Antigen preservation after decalcification: can we have a guideline for everyday bone-related laboratory routine?

Introduction: The processing of the bone tissue presents multiple problems related to decalcification and it is often based on the experience of the operator. The aim of this study is to test bone tissue antigen preservation after different decalcification methods in order to verify which is the most suitable to obtain guidelines for laboratory routine. Materials and Methods: Medial condyle of the left femur of swine, sheep, dogs, mice and rats were fixed and decalcified with two different solutions: A, 1.85% hydrochloric acid, 4% formic acid; B, 7.5% citric acid, 25% formic acid. Integrity of tissue antigenicity were evaluated by means of two commercial antibodies for collagen I by immunofluorescence (IF). IF peak intensity was analysed. Results: Antigen preservation was observed in both treatments in dog, sheep, and swine. Indeed, the bone sections from mouse and rat lost their link ability for the antibodies. IF peaks revealed to be higher with B solution than A in sheep (P<0.01), while no differences were found in swine and dog. Furthermore, IF peaks for one of the two antibodies revealed to be higher in sheep and swine (P<0.01), but no differences among the two antibodies were found in dog. Conclusion: B solution seems to better preserve antigenicity of bone tissue in sheep and one of the two antibodies works better in sheep and swine. With this research we intended to define guidelines for trabecular bone decalcification based on the animal species and antigen preservation which is a basic tool for tissue engineering.