Bone Formation Following Implantation of Bone Biomaterials Into Extraction Sites For figures, tables and references we refer the reader to the original paper. MATERIALS AND METHODS Eight non-smoking subjects, seven females and one male, ranging in age from 37 to 63 years (mean, 53 years), and in need of at least three tooth extractions because of advanced periodontitis volunteered to participate in this study, which was conducted at Catholic University Leuven between December 2003 and December 2005. Ethics committee approval was obtained. The subjects were thoroughly informed about their rights and the procedures involved, and all signed informed consent. Systemic health exclusion criteria included cardiac disease, smoking, psychological disorders, such as depression and anxiety, and a history of diabetes. Tooth extractions took place in the maxilla and in the mandible. After thoroughly cureting the extraction sites to remove any remnant of the periodontal ligament and/or inflammatory tissue, the experimental treatments, consisting of at least two bone biomaterials and one control, were applied randomly in each subject. Three bone biomaterials were evaluated: a synthetic sponge based on polylactic–polyglycolic acid technology‖ (FIS); bovine bone deproteinized by several hours of heat and alkaline treatment¶ (BIO; high-porosity coarse granules [Ø 1 to 2 mm] were used); and a natural coral derivative physically and chemically transformed into a calcium carbonate ceramic# (COR; the variety with 50% porosity was used). All biomaterials were dispensed on the operating table in a dappen dish and were reconstituted with the subject's blood prior to application to the extraction socket. The bone biomaterials were meticulously packed to completely fill the extraction sites, and the locations of the extraction sites were carefully noted (Table 1). A non-resorbable ePTFE GBR device** was placed to cover the experimental and control sites. A mucoperiosteal-releasing incision was performed to allow full closure of the soft tissues. Postoperative care consisted of a systemic antibiotic (amoxicillin, 500 mg, three times per day for 7 days), chlorhexidine 0.2% rinse (1 minute twice a day for 7 days), and pain medication (acetaminophen, 500 mg maximum, four times per day as needed). The GBR devices were removed after 2 months. Healing was allowed to progress for another 4 months for a total healing period of 6 months, at which time a trephine biopsy (Ø 1.7 mm) was obtained from each extraction site. Table 1. Distribution of Implanted Biomaterials and Controls Among Subjects and Sites (corresponding to the extracted tooth) An implant placement drill guide was used to trace the extraction sites. The extraction sites were generally recognizable at surgical reentry because of visible biomaterial and/or surface texture. A trephine biopsy was obtained with a trephine drill, with an outer diameter of 2 mm, from the center of each extraction site, coinciding with the osteotomy for implant placement. The biopsies were immediately fixed in a buffered formaldehyde/ethanol solution (one part formaldehyde neutralized with CaCO3 [50 g/l], two parts 80% ethanol). The fixed biopsies were dehydrated in a graded ethanol series and embedded in methylmethacrylate resin. Serial sections, 80 to 100 μm in thickness, were obtained using a sawing microtome†† and were ground and polished‡‡ to yield undecalcified sections with a final thickness of 20 to 40 μm. Three central serial sections from each biopsy were stained using Stevenel's blue and van Gieson's picrofuchsin.14 With this method, osteoid tissue stains green, whereas mineralized bone stains red. Histomorphometric analysis of the three central sections was performed using a standard light microscope interfaced with a PC-based image analysis system.§§ A magnification of ×12 was chosen to capture the entire biopsy on the screen for the histomorphometric analysis. To identify cell and tissue morphology, ×40, ×400, and ×1,000 magnifications were used. The software program allowed discrimination between mineralized bone, osteoid tissue, bone biomaterial, and fibrovascular or fatty marrow. Endosseous oral implants‖‖ were placed into the biopsy sites following the manufacturer's standard protocol (Table 2). Insertion torque was measured.¶¶ Gingival flaps were closed to cover the implants for primary intention healing after placement of the cover screws. Abutment surgery took place 4 to 6 months post-implant placement. Healing abutments## were adapted to the implant, and electronic mobility testing device measurements*** were performed to assess the rigidity of the implant–bone continuum. After this periodontal surgery, the subjects received further prosthetic treatment at the Department of Prosthetic Dentistry. Table 2. Implants and Implant Site Characteristics The histomorphometric analysis evaluated the fraction of the biopsy occupied by bone or bone biomaterial. Only completely mineralized bone was measured as bone, and osteoid tissue and marrow spaces were not included in this measurement. Summarizing statistics (group means ± SD) were calculated based on subject means for the three central sections. Analysis of differences among experimental groups built in a nested design used a linear mixed model with fixed effects to investigate the covariance among the different materials, taking into account the subject and site characteristics. The Pearson or Spearman bivariate correlation between implant biomechanical and histomorphometric measurements was used to elucidate whether residual bone biomaterial at 6 months would influence primary stability or osseointegration of the implants. Descriptive statistics were used to verify the covariance in biomechanical recordings among the sites where biomaterials were placed compared to control sites. RESULTS Biopsies were obtained uneventfully from all 36 sites (Table 1). Clinically, with the exception of some loose biomaterial particles on top of the extraction sites, the sites appeared filled with bone, leaving no irregularities. Some sites were not chosen for implant placement because of esthetic or biomechanical considerations. Thus, 25 of 36 sites were used for implant placement (Table 2). Histologic Observations Sections of extraction sites implanted with the FIS biomaterial showed limited material residues. The remaining biomaterial was barely recognizable and was often surrounded or replaced by nonstructured fibrous tissue. Some FIS particles were located close to newly formed bone and osteoid tissue formation; however, these particles were never observed in contact with osteoid tissue or mature bone. The FIS particles appeared to be bioabsorbed by giant cells, resulting in fragmentation of the biomaterial (Fig. 1). Figure 1. Photomicrograph of extraction site implanted with FIS. Residual biomaterial is detected within the marrow spaces in a site largely filled with bone. Multinucleated giant cells with pseudopodia (arrows) surround the biomaterial. (Stevenel's blue and van Gieson's picrofuchsin; original magnification ×400.) A layer of osteoid tissue adjacent to mineralized new bone was observed along the BIO particle surfaces. Bone apposition with osteoblast lining was frequently observed on the top surface of the particles (Fig. 2). The BIO particles appeared colonized by numerous cells that were integrated in the newly formed bone. Osteoclasts or resorption lacunae reflecting osteoclast activity were not observed. Figure 2. Photomicrograph of extraction site implanted with BIO showing BIO particles embedded in fibrovascular tissue and limited new bone formation. BIO particles (dashed arrows) without apparent signs of resorption and newly formed bone (solid arrows) and osteoid tissue (dotted arrows) along the BIO surface are seen. (Stevenel's blue and van Gieson's picrofuchsin; original magnification ×400.) The COR biomaterial was frequently observed in clusters. COR particles and newly formed bone trabeculae were usually separated by fibrovascular tissue. In contrast to BIO, the COR particles underwent resorption and were eventually replaced by newly formed bone rather than being surrounded by bone. Resorption of the COR biomaterial, still ongoing at 6 months post-implantation, appeared to be induced by giant cells (Figs. 3 and 4). Figure 3. Photomicrograph from extraction site implanted with COR. The core biopsy shows residual COR biomaterial (dashed arrow) and extensive new bone formation. The solid arrow indicates the location where Figure 4 was taken. (Stevenel's blue and van Gieson's picrofuchsin; original magnification ×400.) Figure 4. Photomicrograph from extraction site implanted with COR. Giant cells (solid arrows) are seen acting at the surface of the COR biomaterial (dashed arrow). (Stevenel's blue and van Gieson's picrofuchsin; original magnification ×1,000.) Biopsies from control sites (Fig. 5) showed newly formed bone having reached nearly complete maturity with osteocytes embedded in the mineralizing new bone, including Haversian systems, within the 6-month healing period. Figure 5. Photomicrograph of a control site. The core biopsy shows newly formed trabecular bone and fibrovascular marrow. Osteocytes (arrows) in lag phase are integrated in newly formed bone. (Stevenel's blue and van Gieson's picrofuchsin; original magnification ×400.) Histomorphometric and Biomechanical Observations Extraction sites implanted with BIO showed the greatest percentage of residual biomaterial. In paired t tests, a comparison was done between BIO and COR (n = 6; 20.2% ± 17.0%; t [t = t test; 54 = the number of degrees of freedom in the test] = 2.3; P <0.05), between COR and FIS (n = 6; 12.0% ± 16.4%; t = −4.2; P <0.001), and between BIO and FIS (n = 8; 5.6% ± 8.9%; t = 6.67; P <0.001) (Fig. 6). The percentage of viable bone was greatest for the control biopsies (n = 5; 29.4% ± 17.1%), followed by FIS (n = 8; 27.9% ± 20.6%), COR (n = 6, 24.0% ± 22.0%), and BIO (n = 6, 20.7% ± 15.8%). Control sites showed statistically significantly more viable bone than sites implanted with BIO (t = −1.97; P = 0.05). Other comparisons were not statistically significant (P >0.1) (Fig. 6). Figure 6. Mean (± SD) area of bone, trabeculae, and osteoid tissue and biomaterial (%) in the core biopsies. This figure is based on summarizing statistics (group means ± SD) calculated based on subject means for the three central sections of each biopsy. The insertion torque measured during implant insertion showed a similar value for all sites for the first one-third of implant insertion, approximating 4.3 Ncm/turn (Fig. 7); however, for the last one- third of implant insertion, controls and sites implanted with BIO produced the highest mean values (>16 Ncm/turn), with controls showing greater variability. Figure 7. Mean (± SD) insertion torque force for the first (OSSEO1) and last third (OSSEO3) of implant insertion at 6 months postextraction/bone augmentation. This figure is based on summarizing statistics (group means ± SD). Summarizing statistics were calculated based on subject means for each measurement performed during implant placement in one of the biomaterials/control sites. The electronic mobility testing device applied at abutment insertion showed similar results for implants placed into sites receiving FIS, BIO, or COR (−2 to −3). The implants placed into control sites showed the lowest values (−4), reflecting the highest rigidity for the implant–bone continuum (Fig. 8). Clinical assessments showed stable implants for all sites at abutment placement. Figure 8. Mean (± SD) electronic mobility testing device values (PTV) at abutment insertion. This figure is based on summarizing statistics (group means ± SD). Summarizing statistics were calculated based on subject means for each measurement performed at abutment placement (second-stage surgery) on the implants placed in one of the biomaterials/control sites. The Pearson correlation showed a statistically significant relationship between the amount of newly formed bone and insertion torque during the last part of implant insertion (r [r = correlation; 64 = the number of data in the analysis] = 0.24; P = 0.05). Furthermore, a statistically significant relationship could be established between residual bone biomaterial and the insertion torque during the first part of implant insertion (r = 0.37; P <0.05). The Spearman correlation between residual biomaterial and electronic mobility testing device recordings at abutment connection was statistically significant (r = 0.24; P = 0.05). DISCUSSION The use of bone biomaterials has been advocated to prevent the collapse of the bone walls of extraction alveoli.15 Although not measured in this study, the contour of the alveolar bone observed at implant placement was within the limits for implant placement without grafting. The goal of this study was to use histomorphometric analysis and torque measurements at implant insertion to assess the resorption, integration, and remodeling of the bone-filling materials. For histomorphometry, the area of the trephine biopsy was taken into account when measuring the amount of remaining bone biomaterial and the magnitude of bone ingrowth in the site 6 months after extraction. The trephine drill has an outer diameter of 2 mm, which is much smaller than any of the extraction sockets, and although drill guides were used to locate each extraction socket, it is possible that the trephine biopsies were not taken in the exact center of each socket. The percentage of bone in the biopsy area indicates the bone-forming capacity of each site, and the percentage of remaining filling material indicates the remodeling capacity after 6 months in vivo. To maintain proper sterility during clinic studies, a calibration of the bone defects was not possible; however, because all biomaterials were randomly applied and most sites were alveoli of singlerooted teeth, the size of the defects should have had no impact in this study. However, the anatomic morphology of extraction sockets is always different. Although the bone biomaterials were carefully applied to the sockets, the packing of the materials was subjective because pressure measurements were not taken at the time of application. The use of ePTFE membranes resulted in better and more predictable ridge dimensions for implant placement than control extraction sites.16 However, exposure of ePTFE membranes often leads to infection and requires their removal, which is why this event occurred at 2 months postextraction in this study. Smoking was shown to significantly decrease alveolar process width. Compared to non-smokers, smokers have lower radiographic bone density in the preexisting apical bone and extraction socket.17 Because smoking may lead to increased alveolar ridge size changes and may delay extraction socket healing, all subjects included in this case series were non-smokers. As other investigators5 showed for FIS, the material quickly disappeared after application, with only 5.6% remaining. The percentage of newly formed bone in biopsy sites filled with FIS was similar to that of control sites, and in both sites, the maturation process of newly formed bone showed ingrowth of blood vessels and osteocytes. Similar to previous studies,18,19 BIO remained in large amounts (20%) at the time of biopsy. No ongoing resorptive activity by osteoclasts was detected, but new bone apposition on the BIO particles was observed, reinforcing previous findings.20 A new finding in this study was that a relatively large amount (12%) of COR remained 6 months after placement in the biopsy sites. Ongoing resorption by osteoclast-like cells on the surface of COR particles at 6 months postextraction may favor further replacement of the material by osteoid tissue. The maturation process in the COR sites may continue after the 6-month healing period. The histomorphometric results for the three bone biomaterials had large standard deviations, which implied a subject-specific reaction to the different filling materials. Future studies with larger sample sizes should clarify this finding. A question that remains is whether the remaining filling material is unfavorable for the implant osseointegration process. For example, ongoing resorption by giant cells, osteoclasts, may lead to further resorption of COR material.21 The presence of giant cells before osseointegration can be cumbersome, although the results showed that full integration occurred in all sites where implants were placed. For polylactic acid, the orthopedic literature reveals that the accumulation of giant cells that leads to the fragmentation of FIS material22 may also lead to local bone resorption. Furthermore, localized decalcification induced by lactic acid and inconsistent alveolar bone healing were observed for polylactic–polyglycolic acid systems.23 For BIO, the remaining material may interfere with bone appositioning onto the implant surface, or it may promote overheating of the bone during the drilling procedure for the implant. Histologically, the present study revealed that bone appositioning only occurs in the BIO sites, whereas FIS and COR were never detected in close contact with newly formed bone. All bone-filling materials primarily serve as space maintainers to prevent fibrous tissue ingrowth into the defect, allowing osteoblasts to colonize the space and induce new bone formation. Thus, the contribution of two of the bone-filling materials, FIS and COR, to bone formation is questionable. The empty control sites showed little osteoid tissue and contained mostly mature bone trabeculae, demonstrating that bone regeneration and maturation occur before 6 months in extraction alveoli left empty. As expected, bone regeneration in GBR sites seems to occur more rapidly than in sites with bone biomaterials.24 Conversely, the lack of signs of resorption of BIO by giant cell activity brings into question the fate of the BIO material and how it influences the biomechanics of the installed implants. A previous study25 showed that remaining BIO particles embedded in the vicinity of implants led to a normal bone–implant interface at the histologic level. In another study,26 BIO material resorption was described as participating in the remodeling process, in contrast to a study27 that showed that BIO did not bioabsorb but moved to other locations in the site. The present study did not document the fate of the bone crest morphology 6 months after healing of the alveoli. However, a previous study5 showed that the use of bone-filling materials favored the maintenance of the original alveolar morphology. Similarly, the present study demonstrated that when the volume of the remaining filling material was added to the newly formed bone, the volumes of the filled sites were generally larger than the empty alveoli. Insertion torque during the first third of implant insertion was the same for BIO, FIS, and COR; however, a positive correlation was found between the amount of remaining filling material and the insertion torque. Similarly, the amount of formed bone positively correlated with insertion torque during the last part of implant insertion. Future studies with larger sample sizes should address the large standard deviations found in this study, which imply a subject-specific reaction to different filling materials. During the last third of implant insertion, higher torque values were recorded for empty alveoli. Torque values were analyzed statistically to compare the relationship between the amount of available bone and the torque; however, the statistical significance of the intergroup differences for the torque of each of the materials could not be measured because a larger number of sites was required for these calculations. Therefore, no statistical evidence from this study can be presented. Because most extraction sockets were deeper than 1 cm, and the implants placed were 13 or 15 mm, the last third of the implant could be placed in native bone. The presence of native bone at the tip of the implant may affect the torque measurements during the last part of implant insertion. The negative electronic mobility testing device values recorded for all sites at abutment placement indicated a sufficient degree of mineralization for all treatments after 6 months, and it should have allowed the implants to carry load-bearing structures without leading to undue micromovements that might have interfered with osseointegration.1 Similar to insertion torque measurements, the statistical significance of intergroup differences in electronic mobility testing device values could not be calculated because a larger sample size was required. CONCLUSIONS The present study confirmed that a relatively high amount of BIO and COR remained after 6 months of healing. COR showed ongoing resorption by giant cells at that time, compared to no cellular activity on the BIO surface. Thus, it is advisable to wait ≥6 months before implant placement when using these filling materials. Fast resorption/diffraction rates were observed for FIS. Invasion with new bone showed the highest rates in empty control and FIS alveoli. The high torque values observed during the final seating of the implants, i.e., the last third of the trajectory, indicated a good primary stability that is promising for subsequent bone apposition. Proper implant rigidity was observed for all sites at the abutment stage, thus indicating a stage of osseointegration that allows load bearing. This case series demonstrated that the prognosis of early implant osseointegration was not influenced by the insertion of the bone biomaterials used in this study. ACKNOWLEDGMENTS The study was supported, in part, in equal amounts by the manufacturers of the bone substitute materials: Geistlich Pharma, Wolhusen, Switzerland; Inoteb, Le Guernol, Saint-Gonnery, France; and Ghimas, Casalecchio, Italy. The authors report no conflicts of interest related to this case series.