Implants can be a treatment option when there is sufficient quantity and quality of bone to provide support for long-term success. In the reconstruction of defects, autogenous bone remains the gold standard for its osteogenic and compatibility properties. However, the disadvantage of secondary surgery and the associated donor site morbidity prompts researchers to develop the ideal bone substitute for optimum bone reconstruction. Parathyroid hormone (PTH1-34) has provided a new option for improvement in bone regeneration. This study used a pig model to evaluate the effectiveness of parathyroid hormone when added to a xenograft, Bio-Oss, in reconstructing mandible defects. Six domestic pigs were used to create 3 posterior mandibular defects measuring 2 × 1-cm bilaterally with a total of 36 defects to simulate tooth extraction sites in humans. The defects were grafted in random order and divided into 3 groups as follows: control (no graft), Bio-Oss without PTH, and Bio-Oss with PTH. Defects were assessed with cone beam computerized tomography (CBCT), micro computerized tomography (microCT), nanoindentation, and histology. Results showed that adding PTH1-34 significantly enhanced the graft construct. CBCT showed a significant increase in the degree of bone mineralization. Nanoindentation showed increased hardness of regenerated bone and accelerated bone mineralization with PTH. MicroCT analysis revealed a trend toward higher bone regeneration and mineralization. The histological analysis showed a positive trend of the increase in cortical bone thickness and mineral apposition rate. In conclusion, the local addition of PTH1-34 to a xenograft has shown promising results to enhance bone regeneration in the reconstruction of mandibular defects.
Dental implants have become an excellent treatment modality since their inception into the modern era of dentistry. Implants are a surgical treatment option when there is sufficient quantity and quality of bone to provide optimum support and predictable long-term restorative success. When there is inadequate quantity and quality of bone, 3-dimensional surgical reconstruction and modifications of the preexisting bony anatomy are required for optimal implant placement.1
Regeneration of bone defects is one of the most challenging topics in the field of implant dentistry. There is no known material that satisfies the requirements to be the ideal graft. Although there is global acceptance that autogenous bone remains the gold standard for its osteogenic and compatibility properties, there is the disadvantage of a secondary surgical site and the associated donor site morbidity.2 Many researchers are focused on testing the efficacy of different bone substitutes to achieve optimum results that will overcome the disadvantages of harvesting autogenous bone.2,3 Several studies have described the use of different graft sizes and porosities in addition to the use of carrier materials and growth factors to achieve the ideal criteria for a graft material.4 A quality bone substitute, which will remain vital to allow better function, as in osseointegration around dental implants or reconstruction of defects following treatment of pathological lesions, is now a target for many clinicians to achieve long-term success.
Parathyroid hormone analogues (PTH1-34) have provided a new effective option for potential improvement in bone regeneration. This hormone is frequently used for the treatment of severe osteoporosis and delayed healing of skeletal fractures.5,6 In this study, we propose that the local addition of PTH to mineralized bone xenograft will enhance bone formation and improve the quantity and quality of the regenerated bone. Since Bio-Oss is one of the most commonly used and studied xenograft materials in implant dentistry, we decided to evaluate the effect of the local addition of PTH to this graft using a large animal model.7
Materials and Methods
Six healthy domestic pigs (Sus scrofa) of either gender, weighing 30 to 35 kg, were procured for our study. All pigs were acclimated to the animal facility for several days before the surgical operation detailed below. The local institutional animal care and use committee (IACUC) approved all live animal procedures.
Creation of osseous defects
All pigs were anesthetized with tiletamine hydrochloride (100 mg/mL at 6 mg/kg body weight intramuscularly [IM]) followed by oral intubation. A state of general anesthesia was maintained with an inhaled mixture of 1%–3% isoflurane in oxygen. A bilateral procedure was completed on all 6 of the pigs as follows: a 4-cm incision was created along the inferior border of the mandible that passed through skin, subcutaneous tissue, and muscle down to the periosteum. The periosteum was incised at the inferior border to expose the mandible from the ramus to the body regions. To prevent variable regeneration potential, the periosteum was excised and discarded. The predetermined dimension of the osseous defects was measured and outlined using a ruler and sterile marker. In an attempt to simulate an alveolar defect following tooth extraction, 3 bicortical defects were created between the body (posterior to the canine) and angle of the mandible (mesial to the last molar). The width of each defect was 2 cm, and the height was 1 cm. All defects were 1 cm above the inferior mandibular border and were created surgically by a reciprocating saw under copious saline irrigation. All sites were irrigated with saline and cleared of any residual soft tissues in preparation for grafting (Figure 1).
Insertion of the graft material
The created defects (n = 36) were then randomly treated by 1 of 3 materials. In one defect, xenograft material (Bio-Oss, small granules) was positioned and filled to capacity as a sole graft. In the next defect, the same xenograft material (Bio-Oss, small granules) was mixed with 20 μg (2.4 mL) of PTH analogue (teriparatide). To prepare for this xenograft-PTH mix, and to prevent the washout of PTH if placed directly on the graft particles, the PTH was first applied to a collagen sponge as a carrier (Collatape, Zimmer Dental, Palm Beach Gardens, Fla), then cut into small pieces, and finally mixed with the xenograft. Same as the previous description, this material was positioned and filled to capacity. Since the membrane is readily very compressible, we believe that firm packing of the graft prevented the membrane from creating any significant spaces between the graft particles. The final defect was used as a control; therefore, no graft material was inserted (Figure 2). An absorbable collagen membrane (Collatape, Zimmer Dental) was used to cover all sites (Figure 3). The soft tissue was reapproximated, and a layered closure was performed in the usual fashion using absorbable sutures (Vicryl 3/0 for deep layers and subdermal Monocryl 3/0 for the skin).
Postoperative pain control was provided by a fentanyl patch (2 μg/kg) applied to the skin of the ear 24 hours before surgery. Two days postoperatively, the patch was removed, and analgesia was provided with flunixin meglumine (2.2 mg/kg IM) once daily for an additional 3 days. Infection control was provided with either penicillin G benzathine/penicillin G procaine (20 000 IU/kg, IM every other day for 1 week) or clavulanate-potentiated amoxicillin (22 mg/kg IM twice daily for 7 days). The pigs were given water-softened chow twice daily and water ad libitum until termination for the remainder of the experimental period. In preparation for future histopathological analysis, fluorescent bone labels calcein and alizarin complexone (12.5 mg/kg) were injected into each pig 10 and 3 days, respectively, before euthanasia.
Acquisition of specimens
All 6 experimental animals were sacrificed at 12 weeks postoperatively. Pigs were anesthetized with telazol (6–10 mg/kg IM). Once a surgical plane of anesthesia was confirmed, a lethal dose of pentobarbital, euthasol, and/or saturated potassium chloride was administered through an intravenous or intracardiac route. All euthanasia procedures were in accordance with the previously mentioned IACUC guidelines obtained prior to starting the study. The mandible was harvested for postmortem imaging and analysis. The soft tissues surrounding the mandible were removed to harvest the operated sites. At this point, 8 of the original 36 defects were excluded from the study because of infection and/or fracture. The final specimen count was 28, in the form of 8 control, 10 xenograft, and 10 xenograft + PTH. The obtained specimens were trimmed and fixed with 4% paraformaldehyde.
Methods of analysis
Cone Beam Computerized Tomography
A clinical cone beam computerized tomography (CBCT) scanner scanned all mandibular specimens at 300- × 300- × 300-μm3 voxel sizes with a scanning energy of 120 kV and 5 mA in a period of 8.9 seconds. This setting was chosen to correlate with scanning conditions commonly used in clinical practice. A heuristic algorithm was used to segment bone voxels from nonbone voxels of those CT images as described in a previous study.8 The gray level of each bone voxel, which is proportional to bone mineral density (BMD), was maintained in the process of bone voxel segmentation. Individual surgical sites were digitally dissected from the segmented CBCT images (Figure 4a). The gray-level histograms of each sites were obtained to determine BMD distribution (Figure 4b). The mean value was computed by dividing the sum of gray levels by the total count of voxels. Standard deviation (SD) shows heterogeneity of the gray-level distribution. Low and high gray levels (Low5 and High5) were determined at the low and high fifth percentiles of voxel counts in the histogram, respectively (Figure 4c). It is worth mentioning that we choose to apply CBCT as a method of analysis since it is currently one of the most widely used techniques in clinical practice.
Micro Computerized Tomography
After CBCT scanning, the individual surgical sites were dissected (8 control, 8 xenograft, and 10 xenograft + PTH) using a low-speed saw (Isomet, Buehler, Lake Bluff, Ill) under saline irrigation. The dissected bone specimens were scanned using a micro computerized tomography (microCT) scanner (SkyScan 1172-D, Kontich, Belgium). Scanning and reconstruction voxel sizes were set at 27- × 27- × 27-μm3 voxel size. The same scanning conditions were used for all specimens (70 kV, 141 μA, 0.4° rotation per projection, 8 frames averaged per projection and 120-ms exposure time). The region of interest was determined and standardized (5 × 5 × 5 mm3) at the graft–native bone interface to avoid unreliable results if it was set entirely over the residual graft particles (Figure 4d). Bone and nonbone voxels were segmented using the same heuristic algorithm used for the CBCT images. The gray levels were converted to degree of bone mineralization (DBM) for each bone voxel using a calibration curve based on known densities of commercial phantoms. The mean, SD, Low5, and High5 of the DBM histogram were obtained as described above (Figure 4e). A morphological code (CTAn, SkyScan, Kontich, Belgium) was used to assess the architectural parameters of bone including trabecular bone fraction (BV/TV), surface-to-volume ratio (BS/BV), thickness (Tb.Th), number (Tb.N), and separation (Tb.Sp).
Fluorescent images of decalcified horizontal sections containing the defect site were captured to quantify cortical plate thickness and the mineral apposition rate at the bone surfaces of the defect sites (Figure 5). The amount of residual xenograft material, mineral apposition zone, inside the defect site was also graded semiquantitatively. All measurements were conducted with the rater blinded of animal and specimen identity.
One-way analysis of variance and a post hoc test (Tukey-Kramer honestly significant difference) were performed to examine differences of the CBCT-based gray-level distribution, microCT-based DBM distribution, and nanoindentation moduli between the surgical sites (control, xenograft, and xenograft + PTH). Student t tests and Wilcoxon rank-sum tests were used to assess the differences between treatment groups for histological analysis. All statistical analyses were performed using SPSS, version 19 (IBM SPSS, Armonk, NY). The significance was set to be P < .05. An independent statistician reviewed all statistical analyses.
The CBCT-based gray levels of mean and High5 of gray levels were significantly higher for the xenograft + PTH group than those of both control and xenograft-only groups (P < .018; Figure 6a; Table 1). The xenograft group had significantly higher mean gray levels than the control group (P = .028). All other values of gray-level parameters were not significantly different from the surgical site groups (P > .062).
All of the microCT-based DBM and morphological parameters were not significantly different between the surgical site groups (P > .05; Figure 6b; Table 2).
The values of nanoindentation modulus (E) of the Bio-Oss + PTH group were significantly higher than those of the control and the xenograft groups (P < .001; Figure 6c). The control group had significantly higher E values than the xenograft group (P < .001).
The control sites (defects) could not be accurately identified from the histological sections and were thus excluded from the analysis. Comparisons between the PTH + xenograft and xenograft-only sites showed that the thickness of the cortical bone tended to be higher at the PTH + xenograft sites (Figures 7a and 8). The same trend was also shown for mineral apposition rate (Figure 7b) and width of the mineral apposition zone (Figure 7c). A moderate amount (one-third to two-thirds of the total defect area) of residual xenograft was present at the PTH + xenograft and xenograft-only sites but tended to be lower at the former sites (P = .665).
The desire of clinicians to use growth factors to facilitate reconstructive surgical procedures, by obviating the need for procurement of autogenous grafts with its associated donor side morbidity, is contrasted by their limited availability for clinical application. Growth factors that are involved in the regeneration and induction of bone tissue have attracted attention as they can possibly facilitate skeletal reconstruction.11 Examples of these factors include platelet-derived growth factor, vascular endothelial growth factor, insulin-like growth factors, and bone morphogenetic proteins. Another growth factor found to greatly influence bone regeneration is PTH.
PTH has been proven to have positive effects in bone regeneration when administered in an intermittent daily dose.12 This had led it to be the only Food and Drug Administration (FDA)–approved therapy in management of postmenopausal osteoporosis.12–14 Teriparatide or PTH1-34, one of the PTH forms, has been numerously studied in treating several skeletal conditions and proved to enhance bone healing.12–20 A landmark study from Anderassen et al22 identified the benefit of daily-administered PTH1-34 on rat tibial fracture healing. Since this report, many studies have shown that intermittent PTH administration improves bone healing by its additional effects of osteogenesis and chondrogenesis.22
In the field of bone regeneration to reconstruct skeletal defects, it was found that PTH1-34 improved graft-host integration, callus, and trabecular bone formation.14,23 Several cellular mechanisms such as proliferation and differentiation showed improvement after the addition of PTH.23
Enhanced bone regeneration resulting from the local addition of PTH to bone graft material in comparison with intermittent daily injections remains undetermined in the literature today. A report by Jung et al24 demonstrated that a local mixture of PTH1-34 with scaffold materials was successful in bone formation. An advantage to this delivery method is the avoidance of painful daily infections, as this would be a deterrent in the dental implant patient.
In our study, we found that the local addition PTH1-34 to the graft construct significantly enhanced the BMD, as evident in our CBCT and nanoindentation results. This is in accordance with the study by Reynolds et al.23 Our nanoindentation results showed that the PTH group had an increased hardness of regenerated bone and accelerated bone mineralization, a situation known to provide a favorable clinical environment for dental implant placement.
Although microCT did not produce a statistically significant difference between the PTH-xenograft and xenograft-only groups, there was a trend toward higher bone regeneration and mineralization in the former. In addition, the native bone was measured as the control group. This explains why the native bone results show a higher degree of bone mineralization than the grafted sites, since native bone is already mature and mineralized.
Our histological analysis confirmed the positive trend of local addition of PTH to the graft construct that was evident by the increased cortical bone thickness in addition to the increased mineral apposition rate in comparison with the other two groups. In addition, the quantity of residual xenograft particles was lower at the PTH grafted sites, which denotes an increased trend of bone regeneration through the bone remodeling–substitution process in which the graft particles are replaced with newly formed bone.
Although pig models are considered to be similar to the human bone regeneration process, they do not simulate human clinical conditions, principally in the postoperative course. In addition to this limitation, PTH is not approved by the FDA for the reconstruction of bone defects. The decision to use the well-established xenograft, Bio-Oss, has inherent limitations, including foreign-body rejection and variable bone resorption rate in comparison with the use of the gold standard autogenous bone, which was not performed in this study. Because of these concerns, the results of the study should be regarded with caution.
In a large animal model (pigs), the local addition of PTH1-34 to a xenograft showed promising results and can positively enhance bone regeneration in the reconstruction of mandibular defects. This enhancement not only improved the mineral density of the regenerated bone but also tended to increase its quantity. These results provide a foundation for implementing a human clinical trial in evaluating the effects of PTH on regeneration of maxillofacial bone defects, which is our future goal.
bone mineral density
cone beam computerized tomography
degree of bone mineralization
Food and Drug Administration
institutional animal care and use committee
micro computerized tomography
Funding was received from the American Academy of Implant Dentistry Foundation and the Ohio State University College of Dentistry. The authors would like to thank Ms Lori Mattox, RVT, RLATG, SRA, senior animal health technician, University Laboratory Animal Resources, for her expertise and help during the research project. We also thank Dr William M. Johnston, MS, PhD, at The Ohio State University for his assistance in performing the statistical analysis of the collected data.