Zen core

To examine the (ultra-) structure and the thickness of the 3 membranes, a histological preparation of the blank materials was conducted, as previously described [11 (link)]. Briefly, the preparation included the histological workup by initial fixation of 3 membrane specimens of each material (sized 10 × 10 mm) in 4% buffered formalin for 24 h followed by dehydration in a series of alcohol and xylene. After paraffin embedding, the blocks were sectioned, resulting in slices with a thickness of 3–5 µm, which were then deparaffinized and histochemically stained via hematoxylin and eosin (H&E). This procedure allowed us to examine and visualize the (ultra-) structures of the biomaterials via a Panthera U light microscope (Motic, Xiamen, China) and a connected Axiocam 105 color camera combined with the software ZEN Core (both: Zeiss, Oberkochen, Germany). Moreover, it allowed the measurement of the membranes’ baseline thicknesses, as described below.

For the qualitative histopathological analysis, the H&E-stained slides were used. The implant beds were viewed microscopically and the local tissue responses as well as their integration and degradation patterns were assessed. Photographs were taken using an Axiocam 105 color camera that was connected to its software ZEN Core (Zeiss, Oberkochen, Germany).

The histological analyses to study the tissue–biomaterial interactions within the implantation beds of the biomaterials and their surrounding tissue were conducted using an Axio Imager M2 (Zeiss, Oberkochen, Germany) based on a protocol according to the DIN ISO 10993-6 as previously described [11 (link),23 (link),33 (link),34 (link),35 (link)]. These analyses focused on the evaluation of the following parameters within the framework of the early and the late tissue response related to the implants: fibrosis; hemorrhage; necrosis; vascularization; and the presence of neutrophils, lymphocytes, plasma cells, macrophages and biomaterial-associated multinucleated giant cells (BMGCs). Finally, microphotographs were taken with an Axiocam 506 color connected to a computer system running the ZEN Core (Zeiss, Oberkochen, Germany) connected to a microscope.