Bone graft incorporation correlates with nearby Wnt-responsive osteoprogenitors.
Genetic models of elevated Wnt signaling provided evidences for higher bone mass.
Combination of WNT protein with allograft accelerates the onset of bone formation.
Compared to autologous bone grafts, allogeneic bone grafts integrate slowly, which can adversely affect clinical outcomes. Here, our goal was to understand the molecular mechanisms underlying graft incorporation, and then test clinically feasible methods to accelerate this process.
Wild-type and transgenic Wnt “reporter” mice were used in a vertical ridge augmentation procedure. The surgery consisted of tunneling procedure to elevate the maxillary edentulous ridge periosteum, followed by the insertion of bone graft. Micro-computed tomographic imaging, and molecular/cellular analyses were used to follow the bone graft over time. Sclerostin null mice, and mice carrying an activated form of β-catenin were evaluated to understand how elevated Wnt signaling impacted edentulous ridge height and based on these data, a biomimetic strategy was employed to combine bone graft particles with a formulation of recombinant WNT protein. Thereafter, the rate of graft incorporation was evaluated.
Tunneling activated osteoprogenitor cell proliferation from the periosteum. If graft particles were present, then osteoprogenitor cells attached to the matrix and gave rise to new bone that augmented edentulous ridge height. Graft particles alone did not stimulate osteoprogenitor cell proliferation. Based on the thicker edentulous ridges in mice with amplified Wnt signaling, a strategy was undertaken to load bone graft particles with WNT; this combination was sufficient to accelerate the initial step of graft incorporation.
Local delivery of a WNT protein therapeutic has the potential to accelerate graft incorporation, and thus shorten the time to when the graft can support a dental implant.
For decades, autologous bone has served as the standard of care for the reconstruction of large bony defects [ ]. Autologous bone grafts (autografts) contain osteoprogenitor cells, a mineralized matrix scaffold, and growth factors that collectively allow the graft to seamlessly integrate at the site of transplantation [ ]. Nonetheless, there are disadvantages to autografting: first, there is persistent discomfort that accompanies the harvesting of bone from the iliac crest [ , ]. Second, there is often a limited amount of autograft that can be harvested [ ]. Third, there is the issue of osteogenic potential: the older the patient, the less osteogenic the autograft becomes [ ]. Finally, there is the issue of resorption: despite initial gains, over time, autografted sites undergo a significant loss in volume [ , ].
Collectively, these disadvantages have made way for the development of bone graft extenders (reviewed in [ ]). Their ease of use and unlimited quantities are two major advantages, but all allogeneic bone graft extenders must undergo deproteinization and decellularization to ensure immune-compatibility [ ], Since they are devoid of stem/osteoprogenitor cells, and pro-osteogenic proteins, new bone tends to form very slowly around allografts [ , ]. This delayed graft incorporation is problematic and contributes to greater variability in clinical outcomes when allografts are used [ ]. We reasoned that if the rate of new bone formation could be accelerated around allografts, then these materials may effectively replace the need for autografts.
WNT proteins are potent, pro-osteogenic factors [ ] that stimulate osteogenesis by activating the osteogenic transcription factor Runx2 [ ], and repressing bone-resorbing pathways via a RANKL-dependent mechanism [ , ]. WNT proteins, however, are lipophilic and thus aggregate in aqueous in vivo environments. To prevent loss of activity, our laboratory developed a liposomal formulation of WNT3A e.g., l -WNT3A [ ]. The liposome interacts with the glycosylated, palmitoylated modifications on WNT proteins, which are essential for Wnt signaling activity [ ]. Liposomal packaging stabilizes the WNT protein, extending its half-life in vivo from minutes to multiple hours [ ]. in vivo , l -WNT3A activates osteoprogenitor cells, leading to their accelerated differentiation into matrix-secreting osteoblasts; consequently, we reasoned that combining this protein therapeutic with a non-resorbing allogeneic bone matrix scaffold might promote the activation of endogenous Wnt-responsive osteoprogenitor cells at the site of grafting, which in turn would accelerate the rate of new bone formation around the allograft.
We tested this possibility in a craniomaxillofacial procedure where allograft materials are used to increase the vertical dimension of an atrophied maxilla or mandible to support the placement of an implant [ ]. A vertical ridge augmentation procedure was performed in the murine edentulous ridge, which lies between the first molar and incisors and is comprised of dense lamellar bone, similar to an atrophied human maxillae or mandible [ ]. This region was large enough to permit the insertion of micro-forceps to create a periosteal tunnel, into which bone graft particles could be inserted. The fate(s) of Wnt-responsive cells around the particles, and the effects of l -WNT3A, could then be monitored over time.
Materials and methods
Experimental protocols followed ARRIVE guidelines and were approved by the Stanford Committee on Animal Research (#13146). Wild-type and Axin2 CreERT2/+ ; R26 mTmG/+ mice (#018867 and #007576) were purchased from Jackson Laboratories. Both genders were used. All mice were between 6–8 weeks old at the initiation of the experiment.
A strain of Wnt reporter mice ( Axin2 CreERT2/+ ;R26 mTmG/+ ) was employed in some experiments. In this strain, Cre expression is under the control of the Wnt target gene, Axin2 [ ]. Cre mediated recombination was induced by intraperitoneal delivery of tamoxifen (4 mg/25 g body weight); thereafter, Wnt-responsive cells are identifiable by expression of green fluorescent protein (GFP). Descendants arising from the initial population of Wnt-responsive cells are also labelled with GFP, which allowed for the unambiguous identification of Wnt-responsive cells in the periosteum. In our experiments, tamoxifen was delivered intraperitoneally for 3 consecutive days; animals were sacrificed 7 days after the last injection.
Daβcat Ot mice and Sost −/− mice were generated in the laboratory of Dr. Teresita Bellido, whose protocol was approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine. Daβcat Ot mice were generated as described [ ], by crossing dentin matrix acidic phosphoprotein 1 ( DMP1 )−8kb-Cre mice with Catnb lox(ex3) mice in which LoxP sites flank exon 3 that encodes for β-catenin degradation. DMP1−8kb-Cre +/ − mice were crossed with Catnb lox(ex3)/lox(ex3) mice to generate Catnb lox(ex3)/+ ; DMP1−8kb-Cre +/ − (daβcat Ot mutant mice) and Catnb lox(ex3)/+ mice (daβcat Ot control) mice. Sost −/− mice carried a targeted disruption of the SOST coding region and were generated as described [ ].
l -WNT3A formulation and Wnt reporter activity assay
l -WNT3A was prepared as described in detail [ ]. In brief, recombinant human WNT3A protein was combined with pre-formed DMPC:cholesterol (90:10) liposomes and incubated at room temperature for 2 h to generate WNT-lipid nanoparticles [ ]. An identical liposomal formulation of phosphate buffered saline e.g., l -PBS was manufactured and used as a control.
The activity of l -WNT3A used here was validated using a cell-based potency LSL assay, in which mouse LSL cells were stably transfected with a Wnt-responsive luciferase reporter plasmid, pSuperTOPFlash (Addgene) containing 3 TCF/LEF binding sites regulating expression of luciferase. When LSL cells are exposed to l -WNT3A, the Wnt protein binds to Frizzled (Fz) receptors on the surface of the LSL cells, which initiates a cascade of Wnt-dependent intracellular events [ ] that leads to the expression of luciferase [ ]. After exposure to the Wnt stimulus, LSL cells were incubated for an additional 18 h at 37 °C, 5% CO 2 in 1 × DMEM (Invitrogen), 10 % FBS (Gibco), and 1% Penicillin/Streptomycin (P/S, Mediatech). Then prior to quantification, cells were washed to remove exogenous Wnt and lysed with Lysis Buffer (Applied Biosystems). Luciferin was added to the culture medium, and underwent cleavage by luciferase; the energy released by this reaction was in the form of light [ ] that was detected by a dual light reader. l -WNT3A potency was defined by comparing the readouts of samples to that of a reference standard consisting of recombinant human WNT3A protein (StemR&D), tested at known concentrations.
Allograft preparation and treatment with l -WNT3A
To allow placement in a mouse surgical site, bone matrix was placed into a mortar and ground with a pestle into smaller particles e.g., ∼15−200 μm. Graft particles were then soaked in either l -WNT3A (concentration =0.3 ng/μL) or l -PBS at 4 °C for 15 min. To measure entrapment and release of active l -WNT3A, particles were removed from their solutions and placed onto LSL cells which were seeded at 50,000 cells/well in a 4-well chamber slide (Thermo) and allowed to recover for 4 h. After 18 h, cells were fixed in 4% PFA then immunostained for luciferase expression (see immunostaining described below). Cell nuclei were counter-stained with DAPI. Negative and positive control wells were treated with l -PBS and l -WNT3A, respectively, at a concentration of 10 μL in 100 μL total culture medium volume.
Quantification of Wnt activity in response to l -WNT3A delivered via bone graft particles was performed using Adobe Photoshop and ImageJ. Three samples co-cultured with l -WNT3A soaked bone graft were included. The first ROI was defined as an annulus around the bone graft particle with a width of 150 μm, while the second was defined as the annulus around the first annulus with the same width. Total cell numbers were counted using particle analysis in ImageJ, while the numbers of luciferase +ve cells were counted manually. Then the percentage of luciferase +ve cells was calculated.
Subperiosteal tunneling and bone graft placement
A vertical ridge augmentation procedure was performed using bone graft particles. Prior to the tunneling and/or grafting procedure, mice were anesthetized, and the mouth was rinsed using povidone‐iodine solution for 1 min. A full-thickness incision was made between the first and second rugae, perpendicular to the maxillary bone surface. Micro-forceps were used to reflect the periosteum towards the maxillary first molar. Bone graft particles, either treated with l -WNT3A or with l -PBS (as above), were gently inserted into the tunnel. The surgical site was closed with non‐absorbable single interrupted sutures (Ethilon nylon suture black monofilament 8‐0, Johnson & Johnson Medical). Mice were fed provided regular hard-food chow (#2918, Envigo) and water ad libitum. Mice were sacrificed at timepoints indicated (see Appendix Table 1 for experimental groups, timepoints, and sample sizes).
Micro-computed tomography (μCT)
Samples were collected after euthanasia. The maxillae were split sagittally with a sharp blade. Tissues were fixed in 4% paraformaldehyde overnight at 4 °C. Three-dimensional μCT scanning and analyses followed published guidelines [ ]. Scanning was performed using a μCT data-acquisition system (VivaCT 40, Scanco) at 10.5 μm voxel size (70 kV, 115 μA, 300 ms integration time). Three-dimensional reconstruction and volume rendering were carried out using Avizo (FEI, Hillsboro, OR), Dataviewer (SkyScan) software and ImageJ (NIH, Bethesda, MD) software. Bone morphometry was evaluated using CTAn software (SkyScan, Belgium). Images were organized using Adobe Photoshop and Adobe Illustrator.
Tissue collection, processing and histology
Following the μCT imaging, samples were transferred to a microwave oven (Ted Pella, Redding, CA), in which a circulating 10 % ethylenediamine tetraacetic acid solution was held for decalcification. After a 2-week demineralization period, specimens were dehydrated through an ascending ethanol series then paraffin-embedded. 8μm‐thick sagittal sections were cut and collected on Superfrost‐plus slides for histology including Aniline blue, Masson’s Trichrome, Movat’s pentachrome, and Picro-sirius red staining, which followed published protocols [ ].
Immunostaining followed published protocols [ ]. In brief, tissue sections were de-paraffinized then permeabilized with 0.5 % TritonX-100. Antigen retrieval was performed using Antigen Unmasking Solution (Vector Labs), following which slides were blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature then incubated with primary antibodies overnight at 4 °C. After washing with PBS, slides were incubated with Cyanine5 conjugated goat anti-rabbit secondary antibody (Invitrogen, A-10523) for 30 min, then mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Labs). Primary antibodies used in this study include anti-luciferase antibody (1:1000, ab21176, Abcam), anti-PCNA (1:5000; ab18197, Abcam), anti-Runx2 (1:1000; ab192256, Abcam), and anti-green fluorescent protein (GFP) (1:400; 2956S, Cell Signaling Technology).
Alkaline phosphatase (ALP) and Tartrate-resistant acid phosphatase (TRAP) activity
To detect ALP activity, tissue sections were treated with ALP-detection solution containing BCIP (5-bromo-4-chloro-3-indolyl phosphate; Roche, #11383221001) and NBT (nitro blue tetrazolium chloride; Roche, #11383213001) according to the manufacturer’s instructions. TRAP activity was observed using a leukocyte acid phosphatase staining kit (catalog #386A-1 KT, Sigma-Aldrich). Tissue sections were processed according to the manufacturer’s instructions.
Histomorphometric measurements were performed using Adobe Photoshop. A minimum of 3 grafted sites/time point were analyzed for each treatment group. For each site, a minimum of three aniline blue-stained sagittal tissue sections, which included the mesial labial root as a landmark, were used to quantify maxillary bone thickness, and augmented thickness. Each tissue section was photographed using a Leica digital image system at 20x magnification. To obtain the augmented height, the region of newly formed bone was selected manually in Photoshop, then the area and width of the selected pixels was recorded using the measurement log. The average augmented thickness was calculated by dividing the area selected by its width. In addition, the area occupied by the bone graft particles was also manually selected and recorded. From these measurements, the composition e.g., particles versus new bone of the total augmented region was determined.
To evaluate Runx2 expression in response to l -PBS versus l -WNT3A treatment, 3 graft sites from each treatment group were analyzed. From each site, at least 3 sagittal tissue sections were analyzed. The lasso tool in Adobe Photoshop was used to select a region of interest (ROI), which was the “pocket” area generated by the tunneling procedure. Within this ROI, the space occupied by bone graft particles was excluded. The number of Runx2 +ve cells and the total number of the other cells (identified by DAPI) were then counted, and the ratio of Runx2 +ve to total cells was then calculated.
To visualize collagen organization, 3 graft sites were selected from the l -PBS group and 3 graft sites from the l -WNT3A group, then 3 sagittal tissue sections from each of the specimens were analyzed. Picro-sirius red stained slides were viewed under polarized light using a Leica digital image system at 20x magnification. The same “pocket” ROI was selected, as described above. With the color analysis tool in Photoshop, the number of orange/red pixels, corresponding to mature collagen fibers, were selected using a uniform color range and counted, and the same was done for green pixels, which corresponded to immature collagen fibers. Then, the total pixels within the same ROI was determined. Mature/immature collagen fibers were expressed as a percentage of their respective pixels/total pixels in the ROI.
Results were presented as mean ± standard deviation. All statistical analyses were performed using the Prism 7.0 (GraphPad Software). Comparisons of immunopositive cell percentage and collagen organization were analyzed using the two-tailed Student’s T-test. Comparisons of augmented maxillary height with non-treated bone grafts were based on a one-way ANOVA followed by Turkey’s post-hoc testing. While, comparisons of augmented maxillary height with l -WNT3A/L-PBS treated bone graft were performed based on a two-way ANOVA with Geisser-Greenhouse correction, followed by Turkey’s post-hoc testing for multiple comparisons. Significance was attained at p < 0.05 (*), p < 0.01(**), and p < 0.001(***). An online tool, designed for calculating the minimum sample size for adequate study power, was employed: clincalc.com/stats/SampleSize.aspx .
Establishing a rodent model of vertical ridge augmentation via tunneling and bone grafting procedures
On atrophic maxillae and mandibles, vertical ridge augmentation is performed when there is insufficient bone volume to support an implant. To mimic this clinical scenario, the rodent edentulous ridge was selected. Its thin, lamellar, dense bone ( Fig. 1 A) resembles the type of bone clinicians usually encounter when performing vertical ridge augmentation [ ]. Bone grafting was accomplished using bovine mineralized matrix particles (see Methods). In most cases (14/16) the bone graft remained on the crest of the edentulous ridge (arrows, Fig. 1 B), stabilized there by a soft tissue envelope produced via a subperiosteal tunneling procedure. In cases where the graft shifted from its original position (N = 2), samples were excluded from further analyses.