Sclerostin and DKK1 Inhibition Preserves and Augments Alveolar Bone Volume and Architecture in Rats with Alveolar Bone Loss

Introduction

Alveolar bone loss—common with aging, menopause, periodontal disease (PD), and edentulism (Jeffcoat 1993; Hansson and Halldin 2012; Tan et al. 2012)—can promote tooth loss and other periodontal and aesthetic complications, including reduced denture fixation and challenges in placing dental implants (Sennerby et al. 1988; Jeffcoat 1993). Surgical alveolar bone augmentation often involves autologous bone grafting (Pagni et al. 2012), which causes donor site pain and other potential complications (Esposito et al. 2008; Herford and Dean 2011). Alternatives include bone graft substitutes, which lack osteoinductive properties (Pagni et al. 2012), and a bone morphogenetic protein (BMP-2) product that carries several potential safety risks and is administered only to exposed alveolar bone (Carreira et al. 2014).

Systemically administered bone-active agents offer the potential for conservative alveolar bone augmentation by stimulating bone formation on existing surfaces, thereby providing new bone stock that is fully contiguous with existing bone and replete with mechanosensitive osteocytes. Systemic parathyroid hormone (1 to 34) was shown to augment alveolar bone in patients with PD (Bashutski et al. 2010), and newer pharmacologic approaches for alveolar bone augmentation include drugs that inhibit sclerostin and/or DKK1. Sclerostin and DKK1 are osteocyte-derived factors that inhibit bone formation and limit bone accrual by suppressing Wnt/β-catenin signaling in osteoblast-lineage cells (Ke et al. 2012). DKK1-overexpressing mice have reduced bone formation and low bone mineral density (BMD) systemically, including alveolar bone (Han et al. 2011; Li et al. 2011). DKK1 upregulation is associated with alveolar bone loss in humans with PD (Napimoga et al. 2014) and rats with periapical disease (Zhang et al. 2014). Conversely, DKK1-deficient mice show increased bone formation and BMD (Morvan et al. 2006), and humans with mutations associated with reduced DKK1 and sclerostin activity have systemically increased BMD, including dense jaw bones (Boyden et al. 2002). DKK1 antibody (DAB) increased bone formation and BMD in the postcranial skeleton of ovariectomized (OVX) mice, rats, and cynomolgus monkeys (Glantschnig et al. 2011; Li et al. 2011), particularly at sites of skeletal injury (Agholme et al. 2011; Li et al. 2011; Ke et al. 2012).

Sclerostin, which is expressed by osteocytes throughout the skeleton (Jager et al. 2010; Kim et al. 2014), limits the accrual and maintenance of bone mass in the cranial and postcranial skeleton (Balemans et al. 2001). Sclerostin upregulation is a key mediator of disuse osteopenia (Robling et al. 2008; Moustafa et al. 2012), and sclerostin inhibition via sclerostin antibody (SAB) inhibits disuse osteopenia (Ke et al. 2012), as does genetic sclerostin deficiency (Lin et al. 2009). Increased sclerostin expression by alveolar bone osteocytes is associated with locally reduced bone formation, and sclerostin upregulation is implicated in alveolar bone loss associated with PD (Napimoga et al. 2014; Kim et al. 2017), orthodontic tooth movement (Nishiyama et al. 2015), and occlusal hypofunction after tooth extraction (Xu et al. 2016). SAB increased bone formation and reversed alveolar bone deficiency in PD models (Taut et al. 2013; Chen et al. 2015; Ren et al. 2015), but its effects in edentulism models were previously unknown.

While BMD gains at postcranial sites of animals or patients receiving SAB are robust relative to other systemically administered antiresorptive or bone-building agents (McClung et al. 2014; Ominsky et al. 2015), the maximum bone-building potential of SAB therapy may be limited by reactive increases in DKK1 expression (Florio et al. 2016; Taylor et al. 2016). In animals, dual DKK1 and sclerostin inhibition caused greater increases in bone formation and BMD as compared with sclerostin inhibition alone (Florio et al. 2016), but effects of this dual inhibition on alveolar bone were not assessed. The current studies investigated the roles of sclerostin and DKK1 in alveolar bone deficiency by examining their expression after tooth extraction and by testing the effects of SAB or SAB+DAB combination therapy in models of established alveolar bone loss driven by estrogen deficiency, tooth extraction, and occlusal hypofunction.

Materials and Methods

All animal procedures were conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International in compliance with U.S. National Research Council and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and in accordance with animal protocols approved by Amgen’s Animal Care and Use Committee.

Results

Effects of SAB and SAB+DAB on Alveolar Bone in Rats with OVX-Induced Bone Loss

Fourteen weeks postsurgery, the mandibular region (M1 to M3) of OVX rats had significantly lower alveolar BVF as compared with sham controls (Fig. 1A, B). Five weeks of SAB or SAB+ DAB therapy, starting 9 wk postsurgery, restored BVF to levels exceeding OVX-Veh or sham controls. Histomorphometry of mandibular alveolar bone indicated significantly greater trabecular thickness with SAB and SAB+DAB versus Veh (Fig. 1B). The SAB and SAB+DAB groups had significantly greater BFR/BS (bone formation rate/bone surface) in alveolar and basal bone versus Veh (Fig. 1C, D), and BFR/BS in basal bone was greater with SAB+DAB versus SAB. The OVX-Veh group showed greater bone resorption (ES/BS [eroded surface/bone surface]) in basal and alveolar bone versus sham, whereas the SAB and SAB+DAB groups had significantly lower ES/BS values versus OVX-Veh rats (Fig. 1E, F). Vertebral and leg BMC was increased from the pretreatment baseline in the SAB and SAB+DAB groups versus Veh controls, and these responses were significantly greater with SAB+DAB versus SAB (Fig. 1G, H).

OPEN IN VIEWER

Figure 1.

Effects of sclerostin antibody (SAB) or SAB + DKK1 antibody (DAB) on mandibles of ovariectomized (OVX) rats. (A) Micro-CT-derived alveolar bone volume fraction (BVF) at the region of M1 to M3. (B) Trabecular thickness in alveolar bone (M1 region). (C, D) BFR/BS (bone formation rate/bone surface) for alveolar and basal bone (M1 region). (E, F) ES/BS (eroded surface/bone surface) for alveolar and basal bone (M1 region). (G, H) Percentage change from treatment baseline in bone mineral content (BMC) of the lumbar vertebrae and whole leg. Data represent mean ± SEM, n = 10. *P < 0.05 vs. Veh (vehicle). #P < 0.05 vs. sham. ^P < 0.05 vs. SAB. Analysis of variance and Tukey’s posttest.

Sclerostin and DKK1 mRNA Expression in Alveolar Bone after Molar Extraction

Sost mRNA was strongly expressed by alveolar osteocytes in the nonextracted and extracted maxilla (Fig. 2D–F). Dkk1 was moderately expressed by osteocytes in the nonextracted maxillae, and Dkk1 expression appeared to be increased within 1 wk after molar extraction (Fig. 2A–C). Dkk1 mRNA remained highly expressed at week 2 postextraction, with an apparent attenuation of Dkk1 expression toward nonextracted levels at weeks 3 to 5 postextraction (Appendix Fig. 2).

OPEN IN VIEWER

Figure 2.

mRNA expression for the genes encoding sclerostin (Sost) and Dkk1 in intact and postextraction maxillae of male rats. In situ hybridization revealed Sost and Dkk1 mRNA expression (punctate purple staining) by osteocytes in alveolar bone of the intact maxilla (A, D). Sost and Dkk1 were also expressed 1 wk (B, E) and 5 wk (C, F) after maxillary molar extractions. The intensity of Dkk1 mRNA expression appeared to be higher after extraction, which is further illustrated in Appendix Figure 2. Sections were stained with hematoxylin; magnification, 63×.

Effects of SAB and SAB+DAB on the Maxilla and Mandible of Rats with an Atrophic Edentulous Maxilla

Micro-CT of extracted maxillae showed a 38% deficit in maxillary alveolar ridge bone volume 9 wk postextraction (P < 0.05 vs. intact controls), primarily due to increased bone volume in the intact group that did not occur in the extracted group (P < 0.05; Fig. 3A). At weeks 2 and 4 of treatment, respectively, the SAB+DAB and SAB groups showed significantly increased alveolar ridge volume as compared with extracted Veh controls, with further increases thereafter. By treatment week 15, the SAB and SAB+DAB groups had 42% and 81% greater alveolar ridge volume, respectively, versus extracted Veh controls (both P < 0.05). The SAB+DAB group had significantly greater bone volume relative to the SAB group from weeks 4 through 15 of treatment, and the SAB+DAB group achieved values similar to intact controls by week 12. Representative sagittal micro-CT images of maxillae after 9 wk of treatment depict alveolar ridge atrophy in the extracted Veh group and significant ridge augmentation with SAB and SAB+DAB (Fig. 3A).

OPEN IN VIEWER

Figure 3.

Effects of sclerostin antibody (SAB) or SAB + DKK1 antibody (DAB) on maxillary alveolar ridge volume (A) and height loss (B) after maxillary molar extractions. (A) Left panel, micro–computed tomography (micro-CT) data for alveolar ridge bone volume; right panels, sagittal micro-CT images of the right maxilla, with images selected for alveolar bone volume closest to the group’s mean value. (B) Left panel, micro-CT data for change in alveolar ridge height. Data represent ridge height for each animal in the extracted groups minus the mean ridge height of the intact control group; right panels, coronal micro-CT images of the right maxilla indicate alveolar ridge height, with images selected for ridge height closest to the group’s mean values. The yellow vertical line indicates the measurement location for ridge height, which corresponds to the intraradicular region of M2. The yellow arrow highlights postextraction bone loss focused on the interradicular region. Data represent means ± SEM for 13 or 14 rats per group (extracted animals) or 5 per group (intact controls). *P < 0.05 vs. Veh (vehicle). #P < 0.05 vs. intact. ^P < 0.05 vs. SAB. Two-way analysis of variance and Dunnett’s posttest.

Vertical resorption of the edentulous maxillary ridge was rapid in Veh controls over the first 9 wk postextraction, with further resorption over the next 15 wk, leading to a final absolute height loss of 0.41 mm (~90% of original ridge height) (Fig. 3B). The SAB+DAB and SAB groups showed significant gains in ridge height within 2 and 4 wk of treatment initiation, respectively, with further gains thereafter. By week 15, the SAB group had recovered around two-thirds of total ridge height loss, while the SAB+DAB group achieved complete height recovery by week 9. Height augmentation with SAB+DAB significantly exceeded that of the SAB group from weeks 4 to 15. Figure 3B shows coronal micro-CT images of maxillae at the M2 level, 19 wk after extractions, with the site of ridge height measurements represented by a yellow line. The extracted Veh control ridge atrophied to a thin saddle-shaped structure, with resorption of all interradicular bone (yellow arrow) and reduced buccal and lingual walls; the SAB and SAB+DAB samples show substantial vertical augmentation of the atrophied ridge.

Fluorescent micrographs in Figure 4 illustrate bone structure and fluorochrome labeling patterns in intact and edentulous maxillae 25 wk postextraction. The intact maxilla (Fig. 4A) exhibits solid (minimally porous) alveolar bone; fluorescent labeling indicates substantial physiologic bone formation along tooth roots and minimal bone formation within alveolar bone or along basal bone surfaces. The extracted Veh control shows alveolar ridge loss, with labels throughout the residual ridge reflecting bone remodeling (Fig. 4B). The SAB and SAB+DAB groups exhibit substantial preservation of alveolar ridge structure (Fig. 4C, D); fluorochrome labeling is evident within alveolar bone, and continuous double-fluorochrome labels along basal bone surfaces (white arrows) suggest increased modeling-based bone formation.

OPEN IN VIEWER

Figure 4.

Maxillary structure and dynamic fluorochrome labeling patterns in the maxillary molar extraction study. The intact maxilla image (A) shows normal basal and alveolar bone structure, with prominent fluorochrome labels on alveolar surfaces facing tooth roots. The tooth-extracted (TE) Veh maxilla (B) shows significant ridge atrophy, with abundant fluorochrome labeling throughout alveolar bone suggesting remodeling-induced alveolar bone loss. The TE SAB and SAB+DAB groups (C, D) show fluorochrome labeling throughout a better-preserved alveolar ridge, with long continuous double calcein labels on basal bone surfaces (white arrows) that likely represent modeling-based bone formation. DAB, DKK1 antibody; SAB, sclerostin antibody; Veh, vehicle.

Loss of occlusive loading after right maxillary molar extraction led to a significant 13.9% deficit in alveolar BVF in the underloaded right mandible of Veh controls as compared with the loaded left mandible (Fig. 5). After 15 wk of treatment, BVF was significantly higher in the underloaded mandible of the SAB and SAB+DAB groups versus extracted Veh or intact controls. The SAB and SAB+DAB groups also showed significantly greater BVF of the loaded left mandible versus extracted Veh controls, with values similar to those of the underloaded right mandible. Representative micro-CT reconstructions of the underloaded mandible show substantial alveolar bone loss in the extracted Veh control, particularly at interradicular and periapical sites (yellow arrow), with obvious alveolar bone augmentation in the SAB and SAB+DAB groups.

OPEN IN VIEWER

Figure 5.

Effects of sclerostin antibody (SAB) or SAB + DKK1 antibody (DAB) on alveolar bone in underloaded mandibles. Upper left: Micro–computed tomography (micro-CT) image of jaw bones from a rat with extracted right maxillary molars (white arrow). Red and green arrows indicate the general location of micro-CT analyses for the underloaded and loaded mandibles, respectively. Lower left: Bone volume fraction for mandibles after 15 wk of underloading (red bars) or normal loading (green bars). Data represent means ± SEM; n = 13 or 14 per group (extracted groups) and n = 5 for intact group. *P < 0.05 vs. corresponding underloaded or loaded vehicle (Veh) controls. ^#P < 0.05 vs. corresponding intact control group. Analysis of variance and Tukey’s posttest. Right: Representative sagittal micro-CT images of underloaded mandibles. The yellow arrow indicates severe alveolar bone loss associated with occlusal hypofunction in a Veh control animal.

Discussion

These studies indicate that SAB and SAB+DAB augmented jaw bones in models of alveolar bone loss caused by estrogen deficiency, partial edentulism, and occlusive hypofunction. These findings extend previous observations that SAB and SAB+DAB promote bone gains at postcranial skeletal sites of osteopenic animals (Ke et al. 2012; Florio et al. 2016). Human and mouse genetics data directly implicate sclerostin and indirectly implicate DKK1 as endogenous factors that limit jaw bone volume and density (Balemans et al. 2001; Boyden et al. 2002; Kuchler et al. 2014), the effects of which are evident prior to skeletal maturity (Boyden et al. 2002; Gardner et al. 2005). SAB and SAB+DAB increased jaw bone formation and mass in 8-mo-old OVX rats, indicating that skeletally mature rat jaw bones are also responsive to sclerostin and DKK1 and to their inhibition.

Alveolar ridge atrophy at edentulous sites can adversely affect aesthetics, mastication, denture fixation, and the ability to place dental implants (Jeffcoat 1993; Sennerby et al. 1988). Alveolar bone augmentation is currently achieved by a variety of invasive procedures (Esposito et al. 2008), whereas SAB and SAB+DAB promoted vertical restoration of the atrophic edentulous maxillary ridge of rats without surgical interventions, bone grafting, or disturbance of overlying gingival tissue. The extraction study did not specifically reveal the mechanisms underlying this augmentation, which could include inhibited ridge resorption, increased bone formation atop the ridge, and/or increased formation on basal bone surfaces. Dynamic histomorphometry in the OVX rat mandible and fluorochrome labeling patterns in the edentulous rat maxilla indicated increased basal bone formation with SAB and SAB+DAB. In the maxilla, this basal bone response could increase ridge height superiorly, potentially reducing the need for maxillary sinus grafting in patients who could not otherwise receive dental implants due to ridge atrophy (Pagni et al. 2012).

SAB and SAB+DAB also reversed alveolar bone loss in mandibles that were underloaded due to loss of occlusion, which is consistent with evidence that sclerostin and DKK1 mediate appendicular disuse osteopenia (Robling et al. 2006; Lin et al. 2009; Xu et al. 2016). Interestingly, SAB and SAB+DAB increased mandibular bone volume to similar absolute levels in the loaded and underloaded mandible, suggesting that SAB and SAB+DAB therapy may effectively mimic physiologic responses to increased skeletal loading. Such natural responses can include downregulation of sclerostin and DKK1, leading to adaptive increases in bone formation and bone mass, in accordance with Wolff’s law (Wolff 1884; Robling et al. 2006; Moustafa et al. 2012).

In the extraction study, SAB+DAB exceeded SAB’s effects in augmenting alveolar bone and reducing bone resorption (serum TRACP-5b). SAB+DAB was also shown to cause greater reductions in resorption surfaces in rat vertebrae as compared with SAB or DAB alone (Florio et al. 2016). Cellular mechanisms for enhanced antiresorptive effects with SAB+ DAB are unclear and could involve greater inhibition of osteoclast formation, function, and/or survival. This possibility is suggested by evidence that SAB+DAB caused greater induction of skeletal OPG mRNA expression versus SAB or DAB alone (Florio et al. 2016), with OPG well established as a potent inhibitor of osteoclast formation, function, and survival (Kostenuik and Shalhoub 2001). In OVX rats, SAB+DAB was not superior to SAB at increasing alveolar BVF, perhaps because BVF in the SAB group (at 94%) was difficult to exceed. As compared with SAB, SAB+DAB increased basal bone formation in OVX rats, suggesting that sclerostin and DKK1 contribute to inhibited jaw bone formation in estrogen-deficient rats. As mentioned, sclerostin inhibition can upregulate DKK1, which appears to limit the maximum potential for SAB-induced bone formation and BMD gains (Florio et al. 2016; Taylor et al. 2016). This report is the first to show that dual inhibition of sclerostin and DKK1 can cause greater bone formation and structural augmentation in rats with alveolar bone loss when compared with sclerostin inhibition alone. A bispecific antibody that inhibits sclerostin and DKK1 caused more robust rat femur fracture healing and greater increases in bone formation markers in cynomolgus monkeys as compared with SAB monotherapy (Florio et al. 2016). The current studies suggest that bispecific sclerostin/DKK1 antibodies may also have utility for augmenting alveolar bone.

This report has several limitations, including the lack of a DAB monotherapy group in the OVX rat and maxillary extraction studies. DAB generally has minimal effects on noninjured bone in older rats (Li et al. 2011), which may reflect the status of jaw bones in the OVX rats and the chronically edentulous rats, but we cannot exclude that DAB alone may augment jaw bones in these models. The clinical relevance of these initial findings is unclear, but if SAB, DAB, or SAB+DAB therapy is eventually shown to increase human jaw bone volume or density, these therapies may have utility for implant site development prior to implant placement. Patients with better initial jaw bone stock show greater odds of implant osseointegration months later (Aro et al. 2012), potentially due to greater primary implant stability (Lioubavina-Hack et al. 2006; Javed et al. 2013). SAB and DAB increased primary stability and osseointegration of implants at postcranial skeletal sites (Agholme et al. 2010; Agholme et al. 2011; Virdi et al. 2015), but their effects on dental implants are currently unknown. SAB therapy was associated with rare cases of osteonecrosis of the jaw in women with postmenopausal osteoporosis (Cosman et al. 2016), although the risk of osteonecrosis of the jaw in other populations, including edentulous individuals, is unknown. Another limitation is that high SAB and SAB+DAB doses were used in the current studies to maintain drug exposure if rats developed antidrug immune responses. This dosing led to dramatic effects on jaw bone architecture that may not be achievable in patients. More modest gains in ridge dimensions might still enable some patients to receive dental implants without invasive ridge augmentation surgery, perhaps as single-stage procedures. Single-stage implants require bone with sufficient strength for immediate load bearing, and we showed that mandibular bone in Sost^-/- mice was stronger than WT controls. Dkk1^-/- mice have generally stronger bones (Wang et al. 2007), but the effect of Dkk1 deficiency or DAB on jaw bone strength has not been assessed. Finally, SAB and SAB+DAB increased bone mass systemically, which may not be desired in patients without high fracture risk, although systemic responses reverse when these treatments are discontinued (Florio et al. 2016; Ominsky et al. 2017). Such reversal may also occur in jaw bones after treatment discontinuation, but Wolff’s law suggests that newly accrued jaw bone may persist as long as it remains loaded—for example, by placing dental implants.

In summary, these studies implicate sclerostin and DKK1 as factors that limit alveolar bone accrual and promote alveolar bone deficiency in estrogen-deficient and edentulous states. Systemic SAB and SAB+DAB preserved and augmented alveolar bone by increasing bone formation and decreasing bone resorption. These noninvasive therapies have the potential to prevent alveolar bone loss and to promote the formation of new bone that is contiguous with original bone, without graft-related donor site morbidity and without disturbing the overlying gingiva.

Author Contributions

M. Liu, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; P. Kurimoto, contributed to design, data acquisition, and analysis, drafted the manuscript; J. Zhang, Q.T. Niu, contributed to data acquisition and analysis, drafted the manuscript; J. Hesterman, contributed to data acquisition and analysis, critically revised the manuscript; M. Stolina, P.C. Dechow, contributed to data analysis, critically revised the manuscript; J.Q. Feng, M.D. Silva, contributed to data analysis and interpretation, critically revised the manuscript; M.S. Ominsky, W.G. Richards, contributed to data interpretation, critically revised the manuscript; H. Ke, contributed to conception and data interpretation, critically revised the manuscript; P. Kostenuik, contributed to data interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material