Histological Analysis of Osteogenic Potential of Biphasic Calcium Phosphate
Condition: Alveolar Ridge Enlargement; Fenestration; Dehiscence
MeSH terms: Hypertrophy
Study type: Interventional
Phase: Phase 4

Summary: 
      Bone healing after tooth extraction may be affected by defects in the buccal wall of the
      alveolus, such as fenestrations and dehiscences. Therefore, it is advisable to perform a
      surgical procedure such as guided bone regeneration after tooth extractions. Different
      biomaterials are used for this purpose. The aim of this study is to investigate the
      qualitative and quantitative histological changes in human biopsies taken after 6 months of
      healing of extraction sockets with buccal wall defects. For this purpose, the defects of 36
      patients (18 per group) will be augmented with injectable biphasic calcium phosphate (I-BCP)
      or bovine xenograft (BX) after extraction. After six months of healing, bone biopsies will be
      taken and processed to the qualitative and quantitative histological analysis.
    

Detailed description: 
      The study was designed as a randomized controlled clinical trial. To participate in the
      study, patients requires to have at least one tooth predisposed to extraction and to have a
      dental implant placed after completion of the healing period. The indications for tooth
      extraction are: chronic periapical process, chronic periodontal disease, or fracture of the
      vertical bone. The inclusion criteria are: age between 18 and 60 years, intact physical and
      mental health, patient must understand the study protocol, patient must sign an informed
      consent. Patients will be excluded from the study if they had at least one of the absolute
      contraindications to implant prosthetic therapy described by Wang and Hwang. Other exclusion
      criteria included the following systemic diseases: uncontrolled diabetes, osteoporosis and
      osteopenia, and vitamin D deficiency, bisphosphonate therapy, glucocorticoid therapy,
      hypothyroidism, uncontrolled cardiovascular disease (hypertension, coronary artery disease,
      congestive heart failure), and the following local factors: use of tobacco products (up to 10
      cigarettes per day) and poor oral hygiene. Pregnant and lactating women will not included in
      the study.

      In addition to the general health criteria mentioned above, the patient has to meet at least
      one intraoral criterion, namely the presence of a buccal bone defect such as fenestration or
      dehiscence. Patients who met the inclusion criteria and were willing to participate, are
      randomly assigned via a web interface (https://www.randomizer.org/).

      Participants and materials Participants will be recruited from the oral surgery office. A
      total of 36 healthy patients will be included in the study. In analyzing the validity of the
      test, the approach of equivalence of the tested methods and noninferiority of the tested
      method compared with the existing method was used. With a significance level α of 0.05, a
      test power of 80%, an expected standard deviation of the predicted result of 10, and an
      equivalence limit of d = 10, it was necessary to include at least 36 subjects in the study,
      18 subjects per group.

      A total of 18 patients in the test group will receive I-BCP (Maxresorb® inject, Botiss GmbH,
      Berlin, Germany). This biomaterial is an alloplastic bone substitute material in the form of
      a paste that is placed into the defect with a plastic syringe. The material consists of an
      aqueous gel containing granules of biphasic calcium phosphate in the composition of 60% HA
      and 40% β-TCP (particle size between 15-50 nm). Patients in the control group will receive BX
      (cerabone®, Botiss GmbH, Berlin, Germany). The material is completely anorganic and consists
      of 100% HA. For this study, the material will be used in granular form (the size of the
      granules is between 0.5 and 1 mm), mixed with physiological solution prior to placement into
      the defect.

      First surgical phase and healing period Before the procedure, the patient will be
      administered a 3% chlorhexidine solution to rinse the oral cavity for one minute. Local
      anesthesia (2% Lidocaine®, Belupo, Koprivnica, Croatia) will be then administered and the
      surgical field will be covered with sterile drapes. A full-thickness mucoperiosteal flap will
      be elevated at the extraction site. To perform the tooth extraction atraumatically, the roots
      of the multirooted teeth will be separated with a drill before extraction. The extraction
      will be performed with atraumatic instruments, with as little trauma to the alveolus as
      possible. The alveolus is then carefully examined and curetted to remove granulations and
      stimulate fresh bleeding in the alveoli. The alveolus will be filled to the edge with I-BCP
      in the test group and BX in the control group. In the end bone grafting biomaterial is
      covered with a resorbable native collagen membrane obtained from porcine pericardium (Jason®
      membrane, Botiss GmbH, Berlin, Germany) in both groups. The mucoperiosteal flap will be then
      adjusted and closed so that the collagen membrane was completely covered with soft tissue
      (5.0 monofilament). The patient will be then prescribed an analgesic (400 mg ibuprofen) and
      instructed to take antibiotics (amoxicillin with clavulanic acid or clindamycin) for the next
      7 days. On the 10th day after surgery, the sutures are removed.

      A 6-month period of regeneration of the bone defect will follow. Based on the radiographic
      examination, the selection and positioning of the appropriate implant will be performed.

      Second surgical phase Six months after extraction, patients come to the office for placement
      of a dental implant (Ankylos®, Dentsply Sirona, Charlotte, USA) in the augmented area. The
      preparation of the patient (rinsing of the oral cavity with an antiseptic solution, local
      anesthesia, sterile covering of the working area) is the same as for the first surgical
      procedure. Subsequently, a full-thickness flap is elevated, and a biopsy of the augmented
      area is taken for histological analysis using a trephine drill (2.5 mm inner diameter;
      Ustomed® intrumente, Tuttlingen, Germany). It is important to emphasize that the inner
      diameter of the trephine drill is smaller than that of the final drill used to prepare the
      implant bed to avoid unnecessary removal of the patient's bone. In addition, each biopsy will
      be taken from the central part of the augmented area to reduce the possibility of an
      incorrect biopsy (e.g., bi-opsy of only the native bone that was not augmented with
      biomaterial).

      Histological analysis The trephine drill will be placed in a hermetically sealed plastic tube
      containing a 4% formaldehyde solution and sent to the histology laboratory for further
      analysis. The tissue will be fixed in 4% formaldehyde solution for two weeks. Subsequently,
      the biopsies will be stored in 25% ethylenediaminetetraacetic acid (EDTA) (Decalcifier soft®,
      Solvagreen®, Karlsruhe, Austria), which is used for gentle decalcification of bone. After
      decalcification, the tissue will be subjected to a standard protocol in a tissue processor
      (MTP, SLEE medical GmbH, Mainz, Germany). This will be followed by the formation of paraffin
      blocks (MPS/P, SLEE medical GmbH, Mainz, Germany). Six consecutive sections of 5 μm thickness
      will be prepared from each biopsy using a rotary microtome (CUT 4062, SLEE medical GmbH,
      Mainz, Germany). The specimens will be then stained with Masson trichrome stain and Movat
      pentachrome stain. Microscopy of the preparations will be performed using a light microscope
      (Leica DMRB, Leica Microsystems GmbH, Wetzlar, Germany) in conjunction with a video camera
      (Axio Imager M2, Zeiss, Oberkochen, Germany). Pathohistological analysis will evaluate the
      tissue response to the implanted biomaterial, i.e., the presence of fibroblasts, blood
      vessels, neutrophils, monocytes/macrophages, and multinucleated giant cells (MGC).

      Histomorphometric processing of the specimens will be performed using the free ImageJ
      software (https://imagej.nih.gov/ij/download.html). The surface of the new bone, the sur-face
      of the biomaterial, and the surface of the soft tissue will be marked on the specimens. The
      surface of the specimen will be then measured, and the following will be calculated from the
      data obtained: percentage of new bone formed, percentage of biomaterial remaining, and
      percentage of soft tissue.

      Statistical analysis Categorical variables will be reported with absolute and relative
      frequencies. Differences between categorical variables were tested with the
      Fisher-Freeman-Halton test.

      Normality of distribution will be tested with the Shapiro-Wilk test. Continuous data will be
      described by the arithmetic mean and standard deviation. Differences in continuous vari-ables
      will be tested with the Mann-Whitney U test (with corresponding difference and 95% confidence
      interval of difference).

      All P values will be two-sided. The significance level was set at alpha = 0.05. IBM SPSS
      Statistical software, version 27.0.1, will be used for the analysis.
    

Intervention name(s): Bone augmentation with I-BCP; Bone augmentation with BX
Intervention type(s): Procedure; Procedure
Intervention description(s): The extraction socket will be filled after tooth extraction with I-BCP, in order to achieve bone regeneration and prevent volume loss after tooth extraction.; The extraction socket will be filled after tooth extraction with BX, in order to achieve bone regeneration and prevent volume loss after tooth extraction.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: bone regeneration; alloplast; bovine xenograft; guided bone regeneration