min. After dehydration of the sections with ethanol, they were washed with distilled water. The tissues were then treated with 2% trypsin (ab970, Abcam, Cambridge, UK) at 37 °C for 15 min and incubated in 3% H2O2 solution for 15 min to inhibit endogenous peroxidase activity. Then, sections were incubated with anti-ALP (sc-166261, Santa Cruz Biotechnology, Inc.), anti OCN (sc-365797, Santa Cruz Biotechnology, Inc.) and anti PCNA (sc-56, Santa Cruz Biotechnology, Inc.) primer antibodies in a 1/100 dilution for 18 h at + 4 °C. They were then given an additional three 5 washes in PBS, followed by incubation with biotinylated Ig G and administration of streptavidin peroxidase (Histostain Plus kit cat no: 85–9043, Invitrogen). After washing the secondary antibody with PBS three times for 5 min, the sections were stained with DAB substrate system containing diaminobenzidine (DAB-plus substrate kit, Invitrogen) to detect the immunoreactivity and then stained with Mayer's hematoxylin (72804E, Microm, Walldorf, Germany) for counterstaining. They were covered with mounting medium (Clear Mount, Mounting Medium Ref: 008,110 Invitrogen, USA) and observed with light microscopy (Olympus BX-43, Tokyo, Japan). Immunostaining for ALP, OCN, PCNA immunostaining intensity was categorized by the following scores: 0 (negative staining), 1 (mild staining), 2 (moderate staining) and 3 (intense staining).

Statistics were analyzed with the aid of SPSS software (version 22, SPSS Inc, Chicago, IL). Tukey’s honestly significantly differenced (HSD) test is used to find differences between groups after the ANOVA test; P < 0.05 was considered a statistically significant difference.

Results

In the samples, no foreign body reaction or necrosis was observed. Successful bone regeneration is observed in all different forms of β-TCP. Osteocytes nuclei which show them viable within the lacunae were evident in the sections where the newly formed bone was seen. Osteoblasts were present throughout the newly formed bone. The mesenchymal density connective tissue, especially in particulate and putty groups, seemed to be going from osteoprogenitor cells to the formation of new bone. The bone graft was distinguished by the empty lacunae and a more basophilic staining intensity (Fig. 2).

Fig. 2.

Hematoxylin & eosin stained view of all groups X100 (A empty, B particle bone graft, C block graft, D putty graft). a vital bone, b graft, c condensed mesenchyme, ob osteoblast, cell arrow: surgical border

The particulate graft group samples were well vascularized with the newly formed bone and a connective tissue with bone marrow. Compared to block and putty groups, significantly higher new bone formation and significantly lower residual graft were observed in the particulate group. The percentage of condensed mesenchyme was significantly higher, and loose connective tissue was significantly lower than control group. However, no significant difference was found compared to block and putty groups.

The block graft group showed significantly lower new bone formation compared to particulate group. However, no significant difference was found compared to putty group. The residual graft was significantly higher in the block group than both the particulate and the putty groups. No significant difference was found between experimental groups in terms of condensed mesenchyme and loose connective tissue.

The putty graft group showed no significant difference in the new bone formation compared to block group; however, it was significantly lower compared to particulate group. No significant difference was found in the residual graft compared to particulate group; however, it was significantly lower than block group.

The cellular immunoreactivity of the samples against ALP, OCN and PCNA was significantly higher in the experimental groups compared to control group. All three parameters were significantly higher in the particulate graft group compared to block and putty groups. However, no significant difference was found in all three parameters between putty and block graft groups (Tables 1, 2).

Table 1.

Mean scores measured for variables

	Control	Particulate	Block	Putty
New bone formation	15.00	56.15	35.38	36.15
Residual graft	0.00	9.85	34.62	14.23
Condensed mesenchyme	11.54	23.85	21.92	29.62
Loose connective tissue	35.00	14.38	8.08	20.00
ALP	0.31	1.23	0.69	0.62
OCN	0.12	0.96	0.62	0.58
PCNA	0.00	0.96	0.62	0.58

Table 2.

Comparisons between groups

	Mean difference	Std. error	p value
New bone formation
Control
Particulate	− 41.154	5.076	0.000*
Block	− 20.385	5.076	0.001*
Putty	− 21.154	5.076	0.001*
Particulate
Control	41.154	5.076	0.000*
Block	20.769	5.076	0.001*
Putty	20	5.076	0.001*
Block
Control	20.385	5.076	0.001*
Particulate	− 20.769	5.076	0.001*
Putty	− 0.769	5.076	0.999
Putty
Control	21.154	5.076	0.001*
Particulate	− 20	5.076	0.001*
Block	0.769	5.076	0.999
Residual graft
Control
Particulate	− 9.846	4.175	0.099
Block	− 34.615	4.175	0.000*
Putty	− 14.231	4.175	0.007*
Particulate
Control	9.846	4.175	0.099
Block	− 24.769	4.175	0.000*
Putty	− 4.385	4.175	0.721
Block
Control	34.615	4.175	0.000*
Particulate	24.769	4.175	0.000*
Putty	20.385	4.175	0.000*
Putty
Control	14.231	4.175	0.007*
Particulate	4.385	4.175	0.721
Block	− 20.385	4.175	0.000*
Condensed mesenchyme
Control
Particulate	− 12.308	4.485	0.041*
Block	− 10.385	4.485	0.109
Putty	− 18.077	4.485	0.001*
Particulate
Control	12.308	4.485	0.041*
Block	1.923	4.485	0.973
Putty	− 5.769	4.485	0.576
Block
Control	10.385	4.485	0.109
Particulate	− 1.923	4.485	0.973
Putty	− 7.692	4.485	0.327
Putty
Control	18.077	4.485	0.001*
Particulate	5.769	4.485	0.576
Block	7.692	4.485	0.327
Loose connective tissue
Control
Particulate	20.615	6.275	0.010*
Block	26.923	6.275	0.000*
Putty	15	6.275	0.093
Particulate
Control	− 20.615	6.275	0.010*
Block	6.308	6.275	0.747
Putty	− 5.615	6.275	0.808
Block
Control	− 26.923	6.275	0.000*
Particulate	− 6.308	6.275	0.747
Putty	− 11.923	6.275	0.242
Putty
Control	− 15	6.275	0.093
Particulate	5.615	6.275	0.808
Block	11.923	6.275	0.242
ALP
Control
Particulate	− 0.923	0.132	0.000*
Block	− 0.385	0.132	0.027*
Putty	− 0.308	0.132	0.105
Particulate
Control	0.923	0.132	0.000*
Block	0.538	0.132	0.001*
Putty	0.615	0.132	0.000*
Block
Control	0.385	0.132	0.027*
Particulate	− 0.538	0.132	0.001*
Putty	0.077	0.132	0.937
Putty
Control	0.308	0.132	0.105
Particulate	− 0.615	0.132	0.000*
Block	− 0.077	0.132	0.937
OCN
Control
Particulate	− 0.846	0.103	0.000*
Block	− 0.5	0.103	0.000*
Putty	− 0.462	0.103	0.000*
Particulate
Control	0.846	0.103	0.000*
Block	0.346	0.103	0.008*
Putty	0.385	0.103	0.003*
Block
Control	0.5	0.103	0.000*
Particulate	− 0.346	0.103	0.008*
Putty	0.038	0.103	0.982
Putty
Control	0.462	0.103	0.000*
Particulate	− 0.385	0.103	0.003*
Block	− 0.038	0.103	0.982
PCNA
Control
Particulate	− 0.962	0.094	0.000*
Block	− 0.615	0.094	0.000*
Putty	− 0.577	0.094	0.000*
Particulate
Control	0.962	0.094	0.000*
Block	0.346	0.094	0.003*
Putty	0.385	0.094	0.001*
Block
Control	0.615	0.094	0.000*
Particulate	− 0.0346	0.094	0.003*
Putty	0.038	0.094	0.976
Putty
Control	0.577	0.094	0.000*
Particulate	− 0.385	0.094	0.001*
Block	− 0.038	0.094	0.976

Statistical analysis = Tukey’s honestly significantly differenced (HSD) test

*p < 0.05

Discussion

β-TCP is a biocompatible synthetic porous material with osteoconductive properties with varying form and porosity depends on the processing condition [11]. The success of β-TCP bone graft materials in implantology has been proven by numerous clinical studies [17].

Particulate bone grafts are shown to provide higher percentage of newly formed bone and faster resorption of the graft material in accordance with our study [13]. Physical properties of bone grafts such as porosity, surface structure and shape have been identified as regulators of tissue reaction, material degradation and regenerative potential [18]. Although particle size that directly affects degradation is also a major factor, there is no consensus on the ideal size. There are studies showing that the application of granules smaller than 100 µm is more successful, as well as studies showing that diameters ranging from 100 to 1000 µm provide a higher level of regeneration [12, 19]. In their study comparing equivalent grafts containing biphasic calcium phosphate with particle sizes in the range of 400–700 µm and 500–1000 µm, Barbeck et al. found that the smaller particle group supports fast implant bed vascularization [12]. The particle and block bone grafts used in our study contain particles of different sizes ranging from 250 to 7000 µm to promote the interlocking of polygonal structures, but this information is not available for putty grafts. It can be thought that the fillers used to obtain the putty form increase the interparticle spaces and show lower efficiency in early-stage new bone formation.

Osteogenic differentiation proceeds through different developmental stages characterized by specific markers including early markers such as ALP and late markers such as OCN [20]. ALP is a key dephosphorylating enzyme expressed by osteoblasts to turnover expressed collagen into a form that is amenable for bone matrix formation, and OCN is a key noncollageneous protein that binds extracellular calcium to bone matrix [21]. In our study, the reason for the immunohistochemical bone marker values detected higher in the particulate bone group can be interpreted as the greater surface area being suitable for a faster microvascularization and the increase in the rate of resorption. The highest residual graft rate in the block graft group indicates that the slowest resorption is in this graft type and low marker values are consistent with this result.

Studies comparing particulate, block or putty grafts are very limited. In their study comparing the particulate and putty forms of the same biphasic calcium phosphate graft with the sinus lifting procedure performed in sheep, Wildburger et al. found that the putty graft provided a higher rate of new bone formation than the particulate graft, but there was a significant decrease in augmentation volume [22]. In the study of Knabe et al. comparing the particulate and putty tricalcium phosphate forms with sinus lifting in 7 patients it was found that the hyaluronic acid containing putty graft provides a higher rate of new bone formation than the particulate graft after 6 months. Although it seems that these results contradict our study, hyaluronic acid is not included in Powerbone® Putty graft, so it is not adequate to compare regeneration potential of different material-based bone grafts. Platelet-rich fibrin (PRF) concentrates are alternatives to obtain putty form from the particulate graft. In the study where Clark et al. applied freeze-dried allograft particles together with advanced PRF to the extraction socket, improved dimensional stability and vital bone formation were found. However, this successful technique requires taking blood from the patient and using special equipment [23]. In another study, beta tricalcium phosphate block and putty grafts were compared in a defect model, higher bone formation was observed in the block graft group in the early period, but there was no significant difference in the long term. The osteoconductive feature of the grafts was similar [14]. In our study, the block and putty graft groups showed similar new bone formation rates and immunoreactivity. According to these findings, β-TCP-based putty graft can be preferred over block graft, considering its manipulation and easy-to-use advantages.

As a conclusion, bone regeneration is significantly affected by the form of β-TCP bone graft. Successful bone regeneration is observed in all different forms of β-TCP compared in our study, and the particulate form was the most successful. The block and putty graft forms showed similar results. Particulate and block bone substitutes are proven products that have been used for many years in oral surgery. Along with its porosity, the pore diameter, particle size and distribution significantly affect their success. Studies comparing different forms of equivalent grafts are very limited. Studies with larger sample sizes comparing different graft forms and types are needed.

Author Contributions

All authors contributed to the study conception and design. Surgical interventions, material preparation, data collection were performed by HA, BT, KK and NU. Histological analysis was performed by FU. The first draft of the manuscript was written by BT, HA and OK. Data analysis was performed by HA, MU and OK. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

The authors did not receive support from any organization for the submitted work.

Data Availability

Additional chart data will be provided by contacting the corresponding author.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Ethical Approval

This study was carried out with the approval of local ethics committee in Ege University Laboratory Animals Application and Research Center (No:2018–046) in accordance with European Directive 2010/63/EU for conducting animal experiments.