The Degradation And Tissue Integration Of Hydroxyapatite Implants Analysed Using Energy Dispersive X-Ray Spectrometry

Cell and tissue interactions with biomaterials at ultrastructural resolutions can be challenging to investigate. Multi-colour electron microscopy using energy dispersive x-ray spectrometry (EDS) is presented here to enhance the interpretation of SEM data and provide additional analytical capabilities in the study of tissue-biomaterial interactions. We used SEM and EDS to examine the degradation of hydroxyapatite implants and subsequent growth of new bone using minipigs as an animal model. Tissue samples were collected at 1 and 3 months after implantation, fixed, embedded in resin, and ground to a thickness of approximately 1mm before being stained using uranyl acetate and lead citrate. EDS data was collected with an Ultim Extreme detector using accelerating voltages of between 5 and 10kV and a beam current of 1na or less. Data was collected as single area scans and large area maps. We found that it was not possible to determine the difference between cells and the implant material using electron signals alone. EDS was instrumental in identifying cells located within voids inside the hydroxyapatite. Cells within the implant at 1 month were stellate, a morphology usually observed in osteocytes embedded in bone matrix. EDS was also used to identify hydroxyapatite particles that were ingested by cells, determine regions of new bone growth, and revealed variations in nitrogen concentration (often used as a marker for peptides) in areas immediately next to the implant. The 3-month-old samples showed a greater quantity of cells inside the implant. The hydroxyapatite was also observed to be more fragmented. There were areas within the 3-month old implants that displayed high concentrations of nitrogen and sodium, while nitrogen levels surrounding the implant material were lower compared to 1-month old samples. EDS provided data that facilitated interpretation of structures.