This study compared cellular responses of maxillary sinuses after augmentation with anorganic bovine-derived hydroxyapatite matrix linked to the cell binding polypeptide P-15 (ABM/P-15) or PepGen P-15 and allogenic freeze-dried cranial bone slabs. Five adult Macaque fascicularis monkeys were used. On one side, the floor of the sinus was augmented with ABM/P-15, while the other side was augmented with 2 cranial bone slabs. Trephine bone biopsies were obtained 6, 12, and 24 weeks postgrafting. Animals were sacrificed 8 months after grafting. Soft X-ray microradiography was used to determine bone density. The volume fraction (Vv) of regenerated bone, the number of mesenchymal cells, and the numbers of proliferating cell nuclear antigen (PCNA)- and alkaline phosphatase–positive cells at different augmentation sites were measured and compared. Basal bone heights were calculated at surgery and compared with total heights of the augmented sinus floors 8 months postgrafting. Bone formation, number of mesenchymal cells, PCNA index, and alkaline phosphatase index were significantly higher for the ABM/P-15 side than for the allogenic bone–augmented side. Both sides ended with a significant increase in bone height. The PCNA index decreased significantly over time (P < .05), while the alkaline phosphatase index increased significantly (P < .05) over time on both sides. Both graft materials have led to significant augmentation of the floor of the maxillary sinus with new bone; however, new bone formation and maturation were faster on the ABM/P-15 sites.
Many edentulous patients present with severe alveolar bone resorption of the posterior maxilla, which is further complicated by pneumatization of the maxillary sinus. The latter is often accompanied by a decrease in the vertical interarch space, which makes placement of conventional prostheses problematic. In severe atrophy of the posterior maxilla, the remaining ridge is often osteopenic, and the trabecular bones lose their interconnectivity, leading to poor-density bone.1 These bone changes will compromise the optimum biomechanical foundation required for implant placement. The placement of implants in bone onlays on top of the atrophied maxilla in the presence of insufficient interarch space is not applicable.2,3 Many dental implantologists have described the surgical procedure for safe access to the maxillary sinus to lift the sinus membrane and to place graft material for sinus floor augmentation.4
In this study, we are presenting bone core biopsy as a useful method for studying the response of the Rhesus monkey's maxillary sinus floor to 2 grafting materials. PCNA, which is a marker for cell proliferation, and alkaline phosphatase, which is a marker for osteoblastic cell differentiation, were used to compare the effectiveness of the used graft material in terms of their ability to induce cellular proliferation and differentiation of the osteoblastic lineage. Morphometric quantitative measurement of the volume of newly formed bone and densitometric evaluation of the bone cores, as well as the final specimens, were employed. The graft materials chosen for this study were the anorganic bovine-derived hydroxyapatite matrix linked to the cell binding polypeptide P-15 (ABM/P-15) (PepGen P-15, Dentsply Friadent Co, Mannheim, Germany) and allogeneic cranial bone slabs. Each monkey had 1 sinus grafted with ABM/P-15 and the contralateral side grafted with 2 allogenic cranial bone slabs. Strong evidence supports the efficacy of ABM/P-15 in the enhancement of osteogenesis of human periodontal bone defects and in human maxillary sinus augmentation using clinical and radiographic assessment methods.5,6 The mechanism of action of ABM/P-15 is believed to enhance collagen and bone formation by facilitating cellular attachment to fibroblasts and osteoblasts with a subsequent increase in cell binding proliferation.6,7 The present study will present experimental evidence in support of this hypothesis. Cranial bone slabs in the contralateral sinus were used for comparison with the ABM/P-15 side. The trephine bone biopsy technique was used to study the fate of the bone grafts at different time periods.
Materials and Methods
The experiment was approved by the Medical College of Georgia Animal Use in Research Committee. Five adult (13–18 years of age) Macaque fascicularis monkeys were used in this study, and they served as their own controls. All animals were immunized against rabies and were free of herpesvirus and tuberculosis.
The anorganic bovine bone hydroxyapatite linked to P-15 (PepGen P-15) was supplied by Dentsply Company as 1.2 cm3 rounded granules (40–60 mesh) in a syringe. Each sinus received approximately 1 g of PepGen P-15.
Preparation of freeze-dried allogenic cranial bone
Fresh-frozen Macaque fascicularis monkey heads were obtained from Yerk's Primate Center in Atlanta, Georgia. The cranial vault was exposed by sharp dissection under aseptic conditions. Cranial bone full-thickness slabs measuring approximately 5 mm × 8 mm were cut from the parietal bones and processed for sterilization and antigenic control.
All surgical procedures were done under general anesthesia. The animals were anesthetized using a combination of ketamine (10–15 mg/kg) and acepromazine (0.1–0.5 mg/kg) injected intramuscularly. These were supplemented by Surital (sodium thiamylal) given intravenously (IV) at a dose of 10–15 mg/kg after an IV line was established. Atropine (0.4 mg) was given IM before surgery. Diagnostic periapical radiographs of posterior maxillary teeth were obtained.
The first surgical procedure involved the surgical extraction of the last 2 maxillary molars bilaterally, followed by alveolectomy and wound closure with Vicryl suture. All animals were put on a soft diet and postoperative antibiotics (250 mg ampicillin) every 6 hours for 5 days. The wounds were left to heal for 6 weeks before the next procedure was performed. Healed sites represented a good model for human atrophied posterior edentulous maxilla.
Procedure II: Bilateral Maxillary Sinus Augmentation
A full-thickness mucoperiosteal flap was made in anesthetized monkeys to expose the lateral surface of the maxilla. A window was made with the aid of a rounded bur and copious saline irrigation. The outlined bone was carefully removed with a small bone curette to expose the membrane lining the sinus. The buccal opening was widened with bone cutters to obtain 10 mm horizontally and 5 mm vertically, with 2–3 mm of bone retained between the inferior margin of the window and the alveolar crest (Figure 1). The sinus membrane was carefully reflected from the alveolar recess using sinus elevation curettes. The reflection of the membrane continued to the anterior wall of the sinus, posteriorly to the maxillary tuberosity, and medially to about one half of the sinus wall. The floor of the sinus was decorticated (∼1 mm) by using a low-speed round bur. Before graft placement, the residual height of the alveolar bone was measured using a caliper. Such measurements served as the baseline preoperative control values and were used for comparison with values obtained from the final specimens (Figure 2).
ABM/P-15 graft (left side)
One gram of ABM/P-15 powder was placed and spread on the sinus floor (Figure 3).
The mucoperiosteal flaps were then closed using Vicryl suture. Postoperative analgesics and antibiotics (250 mg ampicillin IM every 6 hours for 5 days) were administered.
Cranial bone graft (right side)
Two full-thickness slabs of allogenic freeze-dried calvarian bone were cut to fit into the sinus floor (Figure 4). The slabs were immersed in normal saline solution for 1 hour for hydration before the operation. The average length was 18 mm, mediolateral width 8 mm, and thickness 1.8 mm. The 2 slabs were held in position against the sinus floor with a bone clamp. A 0.8-mm-diameter, 8-mm-long titanium screw from the oral side was drilled through the alveolar bone and the 2 slabs.
Procedure III: Core Biopsy
Three core biopsies were obtained from each sinus of each monkey at 3 time intervals—6, 12, and 24 weeks after sinus augmentation—using a 2-mm-diameter trephine bur (Figures 5 and 6). The first core biopsy was obtained through the gingival, and the 2 other cores were obtained after a reflection of mucoperiosteal flap. All biopsies were taken under copious irrigation with chilled saline. The cores were retrieved intact from the trephine, and the gingival end was marked with eosin stain and then fixed in 4% buffered formalin. The surgical site was irrigated with normal saline and then was sutured using Vicryl resorbable suture material. The bone cores were photographed and then were subjected to contact soft X-ray microradiography (Figure 6). The cores were decalcified in ethylenediaminetetraacetic acid (EDTA) and stained using hematoxylin and eosin stain and modified Mason stain for light microscopic and histomorphometric studies. Unstained sections were used for immunohistochemical staining for PCNA (a marker for cell proliferation) and alkaline phosphatase (a marker for osteoblast cell differentiation).
Procedure IV Animal Euthanasia
Eight months postgrafting, each deeply anesthetized monkey was perfused with 4% buffered formalin. The maxilla was dissected and separated in a way to maintain the medial wall of the sinus intact. Coronal 5- to 8-mm-thick sections were made using an Isomet low-speed saw (Buehler Ltd, Lake Bluff, Ill). Each section was examined and photographed under the dissecting microscope. The residual bone height and the augmented floor of the sinus were measured using a polygauge. Half of the sections were decalcified in EDTA solution for 8–12 weeks, and the other half were left undecalcified and were processed for methyl methacrylate (MMA) embedding. The MMA blocks were sectioned at a thickness of 10 micrometers using a Polycut S microtome (Reichert Scientific Instrument, Buffalo, NY). The sections were then stained with modified Mason stain. After enough thin sections were obtained, thicker sections (500 micrometers) were obtained for soft X-ray microradiography.
Contact soft X-ray microradiography
All core bone biopsies and one 500-micrometer-thick calcified section from each of the final specimens were subjected to contact soft X-ray microradiography (20 kV and 2.9 mA for an exposure time of 5 minutes). To determine the density of the grafted sinus floor and to compare it with normal bone density in the same specimen, a rectangular standardized region of interest was drawn on different areas of the radiographic image using Image software (National Institutes of Health, Bethesda, Md). The average bone density expressed as gray level was calculated for normal basal bone and sinus floor. The statistical comparison was done using Statistical Analysis System (SAS) software (SAS Institute, Cary, NC).
Two types of immunostaining techniques were used to localize PCNA and alkaline phosphatase activity as a measure of cell proliferation and cell differentiation, respectively.
PCNA- and alkaline phosphatase–positive cells had their nuclei stained deep brown. PCNA and alkaline phosphatase indices were determined by counting the numbers of positive cells as percentages of total cells counted per unit area of the tissue examined using Image Pro Plus software (Media Cybernetics, Silver Spring, Md). For the histomorphometry, 5 stained sections from the final specimens, or 3 slides per core biopsy per sinus, were used. The areas of new bone (contain bone lacunae filled with osteocytes) in a randomly selected unit area of the grafted sinuses vs the areas of bone grafts were calculated. The same was done for the bone cores. Values were expressed as percent volume fraction (Vv). Data were collected using Image Pro Plus software, and data were transferred to Microsoft Excel for graphing and statistical analysis.
Continuous variable outcome measures were analyzed using 2-way analysis of variance followed by mean comparisons (n = 5/group). Discrete outcome variables were analyzed using nonparametric analysis of variance. Multiple comparison tests were done using least squares means procedures and SAS.
Gross morphologic appearance of core biopsies
Under the dissecting microscope, the core biopsies had no distinct interface between the host basal bone and the grafted floor of the sinus. The area of the graft showed the ABM granules interconnected by hard and soft tissue. No sign of infection or tissue necrosis was noted in any of the bone cores at a gross or histologic level. No noticeable differences between the 6-week, 12-week, and 24-week core biopsies were observed at the gross level.
Allogenic Cranial Bone Side
Under the dissecting microscope, all cores showed a distinct, but intimate, interface between the maxillary basal bone and the area of the graft. No sign of infection or tissue necrosis was noted in any of the cores at a gross or histologic level.
Contact soft radiographs of core biopsies
The grafted areas appeared radiopaque with a clear distinction between the basal bone and the graft. The basal bone next to the ABM/P-15 side appeared more radiopaque than that next to the allogenic bone side (Figure 6).
Gross morphology of the augmented maxillary sinuses 8 months postgrafting
The sinuses were split in the coronal direction and were observed under a dissecting microscope. The sinus membrane was well adapted to the graft area, which was composed of bone and ABM particles. Bone from the basal bone could be traced up into the grafted area. The areas from which the bone cores were taken could not be found and apparently completely healed. Also the windows that were created in the lateral walls of the sinuses to introduce the grafts were completely healed.
Allogenic Cranial Bone Side
An intimate interface was found between the 2 slabs and between the slabs and the alveolar bone. No sign was found of the buccal defects that were created in the anterolateral surfaces of the maxillae at the time of bone grafting.
Contact soft X-ray microradiographs of final specimens
The ABM/P-15 granules were still evident at the floor of the sinus and appeared much denser than the surrounding bone (Figure 7). The cranial bone graft sides showed the 2 slabs connected to each other and to the floor of the sinus with radiopaque bone. Newly formed bone could be observed between the sinus membrane and the bone slabs (Figure 8).
Assessment of augmented bone heights
The maximum height of the atrophied edentulous alveolar basal bone measured at the time of surgery was 3.5 mm, and the minimum height was 1.5 mm, with a mean of 2.56 mm (±0.8). The maximum bone height measured from the allogenic cranial bone sides was 13.5 mm, and the minimum was 8.3 mm, with a mean of 10.87 mm (±2.7). At the ABM/P-15–grafted side, the maximum height was 16.2 mm, and the minimum was 9.7 mm, with a mean of 13.17 mm (±2.83). For both grafted sides, total bone heights achieved 8 months after grafting were significantly higher than the height of the basal bone at the time of surgery (P < .01). Although achieved bone heights on the ABM sides were higher than those on the allogenic bone sides, the difference was not statistically significant (P = .94) (Figure 9).
Qualitative histologic study
The graft was composed of ABM particles, connective tissue, and new bone. The connective tissue around the ABM particles was composed at 6 week bone cores of undifferentiated mesenchymal cells and collagen fibers, with no sign of inflammation. New bone surrounded many of the ABM particles (Figure 10). Blood vessels were seen within ABM particles. At 12 weeks, the bone cores showed more evidence of bone remodeling such as resting lines and maturation into lamellar bone (Figure 11). Multinucleated osteoclasts were observed around some ABM particles, indicative of graft resorption (Figure 12). Osteoblasts were also seen invading the graft materials. At 24 weeks, the bone cores showed more bone surrounding the ABM particles. The bone appeared more mature and lamellated (Figure 13). Evidence of graft resorption was seen as osteoclasts surrounded the small ABM particles. Bone formation was also seen within the ABM particles.
Allogenic Bone Graft Side
Cranial bone slabs can be distinguished easily by their empty osteocytic lacunae. At 6 weeks, a narrow layer of new cellular bone was found on the surface of some areas of the graft with a distinct resting line. Connective tissue rich in mesenchymal cells and collagen fibers was found next to the layer of new bone. By 12 weeks, more evidence of bone formation around the graft and tunneling of the slabs with blood vessels and active osteoblasts were seen. Remodeling of the acellular graft is aimed at replacing the graft with new vital bone and is known as creeping substitution. Such processes were more evident at the 24-week core biopsy. Also, after 24 weeks, the bone appeared more mature, and the number of osteoblasts lining the remodeling tunnels vastly increased.
Sinuses 8 months postgrafting
At low magnification, the sinus membrane appeared as pseudostratified ciliated columnar epithelium and was well adapted to the graft materials. ABM particles next to the membrane were surrounded by healthy vascular connective tissue, while the deeper particles were surrounded by vital bone (Figure 14). The bone appeared mature and rich in osteocytes and has many active osteoblasts on its surface. Small ABM particles were seen within the bone. In the modified Mason stained undecalcified sections, the mineralized bone appeared blue, while osteoid on the bone surface appeared red, indicative of continuing osteogenesis 8 months after grafting (Figure 15). Bone and connective tissues were seen within ABM particles. Multinucleated osteoclasts were present around some ABM particles, with saucerization of the outer surface of the particles denoting resorption activity.
Allogenic Bone Graft Side
The cranial bone slab 8 months postgrafting was still evident at low magnification. At high magnification, the graft was invaded by a large number of tunnels containing blood vessels and active osteoblasts forming new bone (creeping substitution). The modified Mason stain showed a layer of osteoid (stained red) underneath the active osteoblasts, indicative of active osteogenesis 8 months postgrafting (Figures 16 and 17).
Number of Mesenchymal Cells
The number of mesenchymal cells was significantly higher in the core biopsies of the ABM/P-15 side in comparison with the cranial bone side (P < .01, 6 weeks; P < .01, 12 weeks; and P < .01, 24 weeks [biopsies]). Although the numbers of mesenchymal cells at the ABM/P-15 sides 8 months postgrafting were higher than those at the cranial bone graft sides, the difference was not statistically significant (P > .05). Also the number of mesenchymal cells decreased significantly (P < .02) in relation to the postgrafting time, regardless of the type of graft material used (Figure 18).
Proliferative cell nuclear antigen index
At the ABM/P-15 side (Figure 19), the PCNA index was highest at the 6 week core biopsy and then continued to decline and reaches its lowest value in the final specimens. The same was true for the allogenic cranial bone side. The PCNA index was higher in the ABM/P-15 side; however, the difference was statistically significant only on the 6-week biopsy (P < .01) and the 24-week biopsy (P < .01). The PCNA index declined significantly (P < .01) in proportion to the time, regardless of the graft material used (Figure 20).
Alkaline phosphatase index
The alkaline phosphatase index (ALP) index (Figure 21) was lowest at the 6-week core biopsies and highest in the 8-month specimens, regardless of the graft material used. The increase with time was statistically significant (P < .01). The ALP index was higher on the ABM/P-15 graft side when compared with the allogenic bone side; however, the difference was statistically significant only in the 8-month specimen (P < .02) (Figure 22).
Volume fraction of new bone
The Vv of new bone progressively increased from its lowest value at the 6 week biopsies until reaching its maximum in the 8-month specimens (P < .01). This observation was true, regardless of which graft material was used. More bone was present in the 6-week (P < .01) and 24-week (P < .01) biopsies and in the 8-month specimens (P < .05) on the ABM/P-15 side when compared with the allogenic cranial bone side (Figure 23).
The mean density of normal maxillary bone at the sinus floor away from the experimental area was 59.66 (±3.0).
Density of the augmented area of the sinus floor
As expected, the density of the grafted area, whether it was ABM/P-15 or allogenic cranial bone, was significantly higher than that of the normal sinus floor bone. It is interesting to note that the densities of the grafts on both sides progressively increased with time (P < .01) (Figure 24). Although the densities of the ABM/P-15 grafts were higher than those of the allogenic bone grafts, the difference was not statistically significant (P > .05).
Density of the basal bones next to the graft
The mean density of the basal bone was higher than the density of the normal bone in all measurements except for the 6-week and 12-week core biopsies on the allogenic bone side. However, the difference was not statistically significant, except in the case of the ABM/P-15 side, in the final specimens 8 months after grafting (P < .04) (Figure 24).
The purpose of the present study was to follow the fate of 2 different graft materials after the floor of the maxillary sinuses was augmented using trephine core biopsies. The animal model we chose was the Rhesus monkey, which has the same maxillary sinus anatomic features and a bone metabolism quite similar to that of humans.1,8 The surgical access to the sinus was made through a window in the lateral wall, as is most commonly done for the human sinus.4 The first graft used was freeze-dried allogenic cranial bone, which from our previous studies, as well as those of others, proved to be a reliable graft when used for ridge augmentation without (1 stage) and with (2 stages) implant placement.9,10 The second graft chosen was the anorganic bone matrix linked to a cell binding polypeptide P-15. Several clinical and experimental research reports have pointed to the enhancement of bone formation when ABM/P-15 was used as filler of periodontal bone defects5,11,12 or in association with endosseous implants.13
Gross examination of the sinuses 8 months after grafting clearly showed integration of the ABM/P-15 graft with the basal bone of the maxilla; in contrast, a marked distinction of the 2 slabs of cranial bone was seen at the floor of the contralateral sinus. This may be due to the known slow rate of resorption of the cranial bone in contrast to other types of bone grafts such as the iliac crest.10 Measurements of bone height achieved 8 months after grafting revealed that both techniques were successful in significantly augmenting the sinus floor (300%–400% more than control operative height). These results are the measure of success of the final outcome of the grafting procedures employed in this study, and they represent the quantitative and qualitative information needed before appropriate dental implants are selected. The number of mesenchymal cells was significantly higher in the ABM/P-15 sites than in the cranial bone sites. This observation confirms previous studies using a cell culture system.14 The significant increase in the PCNA index at the 6 week bone biopsy further supports the role of P-15 in stimulating cell proliferation. Enhancement of bone formation after ABM/P-15 is used as a graft material in sinus floor augmentation6 and in periodontal bone defects5,11,12 in humans and around the cervical portions of implants in dogs13 has been reported and is consistent with the present experimental results. This was interpreted to be the result of stimulation of cell differentiation of mesenchymal cells into bone-forming cells by P-15 linked to the ABM particles. These results support those of Bhatnagar and Qian,14–18 who studied the ability of P-15 in the promotion of fibroblast cell attachment to ABM in vitro. P-15 was found to be 18 times more effective than collagen in cell binding. Cells growing on the dish surface with P-15 stained deeply for alkaline phosphatase. These studies also showed that cells on P-15–coated ABM particles synthesized more than twice as much protein and DNA than did cells on the uncoated particles. Bone density measurements revealed as expected that as early as 6 weeks, the ABM/P-15, which is primarily made of biological hydroxyapatite, was much denser than both the host maxillary bone and the cranial bone graft. Over time, the density of both graft sites increased, perhaps reflecting the observed remodeling activity that tended to replace the graft materials with new bone. The density of the maxillary host bone under the graft also progressively increased over time, regardless of the graft material used. However, the density of the maxillary bone adjacent to the ABM/P-15 was significantly higher than that of the normal maxillary bone and the maxillary bone in juxtaposition to the cranial bone graft. This may indicate that ABM/P-15 is a better mineral reservoir than allogenic cranial bone.
In conclusion, our results clearly show that ABM/P-15 is a strong osteoconductive material that is able to attract cells to the site of the graft and stimulate their differentiation into adherent active osteoblasts, which eventually results in significant accelerated osteogenesis. The trephine bone biopsy is a dependable technique that allows determination of the amount of new bone in the grafted area and evaluation of the cellular response of the host. More investigation is needed to evaluate the fate of the augmented bone under occlusal stress after insertion of functional implants.
The authors wish to acknowledge the grant support received from the Egyptian Cultural and Educational Bureau. The PepGen P-15 graft materials were donated by Dentsply Company, which the authors appreciate. The monkey calvaria were gratefully received from Yerk's Primate Center in Atlanta, Ga. The authors also wish to thank Ms Vera Larke for her indispensable skillful technical help throughout the project, Ms Linda Cullum for typing the manuscript, and Carl Russell, DDS, PhD, for his valuable assistance with the biostatistics.