β-TCP/HA with or without enamel matrix proteins for maxillary sinus floor augmentation: a histomorphometric analysis of human biopsies

Ten consecutive patients (age range 35–75 years) with the need of bilateral maxillary sinus floor augmentation prior to the placement of four dental implants (two in each side of posterior maxilla) were selected for the study. The main inclusion criterion was a vertical dimension of the residual alveolar bone between 3 and 5 mm in the sites selected for implant placement in the posterior maxilla, as assessed on a cone beam CT. Only patients with no need for additional bone augmentation (i.e., lateral or vertical) were included. The patients did not suffer from any systemic disease that might interfere with bone healing (e.g., uncontrollable diabetes; osteoporosis) and did not smoke more than 10 cigarettes per day. Sample size calculation was based on the statistical mean and standard deviation of percent new bone formation within the augmented maxillary sinus, reported previously in a similar study including histomorphometric evaluation [15].

All patients received systemic antibiotics (amoxicillin 500 mg, every 8 h for 7 days) and anti-inflammatory drugs (nimesulide 100 mg twice daily for 5 days), starting all the medication 1 h before surgery. Patients were also prescribed analgesics (paracetamol 750 mg, max. four times a day) if there was pain. Chlorexidine digluconate 0.12% mouth rinses, four times daily, were also prescribed for 14 days post-operatively.

Surgery was planned using cone beam CT images (i-CAT, Image Sciences International, USA) with 0.25 mm voxel size, in 1-mm-thick sections, generated every 1 mm in the region of interest (posterior maxilla). After extra and intraoral disinfection of the operating field, local anesthesia was administered using lidocaine hydrochloride 2% with epinephrine 1:100.000 (DFL Industry and Trade, Rio de Janeiro, Brazil). Maxillary sinus floor augmentation with a lateral window technique was performed, and each of the sinuses received either β-TCP/HA (Straumann® BoneCeramic, Basel, Switzerland – BC group) or β-TCP/HA manually mixed using a periosteal elevator with EMD (Straumann® Emdogain, Basel, Switzerland), in a proportion of 1 g of BC for 0.3 ml of EMD (BC + EMD group), in a random fashion (by tossing a coin) and using a split-mouth design. In both groups, a very limited amount of sterile ;physiological saline solution (NaCl 0.9%) was added to the graft material mixture, insufficient amount to provide the consistency needed to ease handling and transferring into the sinus. No membrane or other material was used for closing the lateral window. After flap repositioning, closure was performed using simple interrupted nylon sutures (4-0, Ethicon, Johnson & Johnson). No radiographic examination immediately after sinus augmentation procedure was undertaken.

Six months after grafting, another CBCT examination was carried out for implant planning. In the sequence, following the previously described antiseptic and anesthetic procedures, two implants with a sand-blasted and acid etching surface were installed in each of the grafted sinuses, i.e., 40 implants in total (32—Neoporous, Neodent, Curitiba, Paraná, Brazil; 8—SLA, Straumann, Basel, Switzerland). A 10-mm-long cylindrical bone biopsy was harvested using a 2-mm internal diameter trephine bur during preparation for the most anterior implant site (i.e., two biopsies were retrieved from each patient). Six months later, the prosthetic rehabilitation of the patient was performed.

Immediately after retrieval, the apical aspect of the harvested biopsies was marked using India ink, to be used as a guide during histological evaluation. The biopsies were routinely processed (maintained in formaldehyde during 2 days, washed, and decalcified using EDTA solution, under continuous shaking, for 2 months) and embedded in paraffin. Six 6-μm-thick sections representing the central aspect of the cylindrical biopsy were obtained from each biopsy. These sections were stained using hematoxylin-eosin and were used for histological and histomorphometric analyses. Images were acquired using a DIASTAR light microscope (Leica Reichert & Jung products, Germany) connected to a Leica Microsystems DFC-300-FX digital camera (Leica Microsystems, Germany). Additional sections were stained using picrosirius-hematoxylin for microscopic examination under polarized light.

From the entire biopsy, only the 6 mm towards the apical aspect was considered as the region of interest (ROI), in order to allow visualization of approximately 80% of grafted bone and 20% of resident bone. Histological evaluation assessed morphological characteristics of the newly formed bone, remaining grafted material, integration of the grafted material with the newly formed bone, soft tissues, and local inflammation. Also, the newly formed bone was assessed regarding the aggregation and organization of the collagen bundles, reflected in the variation in birefringence intensity. The relative amounts (%) of bone, soft tissues, and “other material” (i.e., remaining grafting material or empty spaces due removal of the grafting material during histological processing, artifacts, and debris), within the ROI were planimetrically estimated using ImageJ (NIH, Bethesda, MD, USA) (Fig. 1).

The data for each tissue component from the three histological sections were averaged to represent the biopsy. Commercially available software (GraphPad Prism 5.0 for Windows, GraphPad Software Inc., USA) was utilized for statistical comparisons between groups and for drawing the graphics. The assumption of normality was checked using D’Agostino & Pearson omnibus test. The data for each evaluated tissue, for BC and BC + EMD groups were analyzed as two paired samples from normal distributions based on a paired t test. Estimates were given with 95% confidence intervals, and statistical significance was set at 5% (p < 0.05).