Dynamic analyses of a soft tissue-implant interface: Biological responses to immediate versus delayed dental implants.

AIM: To qualitatively and quantitatively evaluate the formation and maturation of peri-implant soft tissues around 'immediate' and 'delayed' implants. MATERIALS AND METHODS: Miniaturized titanium implants were placed in either maxillary first molar (mxM1) fresh extraction sockets or healed mxM1 sites in mice. Peri-implant soft tissues were evaluated at multiple timepoints to assess the molecular mechanisms of attachment and the efficacy of the soft tissue as a barrier. A healthy junctional epithelium (JE) served as positive control. RESULTS: No differences were observed in the rate of soft-tissue integration of immediate versus delayed implants; however, overall, mucosal integration took at least twice as long as osseointegration in this model. Qualitative assessment of Vimentin expression over the time course of soft-tissue integration indicated an initially disorganized peri-implant connective tissue envelope that gradually matured with time. Quantitative analyses showed significantly less total collagen in peri-implant connective tissues compared to connective tissue around teeth around implants. Quantitative analyses also showed a gradual increase in expression of hemidesmosomal attachment proteins in the peri-implant epithelium (PIE), which was accompanied by a significant inflammatory marker reduction. CONCLUSIONS: Within the timeframe examined, quantitative analyses showed that connective tissue maturation never reached that observed around teeth. Hemidesmosomal attachment protein expression levels were also significantly reduced compared to those in an intact JE, although quantitative analyses indicated that macrophage density in the peri-implant environment was reduced over time, suggesting an improvement in PIE barrier functions. Perhaps most unexpectedly, maturation of the peri-implant soft tissues was a significantly slower process than osseointegration.

The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study.

OBJECTIVES: Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF-κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte-derived RANKL in bone graft remodeling. MATERIALS AND METHODS: In Tnfsf11fl/fl ;Dmp1-Cre mice without osteocyte-specific RANKL as well as in Dmp1-Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro-computed tomography of the grafts and the donor regions were performed 28 days after grafting. RESULTS: Histology revealed marked resorption of subcutaneous control Dmp1-Cre grafts and new bone formation around subperiosteal Dmp1-Cre grafts. In contrast, Tnfsf11fl/fl ;Dmp1-Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro-computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1-Cre grafts (p < .01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl ;Dmp1-Cre grafts (p = .17). Residual graft volume (p = .08) and thickness (p = .13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl ;Dmp1-Cre and Dmp1-Cre mice and restricted to the defect margins. CONCLUSIONS: The results suggest an active function of osteocyte-derived RANKL in bone graft remodeling.

Mechanical-induced bone remodeling does not depend on Piezo1 in dentoalveolar hard tissue.

Mechanosensory ion channels are proteins that are sensitive to mechanical forces. They are found in tissues throughout the body and play an important role in bone remodeling by sensing changes in mechanical stress and transmitting signals to bone-forming cells. Orthodontic tooth movement (OTM) is a prime example of mechanically induced bone remodeling. However, the cell-specific role of the ion channels Piezo1 and Piezo2 in OTM has not been investigated yet. Here we first identify the expression of PIEZO1/2 in the dentoalveolar hard tissues. Results showed that PIEZO1 was expressed in odontoblasts, osteoblasts, and osteocytes, while PIEZO2 was localized in odontoblasts and cementoblasts. We therefore used a Piezo1floxed/floxed mouse model in combination with Dmp1cre to inactivate Piezo1 in mature osteoblasts/cementoblasts, osteocytes/cementocytes, and odontoblasts. Inactivation of Piezo1 in these cells did not affect the overall morphology of the skull but caused significant bone loss in the craniofacial skeleton. Histological analysis revealed a significantly increased number of osteoclasts in Piezo1floxed/floxed;Dmp1cre mice, while osteoblasts were not affected. Despite this increased number of osteoclasts, orthodontic tooth movement was not altered in these mice. Our results suggest that despite Piezo1 being crucial for osteoclast function, it may be dispensable for mechanical sensing of bone remodeling.

Wnt/ß-Catenin Signaling in Craniomaxillofacial Osteocytes.

PURPOSE OF REVIEW: There is a growing appreciation within the scientific community that cells exhibit regional variation. Whether the variation is attributable to differences in embryonic origin or anatomical location and mechanical loading has not been elucidated; what is clear, however, is that adult cells carry positional information that ultimately affects their functions. The purpose of this review is to highlight the functions of osteocytes in the craniomaxillofacial (CMF) skeleton as opposed to elsewhere in the body, and in doing so gain mechanistic insights into genetic conditions and chemically-induced diseases that particularly affect this region of our anatomy. RECENT FINDINGS: In the CMF skeleton, elevated Wnt/ß-catenin signaling affects not only bone mass and volume, but also mineralization of the canalicular network and osteocyte lacunae. Aberrant elevation in the Wnt/ß-catenin pathway can also produce micropetrosis and osteonecrosis of CMF bone, presumably due to a disruption in the signaling network that connects osteocytes to one another, and to osteoblasts on the bone surface.

A WNT protein therapeutic accelerates consolidation of a bone graft substitute in a pre-clinical sinus augmentation model.

AIM: Autologous bone grafts consolidate faster than bone graft substitutes (BGSs) but resorb over time, which compromises implant support. We hypothesized that differences in consolidation rates affected the mechanical properties of grafts and implant stability, and tested whether a pro-osteogenic protein, liposomal WNT3A (L-WNT3A), could accelerate graft consolidation. MATERIALS AND METHODS: A transgenic mouse model of sinus augmentation with immunohistochemistry, enzymatic assays, and histology were used to quantitatively evaluate the osteogenic properties of autografts and BGSs. Composite and finite element modelling compared changes in the mechanical properties of grafts during healing until consolidation, and secondary implant stability following remodelling activities. BGSs were combined with L-WNT3A and tested for its osteogenic potential. RESULTS: Compared with autografts, BGSs were bioinert and lacked osteoprogenitor cells. While in autografted sinuses, new bone arose evenly from all living autograft particles, new bone around BGSs solely initiated at the sinus floor, from the internal maxillary periosteum. WNT treatment of BGSs resulted in significantly higher expression levels of pro-osteogenic proteins (Osterix, Collagen I, alkaline phosphatase) and lower levels of bone-resorbing activity (tartrate-resistant acid phosphatase activity); together, these features culminated in faster new bone formation, comparable to that of an autograft. CONCLUSIONS: WNT-treated BGSs supported faster consolidation, and because BGSs typically resist resorption, their use may be superior to autografts for sinus augmentation.

An Osteotomy Tool That Preserves Bone Viability: Evaluation in Preclinical and Clinical Settings.

The main objectives of this work were to assess the efficiency, ease-of-use, and general performance of a novel osseoshaping tool based on first-user clinical experiences and to compare these observations with preclinical data generated in rodents using a miniaturized version of the instrument. All patients selected for the surgery presented challenging clinical conditions in terms of the quality and/or quantity of the available bone. The presented data were collected during the implant placement of 15 implants in 7 patients, and included implant recipient site (bone quality and quantity) and ridge evaluation, intra-operative handling of the novel instrument, and the evaluation of subsequent implant insertion. The instrument was easy to handle and was applied without any complications during the surgical procedure. Its use obviated the need for multiple drills and enabled adequate insertion torque in all cases. This biologically driven innovation in implant site preparation shows improvements in preserving vital anatomical and cellular structures as well as simplifying the surgical protocol with excellent ease-of-use and handling properties.

Hormone sensitive lipase ablation promotes bone regeneration.

There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial µCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

Multiscale analysis of craniomaxillofacial bone repair: A preclinical mini-pig study.

BACKGROUND: The rate of reparative osteogenesis controls when an implant is sufficiently stable as to allow functional loading. Using a mini pig model, the rate of reparative osteogenesis in two types of implant sites for example, an osteotomy versus a fresh extraction socket were compared. METHODS: Eight adult mini pigs were used for the study. In phase I, three premolars were extracted on one side of the oral cavity; 12 weeks later, in phase II, osteotomies were produced in healed extraction sites, and contralateral premolars were extracted. Animals were sacrificed 1, 5, and 12 weeks after phase II. Bone repair and remodeling were evaluated using quantitative micro-computed tomographic imaging, histology, and histochemical assays coupled with quantitative dynamic histomorphometry. RESULTS: One week after surgery, extraction sockets and osteotomy sites exhibited similar patterns of new bone deposition. Five weeks after surgery, mineral apposition rates (MARs) were elevated at the injury sites relative to intact bone. Twelve weeks after surgery, the density of new bone in both injury sites was equivalent to intact bone but quantitative dynamic histomorphometry and cellular activity assays demonstrated bone remodeling was still underway. CONCLUSIONS: The mechanisms and rates of reparative osteogenesis were equivalent between fresh extraction sockets and osteotomies. The volume of new bone required to fill a socket, however, was significantly greater than the volume required to fill an osteotomy. These data provide a framework for estimating the rate of reparative osteogenesis and the time to loading of implants placed in healed sites versus fresh extraction sockets.

Clinically relevant preclinical animal models for testing novel cranio-maxillofacial bone 3D-printed biomaterials.

Bone tissue engineering is a rapidly developing field with potential for the regeneration of craniomaxillofacial (CMF) bones, with 3D printing being a suitable fabrication tool for patient-specific implants. The CMF region includes a variety of different bones with distinct functions. The clinical implementation of tissue engineering concepts is currently poor, likely due to multiple reasons including the complexity of the CMF anatomy and biology, and the limited relevance of the currently used preclinical models. The 'recapitulation of a human disease' is a core requisite of preclinical animal models, but this aspect is often neglected, with a vast majority of studies failing to identify the specific clinical indication they are targeting and/or the rationale for choosing one animal model over another. Currently, there are no suitable guidelines that propose the most appropriate animal model to address a specific CMF pathology and no standards are established to test the efficacy of biomaterials or tissue engineered constructs in the CMF field. This review reports the current clinical scenario of CMF reconstruction, then discusses the numerous limitations of currently used preclinical animal models employed for validating 3D-printed tissue engineered constructs and the need to reduce animal work that does not address a specific clinical question. We will highlight critical research aspects to consider, to pave a clinically driven path for the development of new tissue engineered materials for CMF reconstruction.

Effects of masticatory loading on bone remodeling around teeth versus implants: Insights from a preclinical model.

OBJECTIVES: Teeth connect to bone via a periodontal ligament, whereas implants connect to bone directly. Consequently, masticatory loads are distributed differently to periodontal versus peri-implant bone. Our objective was to determine how masticatory loading of an implant versus a tooth affected peri-implant versus periodontal bone remodeling. Our hypothesis was that strains produced by functional loading of an implant would be elevated compared with the strains around teeth, and that this would stimulate a greater degree of bone turnover around implants versus in periodontal bone. MATERIALS AND METHODS: Sixty skeletally mature mice were divided into two groups. In the implant group, maxillary first molars (mxM1) were extracted, and after socket healing, titanium alloy implants were positioned subocclusally. After osseointegration, implants were exposed, resin crowns were placed, and masticatory loading was initiated. In the control group, the dentition was left intact. Responses of peri-implant and periodontal bone were measured using micro-CT, histology, bone remodeling assays, and quantitative histomorphometry while bone strains were estimated using finite element (FE) analyses. CONCLUSIONS: When a submerged osseointegrated implant is exposed to masticatory forces, peri-implant strains are elevated, and peri-implant bone undergoes significant remodeling that culminates in new bone accrual. The accumulation of new bone functions to reduce both peri-implant strains and bone remodeling activities, equivalent to those observed around the intact dentition.

Targeting Notch Inhibitors to the Myeloma Bone Marrow Niche Decreases Tumor Growth and Bone Destruction without Gut Toxicity.

Systemic inhibition of Notch with γ-secretase inhibitors (GSI) decreases multiple myeloma tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine multiple myeloma, treatment with BT-GSI decreased tumor burden and prevented the progression of multiple myeloma-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-resorptive properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of multiple myeloma and associated bone disease. SIGNIFICANCE: Development of a bone-targeted Notch inhibitor reduces multiple myeloma growth and mitigates cancer-induced bone destruction without inducing the gastrointestinal toxicity typically associated with inhibition of Notch.

Biology of sinus floor augmentation with an autograft versus a bone graft substitute in a preclinical in vivo experimental model.

OBJECTIVES: Compared to autografts, bone graft substitutes are slower to consolidate. If we understood why, this might open strategies to accelerate new bone formation and thus shorten the time to implant placement. In this study, we aimed at comparing autologous bone graft with a bovine bone graft substitute in a preclinical sinus lift model. MATERIALS AND METHODS: The mouse posterior paranasal sinus served as a recipient site for grafting. Autograft from the oral cavity was compared against bone graft substitute using molecular, cellular, and histological analyses conducted on post-grafting days (PSD) 0, 9, 18, and 120. RESULTS: Either autografts or bone graft substitutes were positioned on the sinus floor and remained in situ throughout the study. At the time of grafting and until day 9, bone graft substitutes were devoid of cells and alkaline phosphatase (ALP) activity while autografts were comprised of viable cells and showed strong ALP (mineralization) activity. Consequently, new bone formed faster in autografts compared to bone graft substitutes (140.21 ± 41.21 µm vs. 41.70 ± 10.09 µm, respectively, PSD9, p = .0143). By PSD18, osteogenesis was evident in autografted and xenografted sites. Osteoclasts identified by tartrate resistant acid phosphatase attached to, but did not resorb the bone graft substitute matrix. Autograft matrix, however, underwent extensive resorption. Transgenic mice revealed that Wnt-responsive osteoprogenitor cells originated primarily from the internal periosteum of the maxillary bone, and not from the Schneiderian membrane. CONCLUSION: Autografts produce new bone sooner, but bovine bone graft substitutes eventually consolidate and then resist resorption. Enhancing osteoprogenitor cell recruitment to a bone graft substitute constitutes a viable strategy for accelerating bone formation in a sinus lift procedure.

Comparative analyses of the soft tissue interfaces around teeth and implants: Insights from a pre-clinical implant model.

AIM: To evaluate the similarities and differences in barrier function of a peri-implant epithelium (PIE) versus a native junctional epithelium (JE). MATERIALS AND METHODS: A mouse model was used wherein titanium implants were placed sub-occlusally in healed extraction sites. The PIE was examined at multiple timepoints after implant placement, to capture and understand the temporal nature of its assembly and homeostatic status. Mitotic activity, hemidesmosomal attachment apparatus, and inflammatory responses in the PIE were compared against a JE. Additionally, we evaluated whether the PIE developed a Wnt-responsive stem cell niche like a JE. RESULTS: The PIE developed from oral epithelium (OE) that had, by the time of implant placement, lost all characteristics of a JE. Compared with a JE, an established PIE had more proliferating cells, exhibited lower expression of attachment proteins, and had significantly more inflammatory cells in the underlying connective tissue. Wnt-responsive cells in the OE contributed to an initial PIE, but Wnt-responsive cells and their descendants were lost as the PIE matured. CONCLUSIONS: Although histologically similar, the PIE lacked a Wnt-responsive stem cell niche and exhibited characteristics of a chronically inflamed tissue. Both features contributed to suboptimal barrier functions of the PIE compared with a native JE.

Pro-osteogenic Effects of WNT in a Mouse Model of Bone Formation Around Femoral Implants.

Wnt signaling maintains homeostasis in the bone marrow cavity: if Wnt signaling is inhibited then bone volume and density would decline. In this study, we identified a population of Wnt-responsive cells as osteoprogenitor in the intact trabecular bone region, which were responsible for bone development and turnover. If an implant was placed into the long bone, this Wnt-responsive population and their progeny contributed to osseointegration. We employed Axin2CreCreERT2/+;R26mTmG/+ transgenic mouse strain in which Axin2-positive, Wnt-responsive cells, and their progeny are permanently labeled by GFP upon exposure to tamoxifen. Each mouse received femoral implants placed into a site prepared solely by drilling, and a single-dose liposomal WNT3A protein was used in the treatment group. A lineage tracing strategy design allowed us to identify cells actively expressing Axin2 in response to Wnt signaling pathway. These tools demonstrated that Wnt-responsive cells and their progeny comprise a quiescent population residing in the trabecular region. In response to an implant placed, this population becomes mitotically active: cells migrated into the peri-implant region, up-regulated the expression of osteogenic proteins. Ultimately, those cells gave rise to osteoblasts that produced significantly more new bone in the peri-implant region. Wnt-responsive cells directly contributed to implant osseointegration. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of implant placed was sufficient to accelerate osseointegration. The Wnt-responsive cell population in trabecular bone, activated by injury, ultimately contributes to implant osseointegration. Liposomal WNT3A protein therapeutic accelerates implant osseointegration in the long bone.

A novel system exploits bone debris for implant osseointegration.

BACKGROUND: Bone debris generated during site preparation is generally evacuated with irrigation; here, we evaluated whether retention of this autologous material improved the rate of peri-implant bone formation. METHODS: In 25 rats, a miniature implant system composed of an osseo-shaping tool and a tri-oval-shaped implant was compared against a conventional drill and round implant system. A split-mouth design was used, and fresh extraction sockets served as implant sites. Histology/histomorphometry, immunohistochemistry, and microcomputed tomography (µCT) imaging were performed immediately after implant placement, and on post-surgery days 3, 7, 14, and 28. RESULTS: Compared with a conventional drill design, the osseo-shaping tool produced a textured osteotomy surface and viable bone debris that was retained in the peri-implant environment. Proliferating osteoprogenitor cells, identified by PCNA and Runx2 expression, contributed to faster peri-implant bone formation. Although all implants osseointegrated, sites prepared with the osseo-shaping tool showed evidence of new peri-implant bone sooner than controls. CONCLUSION: Bone debris produced by an osseo-shaping tool directly contributed to faster peri-implant bone formation and implant osseointegration.

Drill Hole Models to Investigate Bone Repair.

Our understanding of the mechanisms underlying fracture healing is rapidly developing and is contributing to new therapeutic strategies to enhance repair. To gain new insights, animal models must also evolve. From initially imprecise, uncontrolled bone defects we now have precise injury models that still capture all of the stages and phases of bone repair yet do so in a highly reproducible manner. The simple mono-cortical defect model allows assessment of bone repair through a cartilage intermediate, e.g., endochondral ossification, as well as direct bone repair, e.g., intramembranous healing. Cellular contributions of the periosteum can be distinguished from contributions originating in the bone marrow. In this chapter, we focus on the advantages of this bone repair model, as well as its limitations.

A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens.

The disconnect between preclinical and clinical results underscores the imperative for establishing good animal models, then gleaning all available data on efficacy, safety, and potential toxicities associated with a device or drug. Mini pigs are a commonly used animal model for testing orthopedic and dental devices because their skeletons are large enough to accommodate human-sized implants. The challenge comes with the analyses of their hard tissues: current methods are time-consuming, destructive, and largely limited to histological observations made from the analysis of very few tissue sections. We developed and employed cryo-based methods that preserved the microarchitecture and the cellular/molecular integrity of mini pig hard tissues, then demonstrated that the results of these histological, histochemical, immunohistochemical, and dynamic histomorphometric analyses e.g., mineral apposition rates were comparable with similar data from preclinical rodent models. Thus, the ability to assess static and dynamic bone states increases the translational value of mini pig and other large animal model studies. In sum, this method represents logical means to minimize the number of animals in a study while simultaneously maximizing the amount of information collected from each specimen.

Formation and regeneration of a Wnt-responsive junctional epithelium.

AIM: To identify the molecular mechanisms mediating the persistent defensive functions of the self-renewing junctional epithelium (JE). MATERIALS AND METHODS: Two strains of Wnt reporter mice, Axin2CreErt2/+ ;R26RmTmG/+ and Axin2LacZ/+ , were employed, along with three clinically relevant experimental scenarios where the function of the JE is disrupted: after tooth extraction, after a partial gingivectomy, and after a complete circumferential gingivectomy. RESULTS: Using transgenic Wnt reporter strains of mice, we established the JE is a Wnt-responsive epithelium beginning at the time of its formation and that it maintains this status into adulthood. After tooth extraction, progeny of the initial Wnt-responsive JE population directly contributed to healing and ultimately adopted an oral epithelium (OE) phenotype. In the traditional partial gingivectomy model, the JE completely regenerated and did so via progeny of the original Wnt-responsive population. However, following circumferential gingivectomy, the OE was incapable of re-establishing a functional JE. CONCLUSIONS: A Wnt-responsive niche at the interface between tooth and oral epithelia is required for a functional JE.

Bone formation around unstable implants is enhanced by a WNT protein therapeutic in a preclinical in vivo model.

OBJECTIVES: Our objective was to test the hypothesis that local delivery of a WNT protein therapeutic would support osseointegration of an unstable implant placed into an oversized osteotomy and subjected to functional loading. MATERIALS AND METHODS: Using a split-mouth design in an ovariectomized (OVX) rat model, 50 titanium implants were placed in oversized osteotomies. Implants were subjected to functional loading. One-half of the implants were treated with a liposomal formulation of WNT3A protein (L-WNT3A); the other half received an identical liposomal formulation containing phosphate-buffered saline (PBS). Finite element modeling estimated peri-implant strains caused by functional loading. Histological, molecular, cellular, and quantitative micro-computed tomographic (µCT) imaging analyses were performed on samples from post-implant days (PID) 3, 7, and 14. Lateral implant stability was quantified at PID 7 and 14. RESULTS: Finite element analyses predicted levels of peri-implant strains incompatible with new bone formation. Micro-CT imaging, histological, and quantitative immunohistochemical (IHC) analyses confirmed that PBS-treated implants underwent fibrous encapsulation. In those cases where the peri-implant environment was treated with L-WNT3A, µCT imaging, histological, and quantitative IHC analyses demonstrated a significant increase in expression of proliferative (PCNA) and osteogenic (Runx2, Osterix) markers. One week after L-WNT3A treatment, new bone formation was evident, and two weeks later, L-WNT3A-treated gaps had a stiffer interface compared to PBS-treated gaps. CONCLUSION: In a rat model, unstable implants undergo fibrous encapsulation. If the same unstable implants are treated with L-WNT3A at the time of placement, then it results in significantly more peri-implant bone and greater interfacial stiffness.