Study design
The present manuscript reports the histological outcomes of a prospective single arm study evaluating the safety and clinical performance of CCXBB blocks when used as replacement bone grafts for lateral bone augmentation prior to staged implant placement. The results of the clinical and radiographic outcomes have been reported in a previous publication [21]. For correlation of the histological with the clinical outcome, respective data of the previous publication have been inserted.
Patient sample
Adults (≥18 years of age) were screened on the bases of having single or multiple teeth absences and a severe horizontal collapse of the alveolar ridge in need of one or more implants for implant supported fixed prosthetic rehabilitation.
Patients were selected on the bases of fulfillment of the following inclusion and exclusion criteria:
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Written informed consent
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Insufficient bone ridge width (<4 mm) for implant placement measured on a cone beam computed tomography (CBCT)
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Sufficient bone height for implant placement
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Healthy oral mucosa and ≥3 mm of attached keratinized mucosa
Patients were excluded if they had any of these conditions:
Inflammatory and autoimmune disease of the oral cavity
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Allergy to collagen
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Diabetes
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History of myeloma, respiratory tract cancer, breast cancer, prostate cancer or kidney cancer requiring chemotherapy or radiotherapy within the past 5 years
Concurrent or previous radiotherapy of head area
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Concurrent or previous immunosuppressant, bisphosphonate, or high-dose corticosteroid therapy
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Smokers
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Pregnant or lactating women
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Women of child bearing age, who are not using a highly effective method of birth control
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Participation in an investigational device, drug, or biologics study within the last 24 weeks prior to the study start
Before final inclusion, patients received meticulous verbal and written descriptions of the interventions and conditions and were requested to sign an informed consent form (directive 95/46/EC on data protection, in accordance with current legal provisions by the European Community).
Experimental product information
CCXBB (Bio-Graft® Geistlich Pharma) is a bone substitute material in a natural block form. The dimensions of the Bio-Graft block are 10 mm in height, 10 mm in length and 5 mm in width. It consists of a natural cancellous bone structure of hydroxyapatite and endogenous collagen type I and III, equine origin and is a class III medical device according to the Medical Device Directive 93/42 EECs’ definition (rule 8 implantable, resorbable device) and 17 (animal origin) in annex lX CE certificate G7 11 04 39446 050 for Geistlich Bio-Graft® was issued in June 2011.
The manufacture of Geistlich Bio-Graft is according to a standardized, controlled process and good manufacturing practices (GMPs). Each batch is manufactured and documented according to standard operating procedures, and the entire process has been validated.
Outcomes variables
The study design and follow-up visits have been summarized in Fig. 1. The primary outcome of this study was to assess the performance of the CCXBB by measuring the final crestal ridge width after 6 months of healing and evaluating its appropriateness for implant placement and the occurrence of adverse effects during healing.
Furthermore, the histological outcomes of this xenogeneic bone replacement graft were evaluated by harvesting a core biopsy of the regenerated area immediately before implant placement (after 6 months of healing), as well as the implant survival of those implants placed in the regenerated bone.
Surgical procedure and clinical measurements
The surgical placement of the CCXBB blocks and the clinical evaluation has been described in detail in a previous publication [21]. In brief, severe alveolar horizontal bone deficiencies were isolated after rising full-thickness mucoperiosteal flaps. Once the horizontal width of the alveolar crest was measured 2 mm below the crest with a bone calliper bone blocks were shaped, pre-drilled and pre-hydrated for 5 min with sterile physiological saline before placement and were fixed with titanium osteosynthesis screws allowing for a stable contact between the block graft and the underlying bone. The spaces between the bone block and the surrounding bone were filled with DBBM particles (Geistlich Bio-Oss®, Geistlich Pharma AG, Wolhusen, Switzerland) and covered with a native collagen membrane (CM) (Geistlich Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) fixed to the underlying bone with titanium tacks (FRIOS Fixation-Set®, SYMBIOS, Mainz, Germany). The muco-periosteal flaps were then coronally advanced and sutured achieving a tension-free primary closure (Fig. 2).
Bone biopsies harvesting procedure
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).
Histological processing
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).
For the immunohistochemical analysis, the semi-thin sections were incubated over night with primary antibodies at 4 °C (Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA). The antibody dilutions used were alkaline phosphatase (ALP) 1:100, osteopontin (OPN) 1:100, osteocalcin (OSC) 1:100, and tatrate resistant acid phosphatase (TRAP) 1:100.
Histological analysis
Qualitative analysis
The obtained semi-thin sections were evaluated with a motorized (Märzhäuser, Wetzlar-Steindorf, Germany) light microscope connected to a digital camera and a PC-based image-capture system (BX51, DP71, Olympus Corporation, Tokyo, Japan). Photographs were obtained at ×5 and ×20 magnifications (Fig. 4).
Histomorphometric analysis
From the obtained images, areas within the biopsies occupied by bone, biomaterial and connective tissue were identified using a pen computer (Cintiq companion, Wacom, Düsseldorf, Germany), coloured (Photoshop, Adobe, San José, CA, USA) and digitally measured using an automated image-analysis system (CellSens, Olympus Corporation) (Fig. 5).
Immunohistochemical analysis
The obtained histological sections were observed in a light microscope using 5x magnification. In the centre of each trephine biopsy, a rectangular region of interest (ROI) with a size of 30,000,000 to 32,000,000 pixels was defined and standardized photographs were obtained. The intensity of the antibody staining in the images was analysed using the software ImageJ, which by evaluating the antibody staining intensity in the area of interest allows for assessing quantitatively the specific marker (ImageJ®, IHC Profiler plugin). With this tool, the specimens were categorized into four groups: high positive (HP), positive (P), low positive (LP), and negative (N). To reduce false positives, only the HP and P values were considered for evaluating the percentage of positiveness for each immunohistochemical marker (Fig. 6).
Statistical analysis
Data were entered into an Excel (Microsoft Office 2011) database and proofed for entry errors. The software package (IBM SPSS Statistics 21.0; IBM Corporation, Armonk, NY, USA) was used for the analysis. A subject level analysis was performed for each outcome measurement reporting data as mean values, standard deviations, medians, 95% confidence intervals (CI), and frequencies. Shapiro–Wilk goodness-of-fit tests were used to assess the normality and distribution of data. Descriptive analysis of the histological and immunohistochemical outcomes was carried out by reporting means and standard deviations and comparisons between these histological outcomes between patients with subsequent implant loss versus patients with successful implant outcomes were evaluated using the paired sample t test or U Mann-Whitney if the distributions were non-normalized. Results were considered statistically significant at p < 0.05.