The long-term fate of some biomaterials is still unknown, and the reports present in the literature are not conclusive as to whether these biomaterials are resorbed over time or not. Different reports can be found with regard to the resorption behavior of anorganic bovine bone (ABB). The aim of the present study was to provide a comparative histological and histomorphometrical evaluation, in the same patient, of 2 specimens retrieved from a sinus augmented with ABB and with anorganic bovine matrix added to a cell-binding peptide (PepGen P-15), respectively, after a healing period of 6 months and after 8 years of implant loading, to evaluate the resorption of both biomaterials. A unilateral sinus augmentation procedure with ABB (50%) and with PepGen P-15 (50%) was performed in a 54-year-old male patient. Two titanium dental implants with a sandblasted and acid-etched surface were inserted after 6 months. During this procedure, 2 tissue cores were retrieved from the sinus with a trephine, before implant insertion. After an additional 6 months, a fixed prosthetic restoration was fabricated. One of these implants, after a loading period of 8 years, fractured in the coronal portion and was removed. Both specimens, one retrieved after a 6-month healing period and the other after an 8-year loading period, were treated to obtain thin ground sections. In the 6-month specimen, the histomorphometry showed that the percentage of newly formed bone was 27.2% ± 3.6%, marrow spaces 35.6% ± 2.3%, residual ABB particles 25.1% ± 1.2%, and residual PepGen P-15 particles 12.1% ± 2.2%. In the 8-year specimen, the histomorphometry showed that the percentage of newly formed bone was 51.4% ± 4.8%, marrow spaces 40% ± 7.1%, residual ABB particles 6.2% ± 0.7%, and residual PepGen P-15 particles 2.4% ± 0.5%. Both biomaterials underwent significant resorption over the course of this study.

Introduction

The long-term fate of some biomaterials is still unknown, and the reports present in the literature are not conclusive as to whether these biomaterials are resorbed over time or not.111  Different reports can be found about the resorption behavior of anorganic bovine bone (ABB), and this topic is still a subject of controversy.2  A group of researchers found that ABB resorbs over time.5  Wallace et al3  reported that the amount of ABB gradually decreased over time and was completely absent in a 20-month sample. The resorption rate of ABB in vivo has been reported to be 2–3 years,4  and Tadjoedin et al5  reported a decrease of about 10% per year. Sartori et al6  found that the potential metabolization of ABB by osteoclasts could be confirmed by the progressive increase in relative bone volume in a 10-year period (29.8% at 8 months vs 86.7% at 10 years). Valentini et al7  reported that the density of ABB decreased by a little more than 10% between 6 and 12 months, suggesting a slow but active resorption. Berglundh and Lindhe8  found that ABB with time became integrated and subsequently replaced by newly formed bone. Zaffe et al9  found that in most patients, there was a scarcity or absence of ABB granules in the biopsies. Artzi et al10  evaluated morphometrically the rate of resorbability of an inorganic bovine bone material in a standardized intrabony defect in dogs at 3, 6, 12, and 24 months. They observed a resorptive phase of ABB up to 6 months but did not notice a continued resorption at up to 24 months examination. According to Yildirim et al,11  the inward growth of bone indicated a slow resorption of ABB. In in vitro studies from our laboratory, it was found that it was possible to generate cells with the characteristics of osteoclasts on the surface of different xenografts and that these cells were able to resorb these different biomaterials.1214  On the other hand, no evident signs of any major ABB resorption was found by other researchers1,15,16 ; Hallman et al1  reported that the mean size of the particles was similar in the 6-month and 3-year specimens. Mordenfeld et al17  did not find, after 11 years, any obvious signs of resorption or decrease in size of ABB particles over time.

The aim of the present study was a comparative histological and histomorphometrical evaluation, in the same patient, of 2 specimens retrieved from a sinus augmented with ABB and with anorganic bovine matrix added to a cell-binding peptide (PepGen P-15), respectively, after a healing period of 6 months and after an 8-year implant loading, to evaluate the resorption of both biomaterials. The peri-implant bone response to the implant surface and the bone-implant contact percentage have already been reported in another study.18 

Materials and Methods

A unilateral sinus augmentation procedure, by a lateral wall approach, with ABB (50%; Geistlich Bio-Oss, Geistlich, Wohlhusen, Switzerland) and PepGen P-15 (50%; DENTSPLY, Tulsa Dental Specialties, Tulsa, OK) was performed in a 54-year-old male patient. The patient had a noncontributory past medical history. At the initial visit, the patient underwent a clinical and occlusal examination, and radiographs were performed (Figures 1 and 2). Two titanium dental implants with a sandblasted and acid-etched surface (DPS implants, DENTSPLY-Friadent, Mannheim, Germany) were inserted after 6 months (Figure 3). During this procedure, 2 tissue cores were retrieved from the sinus with a trephine, before implant insertion. After an additional 6 months, a fixed prosthetic restoration was fabricated. One of these implants, after an 8-year loading period, fractured in the coronal portion (Figure 4) and was removed with a 5-mm trephine bur. Both specimens, one retrieved after a 6-month healing period and the other after an 8-year loading period, were treated to obtain thin ground sections.

Figures 1–4.

Figure 1. Panoramic radiograph showing pretreatment sinus anatomy before the regeneration procedure was performed. Figure 2. X-ray showing the sinus after the sinus-lifting procedure. Figure 3. X-ray showing 2 titanium dental implants inserted 6 months after the sinus-lifting procedure. Figure 4. X-ray showing an implant fractured in the coronal portion after an 8-year loading period.

Figures 1–4.

Figure 1. Panoramic radiograph showing pretreatment sinus anatomy before the regeneration procedure was performed. Figure 2. X-ray showing the sinus after the sinus-lifting procedure. Figure 3. X-ray showing 2 titanium dental implants inserted 6 months after the sinus-lifting procedure. Figure 4. X-ray showing an implant fractured in the coronal portion after an 8-year loading period.

Specimen processing

Both specimens were washed in saline solution and immediately fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15 M cacodylate buffer at 4°C and pH 7.4, to be processed for histology. The specimens were treated to obtain thin ground sections with the Precise 1 Automated System (Assing, Rome, Italy).19  The specimens were dehydrated in an ascending series of alcohol rinses and embedded in a glycolmethacrylate resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). After polymerization, the specimens were sectioned, along their longitudinal axis, with a high-precision diamond disk at about 150 μm and ground down to about 30 μm with a specially designed grinding machine (Exakt, Norderstedt, Germany). A total of 2 slides were obtained for each specimen. The slides were stained with acid fuchsin and toluidine blue. The slides were observed in normal transmitted light under a Leitz Laborlux microscope (Leitz, Wetzlar, Germany) and polarized-light microscopy (Leitz, Wetzlar, Germany).

Percentages of newly formed bone, residual grafted materials, and marrow spaces were measured by histomorphometric analysis, which were carried out using a light microscope (Laborlux S, Leitz, Wetzlar, Germany) connected to a high-resolution video camera (3CCD, JVC KY-F55B, JVC, Yokohama, Japan) and interfaced to a monitor and PC (Intel Pentium III 1200 MMX, Intel, Santa Clara, CA). This optical system was associated with a digitizing pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histometry software package with image-capturing capabilities (Image-Pro Plus 4.5, Media Cybernetics Inc, Immagini & Computer Snc, Milano, Italy).

Results

Six-month specimen

It was possible to observe residual particles of both biomaterials surrounded by newly formed bone. The presence of newly formed bone tissue was indicated by the high staining affinity for acid fuchsin. Some particles of ABB were surrounded by mineralized tissues, and the biomaterial particles had served as an osteoconductive scaffold (Figure 5). Some ABB particles were bridged by newly formed bone. Only a few particles of PepGen p-15 were in contact with newly formed bone, while other particles were encapsulated by fibrous soft tissues. No inflammatory cells or foreign-body reaction cells were present around the biomaterial particles. No gaps were present at the bone-particle interface, and the bone was always in close contact with the particles. Osteoblasts could be seen depositing osteoid matrix directly on the ABB particles (Figure 6). No osteoclasts were observed. Marrow spaces contained marrow stromal cells, adipocytes, and small blood vessels.

Figures 5–8.

Figure 5. Specimen at 6 months. It is possible to observe the presence of particles of anorganic bovine bone (ABB; lighter particles) and PepGen P-15 (darker particles). Newly formed bone appears to be strongly stained in red with acid fuchsin, and it presents wide osteocyte lacunae. The newly formed bone is mainly in contact with the smaller and larger ABB particles. Some of these ABB particles are completely surrounded by newly formed bone. Only a few particles of Pepgen P-15 are in contact with newly formed bone. Native bone is present on the left of the figure. Acid fuchsin-toluidine blue ×20. Figure 6. Specimen at 6 months. Rims of osteoblasts can be observed deposing osteoid matrix directly on the graft materials surfaces. No osteoclasts are visible. No foreign-body cells and no inflammatory cell infiltrate are present. Acid fuchsin-toluidine blue ×200. Figure 7. Specimen at 8 years. Biomaterial particles are embedded in a compact, mature bone tissue. Most of the ABB particles are completely surrounded by the bone. Some of the PepGen P-15 particles are completely surrounded by bone, while other PepGen P-15 particles are in contact or are lined by small marrow spaces. Acid fuchsin-toluidine blue ×40. Figure 8. Specimen at 8 years. Apical portion of the implant. Small residual particles of both biomaterials are completely embedded in compact, mature bone. Areas of remodeling are present, with some osteons present on the implant surface. Acid fuchsin-toluidine blue ×40.

Figures 5–8.

Figure 5. Specimen at 6 months. It is possible to observe the presence of particles of anorganic bovine bone (ABB; lighter particles) and PepGen P-15 (darker particles). Newly formed bone appears to be strongly stained in red with acid fuchsin, and it presents wide osteocyte lacunae. The newly formed bone is mainly in contact with the smaller and larger ABB particles. Some of these ABB particles are completely surrounded by newly formed bone. Only a few particles of Pepgen P-15 are in contact with newly formed bone. Native bone is present on the left of the figure. Acid fuchsin-toluidine blue ×20. Figure 6. Specimen at 6 months. Rims of osteoblasts can be observed deposing osteoid matrix directly on the graft materials surfaces. No osteoclasts are visible. No foreign-body cells and no inflammatory cell infiltrate are present. Acid fuchsin-toluidine blue ×200. Figure 7. Specimen at 8 years. Biomaterial particles are embedded in a compact, mature bone tissue. Most of the ABB particles are completely surrounded by the bone. Some of the PepGen P-15 particles are completely surrounded by bone, while other PepGen P-15 particles are in contact or are lined by small marrow spaces. Acid fuchsin-toluidine blue ×40. Figure 8. Specimen at 8 years. Apical portion of the implant. Small residual particles of both biomaterials are completely embedded in compact, mature bone. Areas of remodeling are present, with some osteons present on the implant surface. Acid fuchsin-toluidine blue ×40.

Histomorphometry showed that the percentage of newly formed bone was 27.2% ± 3.6%, marrow spaces 35.6% ± 2.3%, residual ABB particles 25.1% ± 1.2%, and residual PepGen P-15 particles 12.1% ± 2.2%.

Eight-year specimen

Biomaterial particles appeared to be embedded in compact, mature bone (Figure 7). Most of the ABB particles and some of the PepGen P-15 particles were completely surrounded by bone. No inflammatory cell infiltrate and no foreign-body reaction were present (Figure 8). No gaps were present at the bone-biomaterial interface. Osteons and cement lines, indicating areas of remodeling, were present.

Histomorphometry showed that the percentage of newly formed bone was 51.4% ± 4.8%, marrow spaces 40% ± 7.1%, residual ABB particles 6.2% ± 0.7%, and residual PepGen P-15 particles 2.4% ± 0.5%.

Discussion

The resorption times and the ultimate replacement of some xenografts by newly formed vital bone are still not fully understood.20  Most reports concern the resorption capability of ABB, while, according to the author's knowledge, no study is present in the literature about the metabolization rate of PepGen P-15. An ideal material should provide a framework for continuous bone resorption and bone deposition.21,22  The osteoclasts' resorption behavior plays a pivotal role in the integration of different bone substitute biomaterials into the bone tissue.21  The ability to form new bone must be balanced by the resorption rate of the biomaterial.22  Opposing views have been reported about the continued long-term presence of the grafted biomaterials particles, and the controversy continues. On one hand, the presence of graft particles in about 25% to 30% of the treated site volume may interfere with normal healing, by disrupting vascularization and depriving cells of nourishment. This could create problems for the osseointegration of implants.10  From a theoretical standpoint, the lack of resorption of a xenograft could produce a negative consequence on the biomechanical properties of the augmented sites and their implant-supporting capabilities, because the augmented area is composed of a composite rather than a homogenous bone structure.1  It is still unknown if the long-term success of implants could be compromised by their insertion into sites where there is still the presence of residual graft particles. Some clinicians prefer implant insertion in areas of only vital bone and not in bone areas regenerated by bone substitute materials.23  However, the structure constituted by bone and grafted particles could, on the other hand, be advantageous and behave as a cancellous bone network around an implant.10,24  In some clinical indications, a low substitution rate may be beneficial, during which the physical support from the graft material maintains the initial dimensions of the augmented volume area and prevents soft-tissue collapse, providing a stress shield against pressure exercised by the overlying gingival or mucosa of the sinus.15,24  Moreover, the almost complete incorporation of the ABB particles in bone creates a dense, hard tissue network.24  It seems likely that this network provides mechanical support to loaded dental implants, which is comparable to or even exceeds that of native bone.24  ABB resorption does not seem to be absolutely necessary to provide predictable osseointegration.25  In the present specimens, a significant decrease in the percentage of both biomaterials over the long term was found. This is in contrast with the studies by Hallman et al,1  Artzi et al,26  and Mordenfeld et al,17  who did not find any sign of resorption of the grafted biomaterials over time. Moreover, the progressive increase in the percentage of the newly formed bone over time could point, as already stated by Sartori et al,6  to a significant resorption of both grafted biomaterials.

Conclusion

Both biomaterials underwent a significant resorption over the period of this study. Both biomaterials were osteoconductive, and their continued presence in the peri-implant bone did not produce any untoward effect. Both biomaterials appeared to be histologically biocompatible in that no inflammatory cell reactions and no foreign-body reactions were observed.

Abbreviation

     
  • ABB

    anorganic bovine bone

Acknowledgment

The work was partially supported by the Ministry of Education, University and Research (M.I.U.R.), Rome, Italy.

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