Morphometric and Histomorphometric Evaluations of High-Purity Macro/Microporous β-TCP in Maxillary Sinus Floor Elevation


 Background: The present study examined the effectiveness of high-purity macro/microporous beta-tricalcium phosphate (HPMM β-TCP) as a bone grafting material for maxillary sinus floor elevation by morphometric and histopathological, histomorphometric evaluations.Methods: Ten maxillary sinus ﬂoor elevation procedures using 100% HPMM β-TCP were performed on 10 patients. Morphometric evaluation was carried out by CT imaging immediately after augmentation and prior to dental implant placement after 7 months.Histopathological and histomorphometric evaluation were carried out by bone biopsy retrieval at the time of dental implant placement 7 months after sinus floor elevation. Results: All 10 sinus floor elevations were successful. Morphometric evaluation by CT images showed that the vertical height and the volume gained by sinus floor elevation decreased 7 months after surgery. Histopathological evaluation by bone biopsy retrieval specimens showed no signs of inﬂammation at the newly formed bone area and the native alveolar bone area. New bone formation was observed at the cranial side from the native alveolar bone. The newly formed bone had a trabecular structure and was in intimate contact with the HPMM β-TCP material. Histomorphometric evaluation by bone biopsy retrieval specimens revealed an average new bone volume of 33.97%±2.79% and an average residual HPMM β-TCP volume of 15.81%±4.52%.Conclusions: In this study, HPMM β-TCP showed the osteoconductive properties for vertical augmentation of the atrophied maxilla by means of a maxillary sinus ﬂoor elevation procedure allowing subsequent dental implant placement after a 7-month healing period.

that the vertical height and the volume gained by sinus oor elevation decreased 7 months after surgery. Histopathological evaluation by bone biopsy retrieval specimens showed no signs of inflammation at the newly formed bone area and the native alveolar bone area. New bone formation was observed at the cranial side from the native alveolar bone. The newly formed bone had a trabecular structure and was in intimate contact with the HPMM β-TCP material. Histomorphometric evaluation by bone biopsy retrieval specimens revealed an average new bone volume of 33.97%±2.79% and an average residual HPMM β-TCP volume of 15.81%±4.52%.
Conclusions: In this study, HPMM β-TCP showed the osteoconductive properties for vertical augmentation of the atrophied maxilla by means of a maxillary sinus floor elevation procedure allowing subsequent dental implant placement after a 7-month healing period.

Background
Dental implant placement in the posterior maxilla often requires surgical intervention of the maxillary sinus oor area because of insu cient bone volume. Maxillary sinus oor elevation performed for placing the dental implants in this region was rst reported by Boyne and James 1 and Tatum 2 . This surgical procedure makes the dental implant placement possible by increasing the alveolar bone height in this region. A wide variety of graft materials have been used to elevate the maxillary sinus oor.
Autogenous bone grafts are considered to be the gold standard, because they are not immunogenic and they have osteogenic, osteoinductive, and osteoconductive properties 1,3−6 . However, there are several disadvantages, including postoperative complications in the donor site such as limping when the graft is taken from the iliac crest, prolonged healing time at the donor site, requirement of general anesthesia and hospitalization, increased cost of treatment, and unpredictable resorption of the new bone by grafting 7,8 .
These disadvantages have led to a search for eligible graft materials that are a biocompatible and osteoinductive or at least osteoconductive alternative to autogenous bone substitutes in the sinus oor elevation.
Anorganic bovine-derived hydroxyapatite (BHA), one of non-resorbable xenograft, has been shown to be a safe and biocompatible bone graft material with osteoconductive properties [23][24][25] . Also, several experimental and clinical studies have shown successful results of BHA graft materials when used for maxillary sinus oor elevation [9][10][11][12][13][14]22 . BHA, prepared from Australian cattle, is processed chemically and through heat treatments to remove proteins and other organic substances to ensure the product has no prion and is, thus, non-antigenic 26 . The resulting particles are similar to human bone particles with regard to physical and chemical composition, ensuring the graft provides a conductive scaffold for bone formation. On the other hand, Scolozzi et al. showed a signi cant association between speci c deproteinized bovine bone used as sinus bone graft material and the development of fungus balls of the maxillary sinus, which was based on the presence of chronic maxillary sinusitis with purulent discharge. It was hypothesized that similar to the mechanism of a foreign body reaction, the residual proteins could stimulate an immune response to the xenograft, and that this immune response could promote the Aspergillus growth within the maxillary sinus leading to fungus ball formation 27 . Thus, to prevent the complication of the postoperative maxillary sinusitis caused by fungus ball formation, a bone-grafting material that is protein-free, has good osteoconductive properties and has less residual substitute after bone formation is required for maxillary sinus oor elevation.
The purpose of this study was to evaluate the quality and quantity of bone formation in a maxillary sinus oor elevation procedure using only HPMM β-TCP with a 7-month healing period. The clinical, radiological, morphometric, and histomorphometric results of a multicenter study are presented.

Patients
The study consisted of 10 patients (3 male, 7 female) who were partially edentulous in the posterior maxilla and required dental implant placement. The mean age of the patients was 57.2 ± 2.43 years (range, 41-67 years). The main criteria for inclusion in the study group were a residual alveolar ridge height of < 5 mm and a width of ≥ 5 mm. Panoramic radiographs were taken, and these radiographs were used to evaluate the residual alveolar ridge height. The surgical phase was planned as a maxillary sinus oor elevation followed by dental implant placement. The exclusion criteria were unsatisfactory oral hygiene, cute maxillary sinusitis, heavy smoking (≥ 10 cigarettes a day), and systemic diseases such as uncontrolled diabetes mellitus. All patients were healthy, with no disease that might affect the treatment outcome. The patients were fully informed about the procedures, including the surgery, graft materials, and dental implants, and written, informed consent was obtained from all of them. The research protocol was approved by the Ethics Committee of Dokkyo Medical University School of Medicine (R- [10][11][12][13][14]. HPMM β-TCP HPMM β-TCP is a monophase β-TCP ceramic generated by the spray-dry method. The synthesized granules are recognized as high purity by X-ray diffraction analysis (Fig. 1). Osteogenic cellular activity requires bone substitutes with porous spaces that allow cellular penetration in order to obtain the most suitable porosity, optimal pore size, and favorable pore structure. HPMM β-TCP is a granular bone substitute with a diameter of 250 to 2000 µm formed as a bunch of grapes. Primary particles, having many micropores with a diameter of 3 µm, are microspheric with a diameter of 50 µm, and there are many interconnecting pores (10-160 µm) between secondary particles of HPMM β-TCP ceramics ( Fig. 2). It is hydrophilic and has a porosity of 75%.
Maxillary sinus oor elevation procedure, dental implant placement, and bone biopsy retrieval For maxillary sinus oor elevation, 9 patients were treated with an outpatient procedure under local anesthesia, while 1 patient was treated under local anesthesia with intravenous sedation. The antibiotic (amoxicillin 1000 mg four times a day) for 7 days, and an oral rinse with chlorhexidine digluconate 0.006% (10 cm 3 three times daily for 1 min) for 2 weeks were prescribed for all patients. Via crestal incision and elevation of a mucoperiosteal ap, the maxillary wall was prepared. Preparation was made in the lateral maxillary sinus wall using a round burr with irrigation with sterile saline. A buccal window together with the carefully elevated sinus membrane was dissected and re ected inward. The sinus membrane was detached from the maxillary walls and moved in cranial direction. In all cases, the sinus membrane was covered with a resorbable collagen membrane (Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland). The space between the lifted sinus membrane and the sinus oor was lled with HPMM β-TCP (Fig. 3). Complete wound closure was performed with 4/0 mono lament sutures. Removal of sutures was performed after two weeks. After a 7-month healing period, elevation of a mucoperiosteal ap was performed for dental implant placement under local anesthesia in all cases. Before dental implant preparations were made, bone biopsies were taken using a trephine drill with an external diameter of 3.3 mm from the grafted area at the planned dental implant positions, with irrigation with sterile saline. The dental implant bodies with a length of 10 or 12 mm and diameter of 4.0 mm could be placed into the implant bed in all patients. Sutures were removed 10 days after dental implant placement. All surgical procedures were performed by one oral surgeon (AF). Attention was paid to prevent premature loading of the dental implants. Attention was paid to prevent premature loading of the dental implant. After a healing period of 6 months, the implants were loaded.

Morphometric evaluation by radiological examination
Panoramic radiographs and CT images were taken before surgery, after the maxillary sinus oor elevation procedure, and prior to dental implant placement. CT images were stored in DICOM format data, which were analyzed in SIMPLANT® Pro 16 (Dentspy Sirona Inc., New York, USA), a reformatting imaging software package that simulates the placement of dental implants. Images were analyzed by an independent experienced dentist without any clinical information (HK). No information about group assignment was provided during the examinations (Fig. 4).

Histopathological and histomorphometric evaluation by bone biopsy
The specimens were placed in 4% formalin solution for 72 hours and then in the 6% nitric acid solution for 12 hours. The specimens were washed for 4 hours. They were dehydrated in an ascending series in alcohol and xylene. Bone cores were nally embedded in para n. Fine cut, 5-µm-thick sections were made from each specimen parallel to the long axis of the cylindrical core using a microtome. Subsequent to depara nization of the sections, they were exposed to a descending series of alcohol and xylene, then washed with water. Finally, the specimens were stained with hematoxylin and eosin (H&E).
Images of the sections were obtained with a digital camera (Olympus DP 80, Olympus, Tokyo, Japan) attached to a microscope (Olympus BX53, Olympus). The obtained images were transferred to a computer, and image analysis software (WinROOF2015 Standard®, Mitani Co., Fukui, Japan) was used for histomorphometric evaluations. Histomorphometric evaluations were performed over the total section of the biopsy, including residual and newly formed bone. Newly formed bone of the biopsy was de ned as the area from the rst visible HPMM β-TCP particle at the cranial side of the native alveolar bone (Fig. 5). The following histomorphometric measurements were performed: (1) new bone formation in the grafted area (percentage of newly formed bone area to total measured area); (2) area of graft particles (percentage of graft particle area to total measured area); and (3) soft-tissue area in the grafted zone (percentage of soft-tissue area to total measured area).

Statistical analysis
The results of the morphometric analyses were calculated as the means ± SEM for each variable. The signi cance of differences between groups was tested by Student's t-test. P values < 0.05 were considered signi cant.

Clinical evaluation
All 10 sinus oor elevations were successful. And all dental implant placements were also successful. The surgical site infection was not observed. The mean healing time after the maxillary sinus oor elevation was 226.7 ± 12.3 days (range, 175-288 days; Fig. 6).

Morphometric evaluation
CT images were evaluated to assess the height of the most increased position in the maxillary grafted materials after sinus oor elevation, as well as the height of the same position prior to dental implant placement. The average height after the maxillary sinus oor elevation was 15.60 ± 0.88 mm. The average height prior to dental implant placement was 11.27 ± 1.09 mm. The rate of change in the average height from the maxillary sinus oor elevation to dental implant placement was 72.61%±6.44%. The average height was signi cantly lower prior to dental implant placement than after sinus oor elevation (Fig. 7).
CT images were evaluated to assess the increased volume of the maxillary bone after sinus oor elevation, as well as prior to dental implant placement. The average volume of the grafted maxillary bone after sinus oor elevation was 2674.39 ± 453.34 mm 3 . The average volume of the grafted maxillary bone prior to dental implant placement was 1516.88 ± 246.71 mm 3 . The rate of change in the average volume from the maxillary sinus oor elevation to dental implant placement was 61.45%±6.01%. The average volume was signi cantly lower prior to dental implant placement than after sinus oor elevation (Fig. 8).

Histopathological evaluation
Histopathological examinations showed no in ltration of in ammatory cells in the tissue close to the grafted material. New bone formation was examined in all 10 specimens. The trabecular bone that formed from the residual bone expanded the grafted space. The new bone consisted of both lamellar and woven bone. Osteocytes were present in the lacunae. In 9 of 10 specimens, the primary trabecular structure was already reinforced by lamellar bone in some areas. The HPMM β-TCP particles were integrated into the circuitry of newly formed trabecular bone. Close bone-to-substitute contact was observed. Osteoblasts were detected next to the characteristic outlines of the newly formed bone (Figs. 9, 10).
Histomorphometric evaluation Table 1 shows the results of the histomorphometric measurements of HPMM β-TCP. Mean new bone formation was 33.97%±2.79%. The mean percentage of residual grafted particle area was 15.81%±4.52%. The mean percentage of soft-tissue area was 50.21%±4.28% (Table 1).

Discussion
In this study, the maxillary sinus oor elevation procedures using HPMM β-TCP showed uneventful healing. Morphometric evaluation by CT images showed that the vertical height and the volume gained by sinus oor elevation decreased 7 months after surgery. The average height after the maxillary sinus oor elevation was 15.60 ± 0.88 mm. The average height prior to implant insertion was 11.27 ± 1.09 mm. The rate of change in the average height from the maxillary sinus oor elevation to implant insertion was 72.61%±6.44%. Histological investigation also showed no signs of in ammation at the newly formed bone area and the native alveolar bone area. Cranial from the native alveolar bone, newly formed mineralized tissue was observed. The newly formed bone had a trabecular structure and was in close contact with the HPMM β-TCP particles. Histomorphometric evaluation revealed an average new bone volume of 33.97%±2.79% and an average residual HMM β-TCP volume of 15.81%±4.52%. The results indicated that HPMM β-TCP, a new bone substitute, was thought to have good osteoconductive properties with proper absorbency characteristics. The amount of HPMMβ-TCP lled in the maxillary sinus bed for elevation should be planed as overcorrection that compensates for the expected resorption.
TCP is biocompatible, osteoconductive calcium phosphate. The biomaterial can provide a scaffold for potential bony ingrowth 33 . TCP resorbs, unlike BHA. It is replaced by newly formed bone 31,32 . The problem is that this replacement does not necessarily occur in a 1:1 ratio. Often less bone is produced as Germany) using the same method as the present study 21 . According to their results, mean new bone formation was 17%±5%, and the mean percentage of residual graft particle area 6 months postoperatively was 31%±4% 21 . The present study showed that HPMM β-TCP has a good balance with bone formation and material absorption. We consider that the reason for these results was both the high purity and the macro/microporous structure of HPMM β-TCP. The greater volume of new bone formation using 100% HPMM β-TCP was con rmed with the lower percentages of residual graft particles in comparison with β-TCPs 21, 36-38 (Table 2).
Histologically, the vital bone was composed of both lamellar and woven bone. Osteocytes were present in the lacunae. The newly formed bone was in intimate contact with the substitute material, outlining the osteoconductive properties of the HPMM β-TCP material. Bone maturation was evident by the presence of lamellar bone. Osteoblasts were detected next to the characteristic outlines of the newly formed bone (Figs. 9, 10). Retrograde infection with viruses and bacteria via the ostomeatal complex from the nasal cavity causes acute paranasal sinusitis. If there is no activity in the bone of the maxillary sinus, paranasal sinusitis will get worse. And the inactive bone of the maxilla has no potential as supporting tissue for dental implants. Therefore, bone activity is important for the alveolar part of the posterior maxilla for dental implants.
BHA is a deproteinized natural bovine cancellous bone with a crystalline structure that is very similar to human bone. The deproteinization process through chemical and heat treatments should eliminate any immunogenicity concerns. However, Schwartz et al. suggested that BHA can contain osteoinductive proteins, such as transforming growth factor-β, bone morphogenic protein, and bone morphogenic protein-2, which can elicit an immune response 39 . One case report presented a foreign body reaction in a patient who had undergone alveolar ridge augmentation with a mixture of BHA and autogenous bone 40 . The most plausible explanation for such a reaction is an interaction between residual proteins within BHA particles with adhesion receptors present on monocyte/macrophage in ammatory cell populations. Scolozzi et al. hypothesized that similar to the mechanism of a foreign body reaction, the residual proteins could stimulate an immune response to the xenograft and that this immune response could promote Aspergillus growth within the maxillary sinus leading to fungus ball formation 27 . Thus, in order to prevent complications such as a postoperative maxillary sinusitis caused by a fungus ball, a proteinfree bone-grafting material with less residual substitute after bone formation and with good osteoconductive properties, such as HPMM β-TCP is ideal for maxillary sinus oor elevation.

Conclusions
In this study, HPMM β-TCP showed the osteoconductive properties for vertical augmentation of the atrophied maxilla by means of a maxillary sinus oor elevation procedure allowing subsequent dental implant placement after a 7-month healing period. Histological and histomorphometric evaluation showed newly formed bone with a trabecular structure. Osteocytes were present in the lacunae. The The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Ethical approval and Consent to participate: The study protocol was approved by the Ethics Committee of Dokkyo Medical University School of Medicine (R- [10][11][12][13][14]. The consents of the patients have been obtained.

Consent for publication
Not applicable.

Competing interests
Not applicable.