Autogenous bone graft with or without a calcium sulfate barrier in the treatment of class II furcation defects: A histologic and histometric study in dogs

Background: The purpose of this study was to histologically evaluate the healing of surgically created Class II furcation defects treated using an autogenous bone (AB) graft with or without a calcium sulfate (CS) barrier. Methods: The second, third, and fourth mandibular premolars (P2, P3, and P4) of six mongrel dogs were used in this study. Class II furcation defects (5 mm in height × 2 mm in depth) were surgically created and immediately treated. Teeth were randomly divided into three groups: group C (control), in which the defect was filled with blood clot; group AB, in which the defect was filled with AB graft; and group AB/CS, in which the defect was filled with AB graft and covered by a CS barrier. Elaps were repositioned to cover all defects. The animals were euthanized 90 days post-surgery. Mesio-distal serial sections were obtained and stained with either hematoxylin and eosin or Masson's trichrome. Histometric, using image-analysis software, and histologic analyses were performed. Linear and area measurements of periodontal healing were evaluated and calculated as a percentage of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance; P<0.05). Results: Periodontal regeneration in the three groups was similar. Regeneration of bone and connective tissue in the furcation defects was incomplete in most of the specimens. Statistically significant differences were not found in any of the evaluated parameters among the groups. Conclusion: Periodontal healing was similar using surgical debridement alone, AB graft, or AB graft with a CS barrier in the treatment of Class II furcation defects.

Bone reformation and implant integration following maxillary sinus membrane elevation: An experimental study in primates

Background: Recent clinical studies have described maxillary sinus floor augmentation by simply elevating the maxillary sinus membrane without the use of adjunctive grafting materials. Purpose: This experimental study aimed at comparing the histologic outcomes of sinus membrane elevation and simultaneous placement of implants with and without adjunctive autogenous bone grafts. The purpose was also to investigate the role played by the implant surface in osseointegration under such circumstances. Materials and Methods: Four tufted capuchin primates had all upper premolars and the first molar extracted bilaterally. Four months later, the animals underwent maxillary sinus membrane elevation surgery using a replaceable bone window technique. The schneiderian membrane was kept elevated by insertion of two implants (turned and oxidized, Brånemark System®, Nobel Biocare AB, Göteborg, Sweden) in both sinuses. The right sinus was left with no additional treatment, whereas the left sinus was filled with autogenous bone graft. Implant stability was assessed through resonance frequency analysis (Osstell™, Integration Diagnostics AB, Göteborg, Sweden) at installation and at sacrifice. The pattern of bone formation in the experimental sites and related to the different implant surfaces was investigated using fluorochromes. The animals were sacrificed 6 months after the maxillary sinus floor augmentation procedure for histology and histomorphometry (bone-implant contact, bone area in threads, and bone area in rectangle). Results: The results showed no differences between membrane-elevated and grafted sites regarding implant stability, bone-implant contacts, and bone area within and outside implant threads. The oxidized implants exhibited improved integration compared with turned ones as higher values of bone-implant contact and bone area within threads were observed. Conclusions: The amount of augmented bone tissue in the maxillary sinus after sinus membrane elevation with or without adjunctive autogenous bone grafts does not differ after 6 months of healing. New bone is frequently deposited in contact with the schneiderian membrane in coagulum-alone sites, indicating the osteoinductive potential of the membrane. Oxidized implants show a stronger bone tissue response than turned implants in sinus floor augmentation procedures. © 2006 Blackwell Publishing, Inc.

Comparison between polyurethanes containing castor oil (Soft Segment) and cancellous bone autograft in the treatment of segmental bone defect induced in rabbits

The aim of this study is to compare polyurethanes containing castor oil (soft segment) in granular form compared to cancellous bone autograft applied to a segmental bone defect. Norfolk adult female rabbits - approximately 13 months of age with a mean body weight of 4.5 kg - are used. In both radial diaphyses, 1 cm osteoperiosteal segmental defects are created. The defect in the left radius is filled with the castor-oil-based polyurethane, and the right one, filled with cancellous bone autograft, collected from the left proximal humerus. The rabbits are euthanazed at 15, 30, 60, and 120 days postsurgery (5 animals/ period), for histological analyses. By radiographic analyses, at these time points, the bone regeneration is more evident and accelerated in the bone defects treated with the cancellous bone autograft. At 120 days postsurgery, the segmental bone defects treated with the cancellous bone autograft are totally reconstituted and remodeled, while the bone defects treated with polyurethane polymer have bone formation of 79%. Histological study shows that the polyurethane acts as a space filler, minimizing the local production of fibrous tissue. No granule degradation, resorption or any inflammatory reaction is detected. Thus, it is possible to conclude that the castor-oil-plant-based polyurethane - in the granule presentation - is biocompatible and osteointegrated, but does not show the same bone regeneration capacity as the cancellous bone autograft. © 2007 SAGE Publications.

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.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: An ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4oC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37°C. In the first day, the specimens were irradiated 9 times and stored at 40°C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Effects of Long-Term FK 506 Therapy on the Alveolar Bone and Cementum of Rats

Cyclosporine (CsA) and tacrolimus (FK 506) exert complex, incompletely understood actions on bone. The objective of the study was to evaluate the effects of long-term tacrolimus therapy on the periodontium. Rats were treated for 60, 120, 180, and 240 days with daily subcutaneous injections of 1 mg/kg body weight of FK 506. After the experimental period, we obtained serum levels of calcium and alkaline phosphatase (ALP). After histological processing, the alveolar bone and cementum, as well as volume densities of bone (Vb) and osteoclasts (Vo), were assessed at the regions of the lower first molar. There was a tendency toward a statistically significant decrease in ALP levels with FK 506; however, serum calcium levels increased during the long periods. At 60, 180, and 240 days of treatment with FK 506, we did not observe Vb and Vo alterations. At 120 days of treatment, there was an evident decrease in Vb, but it did not show alveolar bone loss. We did not observe any alterations of cementum among rats treated with FK 506. It may be concluded that FK 506 administration did not induce side effects on the periodontium. © 2009 Elsevier Inc. All rights reserved.

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.

Healing outcomes at implants installed in sites augmented with particulate autologous bone and xenografts. An experimental study in dogs

Aim: To evaluate the integration of implants installed at the interface of pristine and grafted tissue augmented with particulate autologous bone or deproteinized bovine bone mineral (DBBM), concomitantly with a collagen membrane. Material and methods: In 6 Labrador dogs, the distal root of 3P3 and 4P4 was endodontically treated and hemi-sected, and the mesial roots extracted concomitantly with the extraction of 2P2. The buccal bony walls were removed, and two box-shaped defects, one larger and one smaller, were created. After 3 months, flaps were elevated, and the defects were filled with particulate autologous bone or DBBM in the right and left side of the mandible, respectively. Collagen membranes were used to cover the grafted areas. Three months later, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites at the interface between grafts and pristine bone. One implant was installed in each of the four defects. After 3 months, biopsies were harvested and ground sections prepared for histological evaluation. Results: The augmentation technique was effective at all sites and all the foreseen implants were installed. In the histological analysis, all implants were integrated in mature bone, at both the buccal and lingual aspects. The most coronal bone-to-implant contact and the top of the buccal bony crest were located at a similar distance between test and control implants. However, these distances were higher at the larger compared with the smaller defects. Especially in the large defect, residual particles of DBBM were found embedded into connective tissue and located outside the bony crest. Conclusions: Particulate autologous bone as well as DBBM particles used to augment horizontally the alveolar bony process allowed for the osseointegration of implants installed after 3 months of healing. © 2012 John Wiley & Sons A/S.

Bone-healing pattern at the surface of titanium implants: An experimental study in the dog

Objective: To study the early sequential stages of osseointegration at implants installed in alveolar bony. Materials and methods: In 12 Labrador dogs, all mandibular premolars and first molars were extracted bilaterally. After 3 months of healing, full-thickness flaps were elevated in the edentulous region of the right side of the mandible. Implants were installed, and the flaps were sutured to allow a fully submerged healing. The timing of the installations in the left side of the mandible and of sacrifices were performed with a schedule that various observation periods to sacrifice from 5, 10, 20, and 30 days were available so that n = 6 was obtained per each healing period. Ground sections were prepared and analyzed. Results: Newly formed bone in contact with the implant surface was found after 10 days of healing and the percentage increased up to 50% after 1 month of healing. A higher percentage was found in the trabecular compared with the cortical bony compartment. Old bone decreased by about 50% during healing, being still present after 1 month (16%). The proportions of bone debris and bone particles were at 27% after 5 days and decreased during healing to 6% after 1 month. Conclusion: Osseointegration (new bone-to-implant contact) developed at various rates for cortical and trabecular compartments, respectively. In the trabecular region, mesenchymal cells were identified, subsequently developing into new bone in contact with the implant surface. In the cortical compartment, however, resorptive processes were observed throughout all periods of healing. The proportion of newly formed bone percentage was lower compared with that of the trabecular area. Old bone was still present after 1 month of healing in both compartments. Bone debris and small bone particles appeared to be involved in initial bone formation. © 2013 John Wiley & Sons A/S.

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.

Impact of diabetes mellitus and metabolic control on bone healing around osseointegrated implants: Removal torque and histomorphometric analysis in rats

Objectives: To evaluate bone healing around dental implants with established osseointegration in experimental diabetes mellitus (DM) and insulin therapy by histomorphometric and removal torque analysis in a rat model. Materials and methods: A total of 80 male Wistar rats received a titanium implant in the tibiae proximal methaphysis. After a healing period of 60 days, the rats were divided into four groups of 20 animals each: a 2-month control group, sacrificed at time (group A), a diabetic group (group D), an insulin group (group I), and a 4-month control group (group C), subdivided half for removal torque and half for histomorphometric analysis. In the D and I groups the DM was induced by a single injection of 40 mg/kg body weight streptozotocin (STZ). Two days after DM induction, group I received subcutaneous doses of insulin twice a day, during 2 months. Groups C and D received only saline. Two months after induction of DM, the animals of groups D, C and I were sacrificed. The plasmatic levels of glucose (GPL) were monitored throughout the experiment. Evaluation of the percentages of bone-to-implant contact and bone area within the limits of the implant threads was done by histomorphometric and mechanical torque analysis. Data were analyzed by anova at significant level of 5%. Results: The GPL were within normal range for groups A, C and I and higher for group D. The means and standard deviations (SD) for histomorphometric bone area showed significant difference between group D (69.34 ± 5.00%) and groups C (78.20 ± 4.88%) and I (79.63 ± 4.97%). Related to bone-to-implant contact there were no significant difference between the groups D (60.81 + 6.83%), C (63.37 + 5.88%) and I (66.97 + 4.13%). The means and SD for removal torque showed that group D (12.91 ± 2.51 Ncm) was statistically lower than group I (17.10 ± 3.06 Ncm) and C (16.95 ± 5.39 Ncm). Conclusions: Diabetes mellitus impaired the bone healing around dental implants with established osseointegration because the results presented a lower percentage of bone area in group D in relation to groups C and I resulting in a lowest torque values for implant removal. Moreover, insulin therapy prevents the occurrence of bone abnormalities found in diabetic animals and osseointegration was not compromised. © 2012 John Wiley & Sons A/S.

Treatment with tacrolimus enhances alveolar bone formation and decreases osteoclast number in the maxillae: A histomorphometric and ultrastructural study in rats

Recent studies have suggested that tacrolimus monotherapy is a beneficial therapeutic alternative for the normalization of cyclosporin- induced bone loss in animal models and humans. The mechanism accounting for this action is unclear at present. In the present study, we attempted to determine the effect of tacrolimus monotherapy on alveolar bone using histological, histomorphometrical and transmission electron microscopy (TEM).Groups of rats (n= 10 each) were treated with either tacrolimus (1mg/ kg/ day, s.c.) or drug vehicle for 60 days. Fragments containing maxillary molars were processed for light microscopy to investigate the alveolar bone volume, trabecular separation, number of osteoclasts and osteoblasts, and transmission electron microscopy to investigate their ultrastructural basic phenotype.Treatment with tacrolimus monotherapy during 60 days may induce increases in alveolar bone volume (BV/ TV,%; P < 0.05) and a non- significant decrease in trabecular separation (Tb. Sp, mm; P > 0.05), represented by a decrease in osteoclast number (N. Oc/ BS; P < 0.05) and maintenance of osteoblast number (N. Ob/ BS; P > 0.05). Osteoblasts were often observed as a continuous layer of active cells on the bone surface. Osteoclasts appeared to be detached from the resorbed bone surface, which was often filled by active osteoblasts and collagen- rich matrix. Moreover, osteoclasts in the treated group were frequently observed as inactive cells (without ruffled border, clear zone and detached from the bone surface).Within the limits of the present study, we conclude that tacrolimus leads to an increase in alveolar bone formation, which probably exerts action on osteoclasts. Tacrolimus could, therefore, play a crucial role in the control of both early osteoclast differentiations from precursors, as well as in functional activation.

Bacterial cellulose-hydroxyapatite composites with osteogenic growth peptide (OGP) or pentapeptide OGP on bone regeneration in critical-size calvarial defect model

This study aimed to evaluate the potential of bacterial cellulose-hydroxyapatite (BC-HA) composites associated with osteogenic growth peptide (OGP) or pentapeptide OGP(10–14) in bone regeneration in critical-size calvarial defects in mice. In this study, the BC-HA, BC-HA-OGP, and BC-HA-OGP(10–14) membranes were analyzed at 3, 7, 15, 30, 60, and 90 days. In each period, the specimens were evaluated by micro-computed tomography (µCT), descriptive histology, gene expression of bone biomarkers by qPCR and VEGFR-2 (vascular endothelial growth factor) quantification by ELISA. Three days post-operative, Runx2, Tnfrsf11b and Bglap bone biomarkers were upregulated mainly by BC-HA OGP and BC-HA OGP(10–14) membranes, suggesting an acceleration of the osteoblast differentiation/activity with the use of these biomaterials. At 60 and 90 days, a high percentage of bone formation was observed by µCT for BC-HA and BC-HA OGP(10–14) membranes. High expression of some bone biomarkers, such as Alpl, Spp1, and Tnfrsf11b, was also observed for the same membranes on days 60 and 90. In conclusion, the BC-HA membrane promoted a better bone formation in critical-size mice calvarial defects. Nevertheless, incorporation of the peptides at the concentration of 10−9 mol L−1 did not improve bone regeneration potential in the long-term.

Bone Ceramic® at implants installed immediately into extraction sockets in the molar region: an experimental study in dogs

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

Immunohistochemical, tomographic, and histological study on onlay bone graft remodeling. Part III: allografts

Objective In the last decades aroused the interest for bone tissue bank as an alternative to autogenous grafting, avoiding donor sites morbidity, surgical time, and costs reduction. The purpose of the study was to compare allografts (ALg) with autografts (AUg) using histology, immunochemistry, and tomographic analysis. Material and methods Fifty-six New Zealand White rabbits were submitted to surgical procedures. Twenty animals were donors and 36 were actually submitted to onlay grafting with ALg (experimental group) and AUg (control group) randomly placed bilaterally in the mandible. Six animals of each group were sacrificed at 3, 5, 7, 10, 20, and 60 postoperative days. Immunolabeling was accomplished with osteoprotegerin (OPG); receptor activator of nuclear factor-k ligand (RANKL); alkaline phosphatase (ALP); osteopontin (OPN); vascular endothelial growth factor (VEGF); tartrate-resistant acid phosphatase (TRAP); collagen type I (COL I); and osteocalcin (OC). Density and volume of the grafts was evaluated on tomography obtained at the surgery and sacrifice. Results The ALg and AUg exhibited similar patterns of density and volume throughout the experiments. The intra-group data showed statistical differences at days 7 and 60 in comparison with other time points (P = 0.001), in both groups. A slight graft expansion from fixation until day 20 (P = 0.532) was observed in the AUg group and then resorbed significantly at the day 60 (P = 0.015). ALg volume remained stable until day 7 and decreased at day 10 (P = 0.045). The light microscopy analysis showed more efficient incorporation of AUg onto the recipient bed if compared with the ALg group. The immunohistochemical labeling picked: at days 10 and 20 with OPG in the AUg group and at day 7 with TRAP in the ALg group (P = 0.001 and P = 0.002, respectively). Conclusions ALg and AUg were not differing in patterns of volume and density during entire experiment. Histological data exhibit more efficient AUg incorporation into recipient bed compared with the ALg group. Immunohistochemistry outcomes demonstrated similar pattern for both ALg and AUg groups, except for an increasing resorption activity in the ALg group mediated by TRAP and in the AUg group by higher OPG labeling. However, this latter observation does not seem to influence clinical outcomes.