Bone regeneration of a polymeric sponge technique—Alloplastic bone substitute materials compared with a commercial synthetic bone material (MBCP+TM technology): A histomorphometric study in porcine skull

Abstract Background Polymeric sponge technique is recommended for developing the desired porosity of Biphasic calcium phosphate (BCP) which may favor bone regeneration. Purpose To investigate the healing of BCP with ratio of HA30/β‐TCP70 (HA30) and HA70/β‐TCP30 (HA70) polymeric sponge preparation, compare to commercial BCP (MBCP+TM). Materials and Methods Materials were tested X‐ray diffraction (XRD) pattern and scanning electron microscope (SEM) analysis. In eight male pigs, six calvarial defects were created in each subject. The defects were the filled with 1 cc of autogenous bone, MBCP+TM (MBCP), HA30, HA70, and left empty (negative group). The new bone formations, residual material particles and bone‐to‐graft contacts were analyzed at 4, 8, 12 and 16 weeks. Results Fabricated BCP showed well‐distributed porosity. At 16 weeks, new bone formations were 45.26% (autogenous), 33.52% (MBCP), 24.34% (HA30), 19.43% (HA70) and 3.37% (negative). Residual material particles were 1.88% (autogenous), 17.58% (MBCP), 26.74% (HA30) and 37.03% (HA70). These values were not significant differences (Bonferroni correction <0.005). Bone‐to‐graft contacts were 73.68% (MBCP), which was significantly higher than 41.68% (HA30) and 14.32% (HA70; Bonferroni correction <0.017). Conclusions Polymeric sponge technique offers well‐distributed porosity. The new bone formation and residual material particles were comparable to MBCP+TM, but the bone‐to‐graft contact was lower than MBCP+TM.


| INTRODUCTION
On account of bone remodeling in dental sockets, the alveolar crests result in progressive changes of their dimensions (Araujo et al., 2015;Chappuis et al., 2017) and qualities (Kuroshima et al., 2017). Consequently, insufficient ridge amounts can occur which challenges dental implant treatment in both esthetics and functional aspects. Bone augmentation is one of the procedures that can resolve this problem (Roden Jr., 2010). Autogenous bone remains considered as the gold standard graft because it consists of all bone formation characteristics which are osteogenesis, osteoinduction and osteoconduction. However, there are some concerns about the surgery at the donor sites that may cause patient morbidity. Moreover, only a limited amount of the graft can be taken and their resorption rate is unpredictable (Marx, 2007;Precheur, 2007;Roden Jr., 2010;Sheikh et al., 2017;Thrivikraman et al., 2017). Other grafts have been developed to fulfill these autograft's disadvantages but these grafts contain just osteoconduction and possible osteoinduction. Allograft and xenograft are common bone substitutions (Marx, 2007;Precheur, 2007;Roden Jr., 2010;Sheikh et al., 2017;Thrivikraman et al., 2017). By contrast, they could cause disease transmission (Boneva et al., 2001;Mirabet et al., 2017). Therefore, alloplastic grafts has been fabricated (Sheikh et al., 2017). The systematic reviews show no significant different outcomes among the bone substitutions in bone augmentation procedures (Cecilia et al., 2018;Corbella et al., 2016;Nawas & Schiegnitz, 2014) and alveolar ridge preservation (De Risi et al., 2015). In addition, the alloplast results in comparable percentage of new bone formation (Papageorgiou et al., 2016;Wu et al., 2016) and implant treatment outcomes in maxillary sinus floor augmentation (Starch-Jensen et al., 2018) to other materials. However, the alloplast yields lower bone-tograft contact than xenograft (Wu et al., 2016).
BCP is considered as an osteoconductive scaffold. Grain size, pore size and porosity play important roles for the scaffold property. Small grain size (<1 mm) scaffolds are preferably resorbed by osteoclast-like cells and have better shapeability while large grain size allows superior space-making capability (Dorozhkin, 2010;Yamada & Egusa, 2017).
Apart from the grain size, pore size is crucial for cell migration and tissue ingrowth. Pore size can be categorized in macroporous, microporous and interconnectivity. Macroporous (>100 μm) is associated with the migration and proliferation of osteoblasts and mesenchymal cells as well as the mineralized bone ingrowth. Microporous (<10 μm) enhances protein adsorption and body fluid exchange. Likewise, interconnectivity assists the vascularity which includes nutrient contribution and waste removal (Hannink & Arts, 2011;Klein et al., 2009;Mate Sanchez de Val, Calvo-Guirado, Gomez-Moreno, Gehrke, et al., 2016;Mate Sanchez de Val, Calvo-Guirado, Gomez-Moreno, Perez-Albacete Martinez, et al., 2016). Higher porosities result in larger surface area which encourage cell adhesion and bone ingrowth but this condition will reduce the mechanical resistance of bone graft Mate Sanchez de Val et al., 2015;Yamada & Egusa, 2017). One study reported that the total porosities should be 70% of the bioceramic volume (Dorozhkin, 2010).
To control the pore size and porosity is a complicated engineering fabrication (Bose et al., 2012;Dorozhkin, 2007). One of the recommended manufacturing methods is the polymeric sponge technique (Aghajani et al., 2017;Naqshbandi et al., 2013;Wang et al., 2017). In this technique, the material is prepared by milling the slurry BCP on the controlled porosity polyurethane template (Ebrahimi et al., 2012;Ebrahimi et al., 2014). Moreover, the property of the graft material is also influenced by the manufacturing process. High However, there has not been any study regarding bone regeneration potential of BCP fabricated by the polymeric sponge method. Therefore, the grafts were to be evaluated in augmentation procedures in experimentally created critical size defects of the adult pigs by histomorphometry.

| Material characterization
The alloplastic materials were fabricated by polymeric sponge method in HA:β-TCP 30:70 (HA30) and 70:30 (HA70) volumetric compositions from the same batches of materials by National Science and Technology Development Agency (NSTDA), Thailand. The sintering temperature was controlled at 600 c followed by increasing to 1000 c with 2 h per each control. In order to identify the phase's composition, the materials were tested by X-ray diffraction (XRD). XRD patterns were obtained on the two ratios of BCP particles in an X-ray diffractometer (PANalytical) using Cu-Ka radiation (40 mA, 30 kV). Scans were achieved with 2θ values from 20 to 60 at a rate of 0.02 /min.
The fabricated materials were compared with MBCP+TM (Biomantlante, France). MBCP+TM is the commercial BCP which composes of HA:β-TCP 20:80 by volume. A scanning electron microscope (JEOL, Japan) was used to study the morphology of all the test particles. The samples were pre-coated using a carbon evaporation coating unit. They were examined using SEM at a working distance of 10 mm, at an acceleration voltage of 5 kV.

| Surgical procedure
The animal experimental study was designed. The Institutional Animal Care and Use Committee, Faculty of Medicine, Chiang Mai University approved the animal experimental study protocol (No. 29/2560). Eight 50-60 kg (4-5 months) domestic male pigs (Sus scrofa domesticus) were acclimatized 1 week then randomized into four groups in order to investigate bone healing at 4 weeks (n = 2), 8 weeks (n = 2), 12 weeks (n = 2) and 16 weeks (n = 2). NPO (with holding oral intake of food and fluids) was decided in each pig at least 12 h before an intramuscular injection of zoletil 5 mg/kg bodyweight and xylazine 2 mg/kg bodyweight premedication. Atropine 0.05 mg/kg bodyweight and midazolam 0.5 mg/kg bodyweight were injected intravenously through an ear vein for the induction. The animal was given an endotracheal intubation and a controlled respiration frequency of 12 per minute with a volume of 10 ml/kg body weight. Isoflurane 1.0%-1.5% was maintained with a mixture of oxygen.
The administration of 10 mg/kg of amoxicillin were given intramuscularly for prophylaxis. The calvarial site was scrubbed with 10% povidone iodine. After local infiltration of 4% articaine with 1:100,000 epinephrine 1.7 ml, a midsagittal incision was created. A subperiosteal dissection and six standardized intraosseous defects using a trephine with copious saline irrigation was performed. Each defect dimension The calvarium were harvested using a small, sharp fissure bur and then fixed in 10% neutral-buffered formalin for at least 2 weeks. Each block was dehydrated using a graded series of alcohol and embedded in methyl-methacrylate for 50 days. Using a low speed diamond saw with copious cooling, a defect was sectioned in a bucco-lingual direction (Donath & Breuner, 1982). The sections were mounted on opaque Plexiglas with acrylic glue and ground to a final thickness of 8-10 μm. Finally, the sections were superficially stained with toluidine blue and basic fuchsin (Buser et al., 2004). Each defect was sectioned into two sample slides.

| Histologic and histomorphometric analysis
To analyze the histology, all sections were illustrated by light microscope (Axio Lab.A1, Zeiss) with the objective lens set at 5×, 10× and 40×. A digital camera (1200D, Cannon) which was connected to the light microscope was used to capture the images and then stored in a computer. Each specimen was investigated for the quality of new bone formation, residual materials, and inflammatory responses. In addition, the compact stereo microscope (Stemi 305, Zeiss) with the objective lens set at 10× was used to analyze the histomorphometry. The histomorphometric values were analyzed by two examiners.
The quantity of new bone formation was calculated by the percentage of the newly formed bone area to the total defect area. % ð Þnew bone formation = newly formed bone area pixel 2 total defect area pixel 2 x 100 The amount of residual material particles was calculated by the percentage of the grafting material particle area to the total defect area.
% ð Þresidual material particles = grafting material particle area pixel 2 total defect area pixel 2 x 100 Bone-to-graft contact of the biomaterial was calculated by the percentage of the length of bone contact to graft surface to the circumference of the graft particle.

| RESULTS
The phases of the materials were presented by the XRD pattern. The increase in peak height and decrease in peak width indicated that the materials exhibit high crystallinity. Two phases can be identified with no new formed phase in two compositions of the materials. The fabricated BCP showed higher peak height and lower peak width ( Figure 2).
The morphologic analysis of the materials was described using the SEM images. The sizes of fabricated BCP in both compositions range from 1000 to 2000 μm. Irregular and rough surface were detected. Its homogenous structures represent highly dense F I G U R E 2 XRD pattern in MBCP group, HA30 group and HA70 group crystallinity. In addition, well distributed pores were found within the materials. The porosity of the fabricated BCP was over 80%, with pore sizes composed of 100-500 μm (macroporosity) and 1-10 μm (microporosity; Figure 3e,g). Moreover, the interconnected pores were noticed with high magnification (Figure 3f,h). While MBCP+TM's granule sizes range from 500 to 1000 μm (Figure 3a). From the view of macrostructure, the surface appeared flatter. With ×500 magnification view, the tiny porosities and the roughness were observed ( Figure 3b). BCP's structure composes of two layers which are the hexagonal plate-like shape form surrounded with the rod shape forms ( Figure 3c). These forms demonstrate small-scale crystallinity. In addition, with ×7000 magnification, the nano-scale porosity arises from the boundary from the droplets of the materials (Figure 3d).
Non-parametric statistics were used in this study, due to the limited number of animals used. The study was strictly concerned about the animal life and followed the principles of the 3Rs (replacement, refinement, reduction). Our study design was minimized the number of animals used and was appropriately analyzed to ensure robust and reproducible findings.
The histology findings were investigated. Two slides of the autogenous samples, one from 8 weeks and the other from 12 weeks were excluded due to poor quality image for analysis. In the autogenous group, the defects showed new osteoid formation which was completely integrated around the remnant grafts in 4 weeks (Figure 4a). The autogenous particles were mainly resorbed and difficult to distinguish from the surrounding bone in 8 weeks.
The laminated bone formation was observed in the defects ( Figure 4b). In 12 and 16th weeks, the Harvesian's canals were seen ( Figure 4c,d).
In group MBCP, the particles showed good distribution. During the first 4 weeks the particles were partially contacted with new woven bone regeneration. The bone formation also occurred within the porous (Figure 5a). In 8 weeks, the lamellar bone was regenerated with more contact bone around the grafts (Figure 5b). The maturation and consolidation of the bone developed over time while the particle sizes were trend to be smaller as seen in 12 and 16 weeks ( Figure 5c,d).
In the fabricated BCP groups, the particles appeared aggregated.
In group HA30, new bone was scarcely regenerated during the first 4 week which was located just around the osteotomy site (Figure 6a).
The osteoid formation was observed with limited contact to the graft in 8 weeks (Figure 6b). The bone maturation and bone-to-graft contact increased over the time (Figure 6b-d). While in group HA70, the woven bone was seen in 16 weeks which showed more quantity in the smaller particles. This group showed restricted contact between bone and graft particles (Figure 7a-e).
In the negative group, the new bone regeneration was very limited. Fibrous tissue growth was mainly observed (Figure 8).
All biomaterials have shown good biocompatibility with no adverse fibrous tissue reaction. Furthermore, the bone remodeling process with osteoclastic and osteoblastic reactivity was observed ( Figure 9).
The histomorphometric analysis was evaluated. The average measures' value of intraclass correlation was 0.878. The measured values of new bone formation and residual material particles are shown in Table 1, while the percentages of bone-to-graft contact are shown in Table 2 (Araujo et al., 2010;Dorozhkin, 2007Dorozhkin, , 2010Yamada & Egusa, 2017). The SEM images also demonstrated even distribution of macro-and microporosity. According to the literature, high porosity enhances cell attachment, cell seeding and cell migration which promotes new bone ingrowth. Moreover, this facilitates the bone graft resorption but too much porosity reduces the mechanical properties (Dorozhkin, 2007(Dorozhkin, , 2010Hannink & Arts, 2011;Mate Sanchez de Val, Calvo-Guirado, Gomez-Moreno, Perez-Albacete Martinez, et al., 2016). Different pore sizes also play different roles (Klein et al., 2009). Macroporosity is considered essential for bone cell colonization and new bone enhancement (Dorozhkin, 2007;Dorozhkin, 2010;Klein et al., 2009). One study revealed good results in using highly macroporous β-TCP in maxillary sinus augmentation (Bettach et al., 2014). Microporosity is crucial for protein adsorption (Dorozhkin, 2007(Dorozhkin, , 2010Klein et al., 2009) and improved growth factor retention (Woodard et al., 2007). An animal study showed significant higher bone formation in microstructured BCP although the remaining graft volume was still higher (Dahlin et al., 2015). Interconnection pores facilitate nutrient transportation, oxygen flow and vasculature (Dorozhkin, 2007(Dorozhkin, , 2010Klein et al., 2009)  were not the only main focus factors. There were other osteoconductive factors that need to be introduced. The crystallinity from high sintering temperature is another factor as described previously. Grain size and morphology also had an effect on the material resorption (Park et al., 2010). In this study, each defect dimension was 5.0 mm in diameter and 2.0 mm in depth following ISO 10993-6: 2007 protocol to exclude the healing capacity of the bone substitution from the osteotomy site.
The sample size was limited due to the animal rights concerns. However, we were able to gather sufficient information within this limitation. The autogenous bone graft trended to show the highest median percentage of new bone formation and exhibited the fastest resorption. This could imply that the autogenous bone graft is still the gold standard for bone regeneration (Cecilia et al., 2018;Corbella et al., 2016;De Risi et al., 2015;Nawas & Schiegnitz, 2014;Papageorgiou et al., 2016;Starch-Jensen et al., 2018).  (Marx, 2007;Roden Jr., 2010). In 4 weeks of the HA30 group, new bone was scarcely regenerated just around the osteotomy site. Since the blood supply comes from this area, the healing potency increased at the osteotomy site (Araujo et al., 2015;Chappuis et al., 2017). In 16 weeks, the woven bone in the HA70 group had a higher quantity of small-sized particles. This may impair bone regeneration because graft resorption has to take place first. However, this is the advantage for space-making capacity in long term bone healing (Dorozhkin, 2010;Roden Jr., 2010).
The other significant factor that needs to be mentioned is the composition between HA and β-TCP. Many studies conclude that lower HA/β-TCP ratio shows more new bone formation area due to the great dissolubility of β-TCP (Jelusic et al., 2017;Jensen et al., 2007;Wang et al., 2013;Yang et al., 2014;Zhang et al., 2005;Zhu et al., 2010). In correspondence with this study, the MBCP group composes of the lowest HA/β-TCP ratio and has a tendency to enhance a higher amount of new bone formation than HA30 and HA70 group which are formulated with a higher ratio of HA respectively. BCP is considered the alternative material. The histology analysis shows good biocompatibility and bone remodeling process among each BCP group. Literature reviews stated that the results in terms of new bone formation and implant survival rate using BCP as a scaffold is comparable to other bone substitutions (Papageorgiou et al., 2016;Starch-Jensen et al., 2018;Wu et al., 2016). Most clinical studies focus on using BCP in the space-making defects like maxillary sinus augmentation (Bettach et al., 2014;Chiu et al., 2018;Christer et al., 2009Christer et al., , 2012Covani et al., 2011;Danesh-Sani et al., 2016;Frenken et al., 2010;Helder et al., 2018;Iezzi et al., 2012;Lee et al., 2018;Mangano et al., 2013;Nishimura et al., 2018;Ohe et al., 2016;Okada et al., 2017;Schmitt et al., 2013;Sebastian et al., 2010;Sverzut et al., 2015;Tosta et al., 2013).
From a systematic review, BCP gains lower bone-to-graft contact than deproteinized bovine bone mineral (DBBM; Wu et al., 2016).
However, one study demonstrated a non-significant difference value (Christer et al., 2012) whereas another study revealed that BCP offers more interfacial bone contact than in DBBM (Yip et al., 2015). No study that evaluated the bone-to-graft contact among the BCP was found. This study shows that the median percentage of bone-to-graft contact increases overtime among each material. The MBCP group gained more interfacial bone contact than the fabricated BCP with ratio of HA30 and HA70 group respectively. This conforms to the new bone formation area and could be explained by the rationale described earlier.
Clinically, the using of biphasic alloplastic graft is difference in each situation, if the purpose of the use is to maintain the space of the recipient site, the higher percentage of hydroxy apatite is required. In contrary, if the purpose of using alloplastic graft is for small defects and need the faster biomaterial resorption, the higher percentage of tricalcium phosphate is prerfer.

| CONCLUSION
Within the limitation of this study, it can be indicated that the different manufacturing methods result in different results of bone-to-graft contact. The polymeric sponge technique can offer well distributed porosity within the graft in terms of macroporosity and microporosity.
However, that desired porosity did not correlate with better bone regeneration, the other factors which are crystallinity, grain size and nano-scale porosity also influence the osteoconductive property.
The autogenous bone graft which remains as the gold standard trends to show the highest new bone formation, while BCP shows good biocompatibility and can be the alternative choice. BCP with higher ratio of β-TCP has a tendency to result in faster resorption and more new bone formation. However, more sample sizes and long term bone healing should be observed.

ACKNOWLEDGMENTS
The authors sincerely thank Dr.

CONFLICT OF INTEREST
All authors declare that there are no conflict of interest in this study.

AUTHOR CONTRIBUTIONS
Punyada Intapibool and Pathawee Khongkhunthian have made substantial contributions to management, analysis and interpretation of data and in collaborating the manuscript. Pathawee Khongkhunthian and Kriangkrai Thongkorn have been participated in the study design and have revised it critically for important intellectual content and also have given final approval of the version to be published. All authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.