Bone-tissue formation and integration of titanium implants: an evaluation with newly developed enzyme and immunohistochemical techniques

Background: Examination of the tissue surrounding retrieved implants involve routine investigations on cut and ground sections. Undecalcified sections with implants in situ are histologically stained followed by qualitative and quantitative observations of the tissue response to the implants by light microscopy. Purpose: A novel technique that allows for the accurate definition and quantification of enzymes involved in bone formation (alkaline phosphatase) and resorption (acid phosphatase) in the tissue is presented. Materials and Methods: Commercially pure titanium and titanium alloy (Ti6A14V) implants were retrieved after 6 and 12 weeks of healing in rabbit bone. In addition, 4-week specimens from commercially pure titanium bone harvest chambers placed in rabbit bone were used. Undecalcified cut and ground sections were produced and evaluated with enzyme and immunohistochemical staining techniques. Results: The titanium implants retrieved after 6 weeks of insertion in rabbit bone revealed a higher activity of both alkaline phosphatase and acid phosphatase activity compared to the implants followed for 12 months. The former samples revealed ongoing bone-tissue remodeling in the interface, whereas the latter ones showed steady-state bone conditions. Applying the new technique allowed for investigation of various bone proteins present in the tissue that had formed inside titanium canals of harvest chambers at various times of follow-up. Conclusions: The combination of routine histologic stainings with enzyme and immunohistochemical technique of cut and ground specimens is a valuable tool in the investigations of retrieved implants from humans and animals. This novel technique now may be used to describe the state of bone regeneration in the interface zone associated with implant research.