Histological and histomorphometrical analysis of a silica matrix embedded nanocrystalline hydroxyapatite bone substitute using the subcutaneous implantation model in Wistar rats

Abstract

The clinical suitability of a bone substitute material is determined by the ability to induce a tissue reaction specific to its composition. The aim of this in vivo study was to analyze the tissue reaction to a silica matrix-embedded, nanocrystalline hydroxyapatite bone substitute.The subcutaneous implantation model in Wistar rats was chosen to assess the effect of silica degradation on the vascularization of the biomaterial and its biodegradation within a time period of 6 months. Already at day 10 after implantation, histomorphometrical analysis showed that the vascularization of the implantation bed reached its peak value compared to all other time points. Both vessel density and vascularization significantly decreased until day 90 after implantation. In this time period, the bone substitute underwent a significant degradation initiated by TRAP-positive and TRAP-negative multinucleated giant cells together with macrophages and lymphocytes. Although no specific tissue reaction could be related to the described silica degradation, the biomaterial was close to being fully degraded without a severe inflammatory response. These characteristics are advantageous for bone regeneration and remodeling processes.

Small-sized granules of biphasic bone substitutes support fast implant bed vascularization

“…6,14,[32][33][34][35][36][37] A first slide of each of the 3 tissue blocks from each animal was stained with hematoxylin and eosin (H&E) for general (histological) evaluation of the material implantation beds. Based on these slides, the tissue block with the best crosssection of the implanted biomaterials and peri-implant tissue was selected for further slide preparation and staining, as well as histological and histomorphometrical analysis.…”

“…The third and fourth sections were stained using special connective tissue staining methods, i.e., Movat's Pentachrome and Azan, to enable a differentiated overview of the different tissue components within the implant beds of the bone substitute material as previously shown. 6,14,[32][33][34][35][36][37] Immunohistochemistry Two further sections of the above-mentioned tissue blocks were used for immunohistochemical detection of murine vessels within the implantation beds of the bone substitutes following established protocols. 6,14,[32][33][34][35][36][37] In brief, epitope retrieval was performed after blocking endogenous peroxidase using a 3% H 2 O 2 -distilled water solution.…”

“…6,14,[32][33][34][35][36][37] In brief, after initial intraperitoneal anesthesia (10 ml ketamine [50 mg(ml] with 1.6 ml xylazine [2%]), 60 mg of the selected bone substitute material was implanted under sterile conditions in a preformed subcutaneous pocket in the animal's subscapular region using a bone curette. Subsequently, the implantation wound was stitched with 5.0 Prolene (Ethicon, New Jersey, USA).…”

“…6,14,[32][33][34][35][36][37] Briefly, the implanted bone substitutes were explanted, fixed in 4% formalin for 24 hours and cut into 3 equal segments. After decalcification in 10% tris-buffered EDTA (Carl Roth, Karlsruhe, Germany) at 37 C for 5-10 d, subsequent dehydration in a series of increasing alcohol concentrations and xylol was carried out, and after paraffin embedding, the preparation of histological sections with a thickness of 3-5 mm was performed with a rotation microtome (Leica, Wetzlar, Germany).…”

“…The outcome of the tissue-biomaterial interaction in the specimens was evaluated by examination of the total implantation bed and its peri-implant tissue. Thereby, the extent of the early (3-15 days) and late inflammatory responses (30 and 60 days) induced by the biomaterials was described following an established research protocol to access the following characteristics 6,14,[32][33][34][35][36][37] : fibrosis, hemorrhage, necrosis, vascularization, presence of granulocytes, lymphocytes, plasma cells, monocytes/macrophages, and TRAP-negative and TRAPpositive multinucleated giant cells (MNGCs). Microphotographs were taken using a DS-Fi1 digital camera and a digital sight control unit (Nikon, Tokyo, Japan).…”