Osteogenesis During Early Healing Around Titanium and Roxolid Implants: Evaluation of Bone Markers by Immunohistochemistry and RT-PCR Analysis in Miniature Pigs: A Pilot Study

Abstract PURPOSE: A novel approach for the study of early bone formation around dental implants in the miniature pig was evaluated. In addition to the traditional histologic and histomorphometric analysis, the expression of the osteogenic genes was analyzed both at the messenger ribonucleic acid (mRNA) and protein level. MATERIALS AND METHODS: Mandibular premolars and the first molar were extracted in six miniature pigs. After 3 months of healing, 36 specially designed bone chamber implants were placed. Three different implant surface configurations were used: titanium SLA, titanium SLActive, and titanium zirconium SLActive (Roxolid). Each hemi-mandible received three randomly allocated implants (one for each surface type) on both sides of the arch, in a split-mouth design. Three animals were sacrificed after 3 days and another three after 2 weeks of healing post-implant insertion. For each animal the right hemi-mandible underwent qualitative histologic and quantitative histomorphometric analysis. The left hemi-mandible underwent immunohistofluorescence (IHF) analysis. β-catenin, Runx2, osteopontin, and osteocalcin were analyzed by IHF; osterix, and osteocalcin mRNA expression was also evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: At 3 days after the implantation, all implants were surrounded by blood clot. No provisional matrix or bone was observed inside the chamber. Infection or degenerative lesions were absent. At 2 weeks, the histomorphometric analysis showed no significant difference between the groups concerning the bone area. qRT-PCR showed that Ti SLActive had the highest osteocalcin mRNA expression followed by Ti SLA and Roxolid SLActive. Osterix mRNA expression was higher on Ti SLA and Roxolid SLActive compared to Ti SLActive. The differences were not statistically significant. IHF was only found positive for osteocalcin at 2 weeks. At 3 days, osteocalcin was detected only on native bone. At 2 weeks, osteocalcin was expressed highest by Ti SLActive followed by Roxolid SLActive and TiSLA; however, there was no statistically significant difference between the groups in the osteocalcin expression level. CONCLUSION: The present methodology allowed evaluation of changes in gene expression during the early phase of osteogenesis that seem to be related to the quality of the surface. Further studies with higher power and more specific antibodies are needed to confirm these preliminary findings.