Periosteum-Derived Cells as an Alternative to Bone Marrow Cells for Bone Tissue Engineering Around Dental Implants. A Histomorphometric Study in Beagle Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs

AimTo compare the influence of autologous or deproteinized bovine bone mineral as grafting material on healing of buccal dehiscence defects at implants installed immediately into the maxillary second incisor extraction socket in dogs.Material and methodsIn the maxillary second incisor sockets of 12 Labrador dogs, implants were installed immediately following tooth extraction. A standardized buccal defect was created and autologous bone particles or deproteinized bovine bone mineral were used to fill the defects. A collagen membrane was placed to cover the graft material, and the flaps were sutured to fully submerge the experimental areas. Six animals were sacrificed after 2 months, and six after 4 months of healing. Ground sections were obtained for histological evaluation.ResultsAfter 2 months of healing, all implants were osseointegrated. All buccal dehiscence defects were completely filled after 2 months irrespective of the augmentation material (autologous bone or Bio-Oss (R)) applied. Bone-to-implant contact (BIC) on the denuded implant surfaces was within a normal range of 30-40%. However, the newly formed tissue at 2 months was partially resorbed (> 50% of the area measurements) after 4 months.ConclusionsApplying either autologous bone or deproteinized bovine bone mineral to dehiscences at implants installed immediately into extraction sockets resulted in high degree of regeneration of the defects with satisfactory BIC on the denuded implant surface.To cite this article:De Santis E, Botticelli D, Pantani F, Pereira FP, Beolchini M, Lang NP. Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs.Clin. Oral Impl. Res. 22, 2011; 430-437.

Alveolar process preservation at implants installed immediately into extraction sockets using deproteinized bovine bone mineral - an experimental study in dogs

Aim To evaluate the soft tissue and the dimensional changes of the alveolar bony crest at sites where deproteinized bovine bone mineral (DBBM) particles, concomitantly with the placement of a collagen membrane, were used at implants installed into sockets immediately after tooth extraction. Material and methods The pulp tissue of the mesial roots of 3P3 was removed in six Labrador dogs, and the root canals were filled. Flaps were elevated bilaterally, the premolars hemi-sectioned, and the distal roots removed. Recipient sites were prepared in the distal alveolus, and implants were placed. At the test sites, DBBM particles were placed in the residual marginal defects concomitantly with the placement of a collagen membrane. No treatment augmentation was performed at the control sites. A non-submerged healing was allowed. Impressions were obtained at baseline and at the time of sacrifice performed 4 months after surgery. The cast models obtained were analyzed using an optical system to evaluate dimensional variations. Block sections of the implant sites were obtained for histological processing and soft tissue assessments. Results After 4 months of healing, no differences in soft tissue dimensions were found between the test and control sites based on the histological assessments. The location of the soft tissue at the buccal aspect was, however, more coronal at the test compared with the control sites (1.8 +/- 0.8 and 0.9 +/- 0.8 mm, respectively). At the three-dimensional evaluation, the margin of the soft tissues at the buccal aspect appeared to be located more apically and lingually. The vertical dislocation was 1 +/- 0.6 and 2.7 +/- 0.5 mm at the test and control sites, respectively. The area of the buccal shrinkage of the alveolar crest was significantly smaller at the test sites (5.9 +/- 2.4 mm2) compared with the control sites (11.5 +/- 1.7 mm2). Conclusion The use of DBBM particles concomitantly with the application of a collagen membrane used at implants placed into sockets immediately after tooth extraction contributed to the preservation of the alveolar process.

Osteogenesis at implants without primary bone contact - An experimental study in dogs

Aim: To evaluate the healing at implants with a moderately rough surface placed and stabilized in recipient sites of dimensions deeper and larger than that of the implants to avoid any contact between parent bone and the implant.Material & methods: In six Labrador dogs, premolars and first molars were extracted bilaterally in the mandible. After 3 months of healing, mucoperiosteal full-thickness flaps were elevated and the premolar area of the alveolar bony crest was selected. Three recipient sites were prepared to place three implants. One implant was used as control. The other two were placed in recipient sites which left a circumferentially and periapical prepared defect of 0.7 mm (small) and 1.2 mm (large), respectively. All implants were stabilized with passive fixation plates to maintain the implants stable and without any contact with the implant bed. After 3 months of submerged healing, the animals were sacrificed. Ground sections were prepared and analyzed histomorphometrically.Results: The BIC% was 5.3% and 0.3% for implants placed in small and large defect sites, respectively, whereas it was 46.1% for control implants. The differences were statistically significant. The width of the residual defects was 0.4 and 0.5 mm at the small and large defects, respectively. An approximately 0.09 mm layer of dense connective tissue (DCT) rich in fibers and fibroblast-like cells was observed adherent to the implant surfaces. The percentage of implant surface covered by DCT was 92.8% and 95.6% at the small and large defects, respectively.Conclusion: Osseointegration was observed at the test sites, and the dimensions of the defects influenced the outcomes. However, the degree of osseointegration at both small and large defects was very low compared with the control sites.

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.

Effects of fibrin sealer and resorbable gelatin on the repair of osseous defects in rat tibia

Gelfoam® - a biologically resorbable gelatin sponge - has the function of restricting hemorrhage, providing platelet rupture, and supporting fibrin threads. Beriplast® - a fibrinogen-thrombin compound - is used to adhere tissues, to consolidate sutures and in hemostasis. The objective of this study was to perform a histological analysis of the effects of haemostatic agents on osseous repair. These materials were inserted into surgical sites in young rat right and left tibiae. After the observation periods of 7, 14, 30 and 45 days, according to the bioethic protocol, the animals were killed, the tibiae were removed and fixed in 10% formalin and decalcified in equal parts of formic acid and sodium citrate solutions. After routine processing, the specimens were embedded in paraffin for microtomy. Analysis of the results demonstrated that the haemostatic agents are effective in controlling hemorrhage; they stimulate osteogenesis, featuring a pattern of osseous tissue formation similar to the control pattern, although the amount of osseous trabeculae was superior, especially in the Gelfoam group in the periods of 7 and 14 days; 30 days after surgery, the delay in tissue healing in the control group in relation to the experimental groups started to decrease, and the control and experimental groups exhibited similar tissue repair after 45 days, when all the groups exhibited secondary osseous tissue.

Histological evaluation of the bone repair using mineral trioxide aggregate combined to a material carrier

The aim of this study was to present a possible carrier for MTA, monoolein gel, with the objective to maintain this material in the place that was inserted and verify if this procedure is able to optimize its action. The data were evaluated by histomorphometric method and submitted to statistical analysis. The histological responses observed in this study indicate that the MTA is a reliable material and should be considered effective in bone periapical defects and the monoolein gel was capable to maintain the MTA in situ.

Bone regeneration in cranioplasty and clinical complications in rabbits with alloxan-induced diabetes

This research evaluated the bone repair process in surgical defects created on the parietal bones of diabetic rabbits using the guided bone regeneration technique to observe the effects of alloxan in the induction of diabetes mellitus. Twenty-four adult rabbits were divided into three study groups: control (C), diabetic (D) and diabetic associated to polytetrafluoroethylene (PTFE) membrane (D-PTFE). For diabetes induction the animals received one dose of monohydrated alloxan (90 mg/kg) by intravenous administration in the auricular or femoral vein. In group D-PTFE the membrane covered both the floor and the surface of the bone defect. In groups D and C, the bone defect was filled up with blood clot. The specimens were fixed in 10% formol and prepared for histomorphometric analysis. The results showed that the 90 mg/kg dose of monohydrate alloxan was sufficient to promote diabetes mellitus when administered in the auricular vein. Bone regeneration was slower in the diabetic group when compared with the control and diabetic-PTFE groups, but there was no significant statistical difference between the two experimental groups (D and D-PTFE). The oral and general clinical complications among the diabetics were weight loss, polyuria, polyphagia and severe chronic gingivitis.

Effect of induced diabetes mellitus on alveolar bone loss after 30 days of ligature-induced periodontal disease

Several studies have shown that diabetics are more susceptible to the development of severe periodontal disease. Currently, the use of animal models can be considered a feasible alternative in radiographic assessments of these two pathologies. The purpose of this radiographic study was to evaluate the effect of induced diabetes mellitus on alveolar bone loss after 30 days of ligature-induced periodontal disease. Sixty-four Wistar rats were randomly distributed into four experimental groups. Diabetes was induced in Groups II and IV, while periodontal disease was induced in Groups III and IV; Group I was used as control. In order to perform the radiographic assessment of the specimens, the rats were killed on the 3rd and 30th days of the study. Radiographic measurements were assessed with ANOVA and Tukey's test to determine statistically significant differences (p < 0.05). It was observed that Groups III and IV featured greater bone loss when compared to Groups I and II. Only the diabetic group with periodontal disease (Group IV) featured statistically significant greater bone loss when compared to the other groups. These results suggested that the alveolar bone loss resulting from the periodontal disease installation is greater when associated to the diabetes mellitus.

Osteoconductivity of modified fluorcanasite glass-ceramics for bone tissue augmentation and repair

Modified fluorcanasite glasses were fabricated by either altering the molar ratios of Na 2O and CaO or by adding P 2O 5 to the parent stoichiometric glass compositions. Glasses were converted to glass-ceramics by a controlled two-stage heat treatment process. Rods (2 mm x 4 mm) were produced using the conventional lost-wax casting technique. Osteoconductive 45S5 bioglass was used as a reference material. Biocompatibility and osteoconductivity were investigated by implantation into healing defects (2 mm) in the midshaft of rabbit femora. Tissue response was investigated using conventional histology and scanning electron microscopy. Histological and histomorphometric evaluation of specimens after 12 weeks implantation showed significantly more bone contact with the surface of 45S5 bioglass implants when compared with other test materials. When the bone contact for each material was compared between experimental time points, the Glass-Ceramic 2 (CaO rich) group showed significant difference (p = 0.027) at 4 weeks, but no direct contact at 12 weeks. Histology and backscattered electron photomicrographs showed that modified fluorcanasite glass-ceramic implants had greater osteoconductivity than the parent stoichiometric composition. Of the new materials, fluorcanasite glass-ceramic implants modified by the addition of P 2O 5 showed the greatest stimulation of new mineralized bone tissue formation adjacent to the implants after 4 and 12 weeks implantation. © 2010 Wiley Periodicals, Inc.

Xenograft enriched with autologous bone marrow in inlay reconstructions: A tomographic and histomorphometric study in rabbit calvaria

Objective. The aim of this study was to evaluate the bone healing after the usage of a scaffold enriched with bone marrow. Study Design. Ten rabbits were divided into 2 groups of 5 animals. Bilateral 12 mm diameter defects were created in the parietal bones. In control group Bio-Oss were inserted in both defects and, in experimental group, Bio-Oss enriched with autologous bone marrow were inserted in both defects. In these two groups, one of the calvarial defects was covered with Bio-Gide. The rabbits were sacrified 8 weeks after surgery and both CT and histomorphometric analysis were done. Results. The CT showed a lower remaining defect area in the experimental group covered with Bio-Gide when compared with control group, with and without Bio-Gide. The histomorphometrics showed no difference between groups regarding the non-vital mineralized tissue area. For vital mineralized tissue area, the experimental group covered with Bio-Gide obtained a higher percentage area when compared with control group, with and without Bio-Gide. For non-mineralized tissue area, the experimental group covered with Bio-Gide obtained a lower percentage area when compared with control group, with and without Bio-Gide. Conclusion. Both autologous bone marrow and membrane can contribute to the enhancement of bone healing. Copyright © 2012 Marcelo de Oliveira e Silva et al.

Wound healing of dehiscence defects following different root conditioning modalities: An experimental study in dogs

Objective: The purpose of this study was to investigate the periodontal healing pattern of dehiscence-type defects following different chemical root conditioning modalities. Materials and methods: Buccal osseous dehiscence defects were created on six teeth of seven dogs. After dental plaque accumulation, defects were treated with sterile saline solution (control group) or one chemical conditioning modality: citric acid (CA group), ethylenediaminetetraacetic acid (EDTA group), tetracycline (TTC group), citric acid + tetracycline (CA + TTC group), or tetracycline + citric acid (TTC + CA group). After 3 months of healing, clinical parameters were evaluated, and the animals were killed. Histological sections were processed, and a computer-assisted histometric analysis was used to evaluate the formation of new cementum, new bone, and epithelial apical migration. Results: All treatments yielded significant improvements in terms of probing depth decrease and clinical attachment level gain compared to baseline values; however, without significant differences among the groups (p > 0.05; one-way ANOVA). The highest amount of new cementum was noted in the EDTA group (3.72 ± 0.83 mm, 77.6 %), while the lowest amount of new bone was observed in the TTC group (0.7 ± 0.94 mm, 14.3 %). However, no statistically significant differences could be observed among the groups regarding epithelial apical migration, new cementum, and alveolar bone formation (p > 0.05). Conclusion: Chemical root surface conditioning did not promote any significant improvement in periodontal healing pattern of dehiscence-type defects in dogs. Clinical Relevance: Chemical root surface conditioning after surgical debridement did not promote positive or negative effects on periodontal healing pattern of dehiscence-type defects. © 2012 Springer-Verlag Berlin Heidelberg.

Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing

This study evaluated the influence of bone marrow aspirate (BMA), low-level laser therapy (LLLT) and their combination on bone healing in surgically created critical-size defects (CSDs) in rat calvaria. 40 rats were divided into four groups: C (control), BMA, LLLT and BMA/LLLT. A 5 mm diameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group BMA, the defect was filled with BMA. In groups LLLT and BMA/LLLT, the defect received laser irradiation (InGaAlP laser), was filled with blood clot or BMA respectively, and irradiated again. Animals were euthanized 30 days postoperatively. Histomorphometric and immunohistochemical analyses were performed. Newly formed bone area (NFBA) was calculated as percentage of the total area of the original defect. Proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) immunohistochemical staining were performed. PCNA-positive, Runx2-positive and OCN-positive cells were quantified. Data were statistically analyzed. Group BMA/LLLT had significantly greater NFBA than groups C, BMA or LLLT. Group BMA presented significantly greater NFBA than control, while group LLLT did not. Group BMA/LLLT presented a significantly higher number of PCNA-positive and OCN-positive cells than any of the other groups. Groups BMA/LLLT and BMA showed a significantly lower number of Runx2-positive cells than groups C or LLLT. The combination of BMA/LLLT yielded significantly greater bone formation in surgically created CSD in rat calvaria when compared to control, or either treatment alone. © 2013 Elsevier B.V. All rights reserved.

Healing of fresh frozen bone allograft with or without platelet-rich plasma: A histologic and histometric study in rats

Purpose: This study histomorphometrically analyzed the effect of autogenous platelet-rich plasma (PRP) on healing of fresh frozen bone allograft (FFBA) in bony defects in rat calvaria. Materials and Methods: A 5mm-diameter defect was created in the calvarium of 30 rats. Animals were divided into three groups: C (defect was filled by blood clot only), FFBA (defect was filled with 0.01mL of FFBA), and FFBA/PRP (defect was filled with 0.01mL of FFBA combined with 100μL of PRP). All animals were euthanized at 30 days postoperatively. Histomorphometry and histology analyses were performed. Data were statistically analyzed (analysis of variance, Tukey, p<.05). Results: FFBA had a statistically smaller new bone area than groups FFBA/PRP and C. No statistically significant differences were observed between groups FFBA and FFBA/PRP with regard to remaining bone graft particle area. Conclusion: It can be concluded that (1) PRP improved the incorporation of FFBA, increasing the amount of new bone formed; (2) PRP has not influenced the resorption of nonviable particles of the FFBA; and (3) presence of remaining FFBA particles might have accounted for the smaller amount of new bone observed in group FFBA when compared with control group. © 2011 Wiley Periodicals, Inc.

Dynamics of bone healing after osteotomy with piezosurgery or conventional drilling - histomorphometrical, immunohistochemical, and molecular analysis

Background: Piezosurgery is an osteotomy system used in medical and dental surgery. Many studies have proven clinical advantages of piezosurgery in terms of quality of cut, maneuverability, ease of use, and safety. However, few investigations have tested its superiority over the traditional osteotomy systems in terms of dynamics of bone healing. Therefore, the aim of this study was to evaluate the dynamics of bone healing after osteotomies with piezosurgery and to compare them with those associated to traditional bone drilling.Methods: One hundred and ten rats were divided into two groups with 55 animals each. The animals were anesthetized and the tibiae were surgically exposed to create defects 2 mm in diameter by using piezosurgery (Piezo group) and conventional drilling (Drill group). Animals were sacrificed at 3, 7, 14, 30 and 60 days post-surgery. Bone samples were collected and processed for histological, histomorphometrical, immunohistochemical, and molecular analysis. The histological analysis was performed at all time points (n = 8) whereas the histomorphometrical analysis was performed at 7, 14, 30 and 60 days post-surgery (n = 8). The immunolabeling was performed to detect Vascular Endothelial Growth Factor (VEGF), Caspase-3 (CAS-3), Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), and Osteocalcin (OC) at 3, 7, and 14 days (n = 3). For the molecular analysis, animals were sacrificed at 3, 7 and 14 days, total RNA was collected, and quantification of the expression of 21 genes related to BMP signaling, Wnt signaling, inflammation, osteogenenic and apoptotic pathways was performed by qRT-PCR (n = 5).Results: Histologically and histomorphometrically, bone healing was similar in both groups with the exception of a slightly higher amount of newly formed bone observed at 30 days after piezosurgery (p < 0.05). Immunohistochemical and qRT-PCR analyses didn't detect significant differences in expression of all the proteins and most of the genes tested.Conclusions: Based on the results of our study we conclude that in a rat tibial bone defect model the bone healing dynamics after piezosurgery are comparable to those observed with conventional drilling. © 2013 Esteves et al.; licensee BioMed Central Ltd.