Twenty-six weeks after the regenerative procedure the patient returned for the re-entry intervention for placement of dental implants. After raising full-thickness flaps, the augmented area was exposed and horizontal crestal width measurements were performed. Then, the surgeon evaluated the bone availability and if implant placement was considered possible, a core bone biopsy was harvested with the use of a trephine, replacing the first drill of the implant bed preparation (2 mm diameter and 10 mm length, Hager and Meisinger® Neuss, Germany).

The retrieved trephine containing the bone biopsy was irrigated with saline to remove the blood and was introduced in a tube containing 10% formalin solution, which was coded and stored until processing. Commercially available titanium dental implants were inserted in accordance with manufacturer guidelines and after 8 weeks of healing, fixed screwed-retained prosthetic restorations were placed (Fig. 3).

One biopsy per patient was processed for ground sectioning according to the method described by Donath and Breuner (1982). In brief, the specimens including the trephines were fixed in neutral-buffered formalin, stored in compartment biopsy cassettes, and appropriately coded for identification. Once fixed, the blocks containing the trephines were dissected, dehydrated with ascending alcohol grades and embedded in a light-curing resin (Technovit 7200 VLC; Heraeus-Kulzer, Wehrheim, Germany). At least two longitudinal sections of each core biopsy were grounded and reduced to a thickness of approximately 40 microns using Exakt cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). All the sections were stained using the Levai-Laczkó technique [22].

The second biopsies were processed for decalcification, included in paraffin, stained with hematoxyline-eosine (H-E) and further processed for immune-histochemical analysis. The biopsies were fixed overnight in 4% neutral buffered formalin. Decalcification was achieved by immersing the specimens in 1 mM EDTA solution and then embedded in paraffin following standard procedures. Semi-thin sections of 4-μm-thick were obtained and stained with hematoxyline-eosine (H-E).