An Innovative Bioceramic Bone Graft Substitute for Bone Defect Treatment: In Vivo Evaluation of Bone Healing

: This study aimed to analyze characteristics of an innovative α -calcium sulfate hemihydrate ( α -CSH) bioceramic and bone healing and regeneration characteristics following its implantation on artiﬁcially created defects of rat models and human jaw defects. The α -CSH bioceramic was characterized using ﬁeld emission scanning electron microscope (FE-SEM), energy-dispersive spectroscopy (EDS), and thermal-imaging instruments. The material was implanted on artiﬁcially created defects in a rat’s right hind leg bone and observed histologically after three days and seven weeks. The material was also implanted in patients with bone defects in the posterior maxillary, then observed immediately and six months post-treatment by panoramic and computed tomography image. The FE-SEM conﬁrm this material is a uniform-shaped short column crystal, while the EDS measurement reveals calcium as the most component in this material. Thermal observation shows temperature change during the setting time is less than 2 ◦ C, and the maximum temperature reached is 31 ◦ C. In the histological analysis, α -CSH bioceramic shows new trabecular bone formation and absorbed material at seven weeks post-treatment. Moreover, panoramic and computed tomography image shows intact bone six months post-treatment. Therefore, this study suggests that the innovative α -CSH bioceramic can be useful in bone defect treatment.

with deionized water (10 mL). The mixture and the magnetic stirring bar were put in a Teflon pressure tube (55 mL,~350 psi), then microwaved (800 W) at 160 • C for 10 min. The synthesized sample was filtered, cleaned with ethanol, dried in an oven at 60 • C for 8 h, and grounded as powder [41].

Material Characterizations
Sample for morphology observation and composition analysis was placed on the copper grid sample holder. Before being loaded into the sample chamber, a platinum thin film with a thickness of 30 nm was sputter-coated on the sample surface to enhance electrical conductivity. Afterward, the sample was observed by field emission scanning electron microscope (FE-SEM; JEOL, JSM-6500F, Tokyo, Japan) at an accelerating voltage of 20 kV, while the chemical composition was analyzed by INCA energy-dispersive spectroscopy (EDS, Oxford instruments, Abingdon, UK). Moreover, temperature variation during the setting time of the sample was measured by thermal imaging infrared camera (Advanced Thermo TVS-500EX, Nippon Avionics Co., Ltd., Yokohama, Japan). Sample with a weight of 0.3 g was mixed with 5-mL normal saline (Sigma-Aldrich, Taipei, Taiwan), and then shaped into a spherical pellet. Subsequently, the temperature of the spherical pellet was recorded at room temperature of 25 • C using the TVS-500EX infrared camera fifteen centimeters from the pellet.

Animal Study
The protocols for animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee for Taipei Medical University under a project identification code of LAC-2015-0073. Twenty-four Sparague-Dawley rats weighing 200-300 g obtained from BioLASCO Co., Ltd. (Taipei, Taiwan) were used in this study. The Sparague-Dawley rats were divided into three groups according to the type of implantation materials, consisting of the testing article (TA; the innovate α-CSH bioceramic) group, commercial product (CP; Merries Uni-Osteo, J.O. Corporation, New Taipei City, Taiwan) group [40], and blank group. The implantation procedure was conducted under general anesthesia using isoflurane inhalation. The implantation of approximately 0.1-g powder of α-CSH bioceramic bone graft substitute testing article (n = 4) and commercial predicate material (n = 4) were randomly implanted in the rat's right hind leg bones after created artificial defects by drilling (diameter: 2 mm and depth: 2 mm), while in the blank group (n = 4), artificially created defects were left without any implanted materials, and radiography was taken immediately after implantation ( Figure 1). The animal was sacrificed according to the observational period (3 days and 7 weeks after treatment). A short-term healing observation was used to investigate its early-stage bone healing and inflammatory response.

Histological Analysis
Following sacrificing, artificially created defects in the rat's right hind leg bones and their implants were subjected to histological analysis. The samples were fixed in 10% neutral-buffered formalin, dehydrated in ethanol, embedded in paraffin wax, trimmed, and stained with hematoxylin and eosin (H&E). Then, the stained slides were evaluated by optical microscopy (Olympus BX51, Tokyo, Japan) under different magnifications.

Histological Analysis
Following sacrificing, artificially created defects in the rat's right hind leg bones and their implants were subjected to histological analysis. The samples were fixed in 10% neutral-buffered formalin, dehydrated in ethanol, embedded in paraffin wax, trimmed, and stained with hematoxylin and eosin (H&E). Then, the stained slides were evaluated by optical microscopy (Olympus BX51, Tokyo, Japan) under different magnifications.

Bone Defect Treatment Analysis in Patient with Height Bone Loss in the Posterior Maxillary
The protocols for clinical investigations were reviewed and approved by the Taipei Medical University-Joint Institutional Review Board under a project identification code of TMU-JIRB 201301009. The GCP has been followed together with ISO 14155 prescriptions during clinical study. Patients (n = 10) with height bone loss in the posterior maxillary were recruited for investigation in this study. Bone defect in the left posterior maxillary exposed under local anesthesia to perform sinus lifting with the Luc-Caldwell procedure. The lateral window was performed on the lateral wall of the maxillary sinus, lateral wall of the sinus in-fractured, membrane lifted from the sinus floor, and implant placed simultaneously. The α-CSH bone graft substitute was filled in the defect area to cover the lateral window ( Figure 2a) and, subsequently, put the concentrated growth factor (CGF) membrane on the filled α-CSH bone graft substitute (Figure 2b), then sutured. The bone regeneration was observed at immediately and 6 months post-treatment by panoramic and computed tomography (CT) image.

Histological Analysis
Following sacrificing, artificially created defects in the rat's right hind leg bones and their implants were subjected to histological analysis. The samples were fixed in 10% neutral-buffered formalin, dehydrated in ethanol, embedded in paraffin wax, trimmed, and stained with hematoxylin and eosin (H&E). Then, the stained slides were evaluated by optical microscopy (Olympus BX51, Tokyo, Japan) under different magnifications.

Bone Defect Treatment Analysis in Patient with Height Bone Loss in the Posterior Maxillary
The protocols for clinical investigations were reviewed and approved by the Taipei Medical University-Joint Institutional Review Board under a project identification code of TMU-JIRB 201301009. The GCP has been followed together with ISO 14155 prescriptions during clinical study. Patients (n = 10) with height bone loss in the posterior maxillary were recruited for investigation in this study. Bone defect in the left posterior maxillary exposed under local anesthesia to perform sinus lifting with the Luc-Caldwell procedure. The lateral window was performed on the lateral wall of the maxillary sinus, lateral wall of the sinus in-fractured, membrane lifted from the sinus floor, and implant placed simultaneously. The α-CSH bone graft substitute was filled in the defect area to cover the lateral window ( Figure 2a) and, subsequently, put the concentrated growth factor (CGF) membrane on the filled α-CSH bone graft substitute (Figure 2b), then sutured. The bone regeneration was observed at immediately and 6 months post-treatment by panoramic and computed tomography (CT) image.   Figure 3 illustrates the morphology and chemical compositions of the α-CSH bioceramic bone graft substitute material that was used in the present study. The FE-SEM image of α-CSH bioceramic revealed a uniform-shaped short column crystal around 10 µm in length ( Figure 3a). The morphology of α-CSH bioceramic was organized by a microwave irradiation treatment on CSD at 160 • C for 10 min, which resulted in changes in the morphology of CSD crystals from tablet-like to a uniform short column and, also, changes in the crystal structure from monoclinic to hexagonal [41]. The EDS measurements ( Figure 3b) recognizing calcium as the most component in this material, with an average content value of 37.51 wt.%, which is very important in bone formation [25,40,41]. In addition to the calcium element, the main composition of sulfate (27.52 wt.%) can also be detected. No other impurity substances were detected in the α-CSH bioceramic. It is noticed that the copper element was generated from the copper grid, which was used as a sample holder for the FE-SEM analysis. min, which resulted in changes in the morphology of CSD crystals from tablet-like to a uniform short column and, also, changes in the crystal structure from monoclinic to hexagonal [41]. The EDS measurements ( Figure 3b) recognizing calcium as the most component in this material, with an average content value of 37.51 wt.%, which is very important in bone formation [25,40,41]. In addition to the calcium element, the main composition of sulfate (27.52 wt.%) can also be detected. No other impurity substances were detected in the α-CSH bioceramic. It is noticed that the copper element was generated from the copper grid, which was used as a sample holder for the FE-SEM analysis.  ). The thermal behavior demonstrated by the α-CSH bioceramic included a quick setting and lower temperature variations during the setting time, which are beneficial properties as a bone graft material. Due to the low temperature, this material is safe to use as a bone graft, because it does not cause protein denaturation that will lead to bone damage [22,35,42,43]. A quick setting also can speed up the healing process as various proteins and growth factors are absorbed in the blood to the affected area [40,44].   (Figure 4b). The thermal behavior demonstrated by the α-CSH bioceramic included a quick setting and lower temperature variations during the setting time, which are beneficial properties as a bone graft material. Due to the low temperature, this material is safe to use as a bone graft, because it does not cause protein denaturation that will lead to bone damage [22,35,42,43]. A quick setting also can speed up the healing process as various proteins and growth factors are absorbed in the blood to the affected area [40,44]. graft substitute material that was used in the present study. The FE-SEM image of α-CSH bioceramic revealed a uniform-shaped short column crystal around 10 µm in length (Figure 3a). The morphology of α-CSH bioceramic was organized by a microwave irradiation treatment on CSD at 160 °C for 10 min, which resulted in changes in the morphology of CSD crystals from tablet-like to a uniform short column and, also, changes in the crystal structure from monoclinic to hexagonal [41]. The EDS measurements (Figure 3b) recognizing calcium as the most component in this material, with an average content value of 37.51 wt.%, which is very important in bone formation [25,40,41]. In addition to the calcium element, the main composition of sulfate (27.52 wt.%) can also be detected. No other impurity substances were detected in the α-CSH bioceramic. It is noticed that the copper element was generated from the copper grid, which was used as a sample holder for the FE-SEM analysis.   (Figure 4b). The thermal behavior demonstrated by the α-CSH bioceramic included a quick setting and lower temperature variations during the setting time, which are beneficial properties as a bone graft material. Due to the low temperature, this material is safe to use as a bone graft, because it does not cause protein denaturation that will lead to bone damage [22,35,42,43]. A quick setting also can speed up the healing process as various proteins and growth factors are absorbed in the blood to the affected area [40,44].

Bone Healing and Regeneration Characteristic of the Materials in the Animal Study
The histologic finding of α-CSH bioceramic under H&E staining is exhibited in Figure 5. On the third day post-treatment, there was no visible inflammatory process in each study group, while the remaining implanted material was observed in both the TA and CP groups. The presence of inflammatory cells, new trabecular bone formation, and the evidence of absorbed implanted

Bone Healing and Regeneration Characteristic of the Materials in the Animal Study
The histologic finding of α-CSH bioceramic under H&E staining is exhibited in Figure 5. On the third day post-treatment, there was no visible inflammatory process in each study group, while the remaining implanted material was observed in both the TA and CP groups. The presence of inflammatory cells, new trabecular bone formation, and the evidence of absorbed implanted materials in both the TA and CP groups were observed at the seventh week post-treatment. However, the number of inflammatory cells that migrated to the defect area was greater in the TA group. These conditions will promote the inflammatory phase leading to the healing process [45,46]. The absorption of α-CSH bioceramic is related to the formation of new bone, because the defect can absorb calcium from this material, which plays an important role in the demineralization process [40,41]. In our previous study, we confirmed that bioceramic α-CSH absorption occurred at the fourth week post-treatment [40]. However, the current study showed that all implanted materials absorbed at the seventh week post-treatment.

Bone Healing and Regeneration Characteristic of the Materials in the Animal Study
The histologic finding of α-CSH bioceramic under H&E staining is exhibited in Figure 5. On the third day post-treatment, there was no visible inflammatory process in each study group, while the remaining implanted material was observed in both the TA and CP groups. The presence of inflammatory cells, new trabecular bone formation, and the evidence of absorbed implanted materials in both the TA and CP groups were observed at the seventh week post-treatment. However, the number of inflammatory cells that migrated to the defect area was greater in the TA group. These conditions will promote the inflammatory phase leading to the healing process [45,46]. The absorption of α-CSH bioceramic is related to the formation of new bone, because the defect can absorb calcium from this material, which plays an important role in the demineralization process [40,41]. In our previous study, we confirmed that bioceramic α-CSH absorption occurred at the fourth week post-treatment [40]. However, the current study showed that all implanted materials absorbed at the seventh week post-treatment.   Figure 6 shows a panoramic image of the patient before and after obtaining a bone graft. A vertical bone loss in the posterior maxillary region (Figure 6a) caused the sinus lifting, and an additional bone graft was required to obtain adequate bone volume before implant placement [12,15,20]. The panoramic image immediately after sinus lifting and the implant of a bone graft material (Figure 6b) indicate that the bone graft material fills the space after sinus lifting. At the six months of follow-up after treatment, the bones in the implanted area were more radiopaque, and all of the bone graft material was absorbed.

Bone Healing and Regeneration Characteristisc of the Materials in the Clinical Application
vertical bone loss in the posterior maxillary region (Figure 6a) caused the sinus lifting, and an additional bone graft was required to obtain adequate bone volume before implant placement [12,15,20]. The panoramic image immediately after sinus lifting and the implant of a bone graft material (Figure 6b) indicate that the bone graft material fills the space after sinus lifting. At the six months of follow-up after treatment, the bones in the implanted area were more radiopaque, and all of the bone graft material was absorbed. The cone-beam CT images before treatment also showed a vertical bone loss in the posterior maxillary region (Figure 7). In the jaw, the maxilla region is the most frequently experiencing vertical bone loss and the most difficult to treat [11,12]. Therefore, both moderate and severe bone loss in this region require a bone graft to treat it [47]. The cone-beam CT images six months follow-up after treatment are presented in Figure 8, which exhibits the presence of ossification in the defect area. Figure 8a shows a cone-beam CT image representing a clinical case six months after bone graft placement in the left maxillary region, while Figure 8b,c represent bone graft placement in the right maxillary region. The presence of bone formation can also be seen in the area around the dental implant apex. Several studies have shown The cone-beam CT images before treatment also showed a vertical bone loss in the posterior maxillary region (Figure 7). In the jaw, the maxilla region is the most frequently experiencing vertical bone loss and the most difficult to treat [11,12]. Therefore, both moderate and severe bone loss in this region require a bone graft to treat it [47].
vertical bone loss in the posterior maxillary region (Figure 6a) caused the sinus lifting, and an additional bone graft was required to obtain adequate bone volume before implant placement [12,15,20]. The panoramic image immediately after sinus lifting and the implant of a bone graft material (Figure 6b) indicate that the bone graft material fills the space after sinus lifting. At the six months of follow-up after treatment, the bones in the implanted area were more radiopaque, and all of the bone graft material was absorbed. The cone-beam CT images before treatment also showed a vertical bone loss in the posterior maxillary region (Figure 7). In the jaw, the maxilla region is the most frequently experiencing vertical bone loss and the most difficult to treat [11,12]. Therefore, both moderate and severe bone loss in this region require a bone graft to treat it [47]. The cone-beam CT images six months follow-up after treatment are presented in Figure 8, which exhibits the presence of ossification in the defect area. Figure 8a shows a cone-beam CT image representing a clinical case six months after bone graft placement in the left maxillary region, while Figure 8b,c represent bone graft placement in the right maxillary region. The presence of bone formation can also be seen in the area around the dental implant apex. Several studies have shown The cone-beam CT images six months follow-up after treatment are presented in Figure 8, which exhibits the presence of ossification in the defect area. Figure 8a shows a cone-beam CT image representing a clinical case six months after bone graft placement in the left maxillary region, while Figure 8b,c represent bone graft placement in the right maxillary region. The presence of bone formation can also be seen in the area around the dental implant apex. Several studies have shown satisfactory results in the sinus lifting treatment accompanied by the placement of bone graft material, and this will affect the survival rate of the dental implant [21].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 13 satisfactory results in the sinus lifting treatment accompanied by the placement of bone graft material, and this will affect the survival rate of the dental implant [21]. In the clinical case presented in this study, a CGF membrane was used to cover the α-CSH bioceramic bone graft. The use of bone graft following with the CGF membrane on dental implants has been widely reported [12,48,49]. The application of the CGF membrane is not only to accelerate bone formation but, also, to aid in soft tissue healing as a consequence of surgical procedures [12,[48][49][50][51]. The CGF membrane is safe for clinical application due to, during the preparations, it does not require a chemical additive [12,48,49,51].
The use of autologous bone graft and platelet-rich fibrin (PRF) as natural sources has also been reported to be successful in treating bone defects [52,53]. Several harvesting methods such as a traditional (open) method or minimally invasive method can be used to harvest autologous bone grafts, although there have been several controversies in the literature both due to its complications and the need for the bone graft required [54,55]. In general, traditional methods have been used to obtain more bone grafts. However, this method causes more complications than minimally invasive methods. Another study showed both the traditional and minimally invasive method that has no significant difference in morbidity rates [54]. Another issue of using a natural source bone graft is in the use of PRF for the treatment of bone defects. Although the use of PRF alone can be successful in bone formation, its osteogenic ability is still limited compared to using bone graft material due to a lack of rigidity and faster degradation [56]. To solve the issues of using natural sources, in this study, we used α-CSH bioceramic as a synthetic bone graft substitute to treat bone defects.
Large bone defects (2-2.5-times larger than the affected bone) would not be ossified naturally [5,25,57]. Hence, α-CSH bioceramic as a substitute bone graft was necessary to treat large bone defects because of its ability to act as a bone formation booster for a short period [18,40,[58][59][60]. The ability of α-CSH bioceramic to facilitate new bone formation is related to the absorption of this material in the defect area [18,23,41,59]. The absorption time of calcium sulfate varies widely from one-three months after treatment [23,40,61]. The rapid absorption of calcium sulfate provides a large supply of calcium ions revealed by a more radiopaque image on the radiograph [19,41,59]. In the absorption process, osteoclasts will absorb calcium sulfate as the absorbing bone, preventing osteoclast activity in the bone and promoting osteoblast activity by increasing the concentration of osteoprogenitor cells that play a role in the bone-formation process [18,19,25,40,41,58,60,62]. Several studies have also reported the success of using calcium sulfate bone graft in patients with bone infections [23,58,63]. Due to the ability of calcium sulphate to create an acid environment that is unfavorable for bacteria, causes this material to be recommended in cases of bone defect treatments as the consequences of osteomyelitis and tumor resection [23,62,63].
Another advantage of using α-CSH bioceramic as the bone graft is a micro/nano-sized powder characteristic that easily mixes with various liquids and adapts well to the various shapes of bone defects [35,61,64]. Furthermore, the setting time of this material is acceptable for use in a wide range of surgical and clinical applications, which allows the clinician to place it in the defect area before it hardens [35,65].
The analysis results that were found above support the α-CSH bioceramic properties for use as a bone graft in the treatment of bone defects. However, further study is needed for clinical studying with a larger sample size to strengthen the evidence of the effectiveness of the α-CSH bioceramic as a bone graft substitute in the treatment of bone defects.

Conclusions
The morphology and composition analysis results indicate that the α-CSH bioceramic bone graft is a uniform-shaped short column crystal containing high calcium, which is important for bone demineralization. In the thermal observation, the α-CSH bioceramic shows the ideal setting time and low temperature during the setting. Histological findings in an animal study indicated that the α-CSH bioceramic is an absorbable material and has a favorable healing process. Moreover, the observation from the clinical application showed the presence of intact bone in the bone defect after being treated with an α-CSH bioceramic. Therefore, the α-CSH bioceramic is useful as a bone graft substitute in bone defect treatments.