Calcification at the Interface Between Titanium Implants and Bone: Observation With Confocal Laser Scanning Microscopy
It has not been previously possible to observe bone formation in undecalcified sections with titanium implants at high magnification because of the difficulty in sectioning bone together with implants. A method for examining the bone-implant interface in undecalcified sections is described in which implants are left in situ and confocal laser scanning microscopy (CLSM) is used to examine both the implant surface and adjacent bone. Pulsing of animals at different times with the fluorescent dyes calcein and alizarin red permitted assessment of temporal patterns of bone formation by CLSM. Reflectivity of the polished implant surface permitted accurate assessment of the position of the implant relative to labeled bone. The analysis showed that bone first formed as thin processes towards and across the implant surface, followed by further bone formation behind these processes. The interface between calcified bone tissue and the implant surface was characterized by a 10-μm space. The CLSM technique enabled detailed observations of new bone formation at the titanium implant interface.Abstract
Confocal laser scanning microscopy images with detection conditions 1 to 6 (see the Table) of an implant 8 weeks after surgery. (A) Both calcein and alizarin red are detected, but the implant is devoid of the reactions in condition 1. (B and C) Calcein and alizarin red are detected together with the implant in detection conditions 2 and 3, respectively. (D) The highly reflective image is limited to the implant surface in detection condition 4. (E) Superimposition of the reflective implant image of 1B (red) with 1D (blue) is seen as purple in detection condition 5. (F) This image was subsequently used to assess calcein and alizarin red fluorescence, together with reflection of the implant surface, by combining images collected for detection conditions 1 and 4
Contributor Notes
Tetsunari Nishikawa, DDS, PhD, is an associate professor and Kazuya Masuno, DDS, MS, is a researcher at the Department of Oral Pathology, Masahiko Mori, DDS, PhD, is a visiting professor, Yasuhiro Tajime, DDS, is a PhD student, Kenji Kakudo, DDS, PhD, is a professor at the Second Department of Oral and Maxillofacial Surgery, and Akio Tanaka, DDS, PhD, is a professor at the Department of Oral Pathology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka, 573-1121, Japan. Address correspondence to Dr Nishikawa (tetsu-n@cc.osaka-dent.ac.jp).