Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization

Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Introduction titanium (Ti) is considered the gold standard biomaterial in oral implantology 1 , due to the material's high biocompatibility, adequate mechanical properties, and osseointegration capacity 1,2 , which lead to longterm performance and high rates of clinical success 1,3 .
Additionally, Ti is also currently regarded as an immunomodulatory biomaterial rather than an inert metal, since Ti implantation in bone is associated with a transitory small degree of inflammation, which seems to contribute to the activation of host pathways that leads to osseointegration 2,4

Titanium implant screws
In an attempt to employ a titanium screw comparable to the one clinically used in Dentistry, a screw with Ø 0.6 mm, titanium-6 aluminum-4 vanadium alloy (NTI-Kahla GmbH Rotary Dental Instruments, Kahla, Thüringen, Germany) and machined titanium surface was used in this study, as previously described in the oral osseointegration model in CD1 mice 10 . The screws were cut at a length of 1.5 mm and sterilized by autoclaving before surgical procedures. Subsequently, the screws were analyzed via scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) before Ti implantation, in order to demonstrate the surface topography and chemical composition of the screws used in this study. The

Development of the surgical protocol
Our focus is this study was to address a pre-clinic murine model of oral osseointegration, previously developed in CD1 mice 10    spaces occupied by the three initial Ti-screw threads, from coronal to apical, on each side of the Ti-screw, as indicated by arrows in Figure 4A. After 3 days, the bone-implant interface was filled mainly by a blood clot and inflammatory infiltrate, as demonstrated through histomorphometry ( Figure 5A, B). The blood clot was evidenced by erythrocytes, surrounded by an eosinophilic and slight matrix of the fibrin network, also permeated by an inflammatory infiltrate with predominance of mononuclear cells ( Figure 4B      implant screw cut at a 1.5 mm length. The implant's placement in this area, without preceding multiple tooth extraction, was previously reported in CD1 mice, which, due to their increased size, were suitable for the insertion of a 2 mm implant 10 . Additionally, the Ti screw used in this study was based on a conventional Ti6Al4V alloy, with a machined surface without any treatments and/or topography alterations, as demonstrated through SEM and X-ray analysis (Figure 2), in order to characterize the osseointegration process per se, as has been frequently used in experimental studies using craniofacial 10 and long bones 16,25 as osseointegration models.
The surgical procedures used in this study were performed following the same principles and procedures used in Dentistry, to avoid lack of primary stability and overheating. Of all titanium implants with adequate primary stability, 77.78% achieved osseointegration, demonstrated through µCT and histological data (Figures 3 and 4), which is in agreement with the success rates previously described in a similar model performed in CD1mice (74% of osseointegration after 21 days) 10   Concurrently with the early upregulation of the MSCs markers, a provisional extracellular matrix is formed and gradually evolves into a highly vascularized granulation tissue (Figures 4 and 5), which will provide further support for cell migration and differentiation.
A similar response was observed in peri-implant sites in mice 10,22 and rats 27 , but the presence of biomaterials was associated with delayed healing dynamics compared to alveolar intramembranous bone healing in the absence of biomaterials 9,10 . Indeed, the earlier granulation tissue formed in the space between the Ti threads and remaining bone works as a preosteoblastic supportive connective tissue 10,22 , as evidenced in this study by an increased area density of blood vessels  In this study, while iNOS (a M1 marker) expression remained low at the osseointegration sites, ARG1 (a M2 marker) was upregulated after Ti implantation, indicating a predominance of a M2-type response.
Indeed, in enhanced osseointegration models observed in long bones in rats, the upregulation of ARG1 and downregulation of iNOS are correlated with a high proportion of M2 macrophages and beneficial bone healing around the Ti surfaces 37 . Accordingly, a marked-up regulation of reparative/regulatory M2type macrophages is also observed after Ti implant placement in humans 30 . Indeed, the M2-type response has been suggested to be critical to wound healing outcomes for expressing several pro-resolutive molecules, including ARG1, IL10 and TGFb1 38 . These data are also compatible with the transitory nature of the inflammatory infiltrate surrounding the Ti surface, which showed a gradual decrease over time in this study ( Figure 4B, C, D and Figure 5).
Following the resolution of inflammation ( Figure   4D), while the expression of inflammatory factors and density of inflammatory infiltrate tend to decrease over time post-implantation, the expression of osteogenic factors and ECM components were gradually increased, in agreement with previous findings in rats 27 . In line with the events of intramembranous bone repair, the granulation tissue is directly replaced by bone over time (Figures 3 and 4), as also previously reported in other animal models of oral osseointegration 10,27 , while Ti osseointegration in long bones is dependent on the formation of hypertrophic cartilage 15 . As the density area of the primary bone matrix significantly increased after 14 days, also followed by expression of Col1a1 and Col21a2 and a gradual maturation of collagen fibers detected through birefringence analysis ( Figure   6), there was a remarkable remodeling process, evidenced by peaks corresponding to MMPs (MMP1, MMP2 and MMP9), RANKL and OPG, and also an increased area density of osteoclasts ( Figure 5H). As also demonstrated in other models 28,30 , all these events will collectively determine bone quality and influence the mechanical properties of osseointegration 37 .
Indeed, the quality of osseointegration is dependent on a highly organized bone matrix and its ECM components, in which collagen plays a crucial role 38 . osseointegration, evidencing elements that could be responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel histological, birefringence and μCT analysis ( Figure   9). Considering all these observations and comparing with previous descriptions of osseointegration, this C57Bl/6 mice oral osseointegration model would be a suitable tool for the assessment of biological events associated with the osseointegration process.