Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

The influence of local administration of simvastatin in calvarial bone healing in rats

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

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: Calvarial bone

Objectives: Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods: Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-κβ ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results: The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P≤0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions: These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. © 2009 John Wiley & Sons A/S.

New LLLT protocol to speed up the bone healing process-histometric and immunohistochemical analysis in rat calvarial bone defect

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Survival rate of osseointegrated implants in atrophic maxillae grafted with calvarial bone: a retrospective study

The aim of this study was to evaluate the clinical survival rate of osseointegrated implants placed in the atrophic maxilla that has been reconstructed by means of autogenous bone grafts harvested from a cranial calvarial site. Further, we sought to analyse the level of pen-implant bone after prosthetic rehabilitation and to determine subjective patient satisfaction with the treatment performed. This study conformed to the STROBE guidelines regarding retrospective studies. Twenty-five patients who had received osseointegrated implants with late loading in the reconstructed atrophic maxilla were included in the study. The survival rate and level of pen-implant bone loss were evaluated. A questionnaire related to the surgical and prosthetic procedures was completed. The observed implant survival rate was 92.35%. The mean bone loss recorded was 1.76 mm in the maxilla and 1.54 mm in the mandible. The results of the questionnaire indicated a high level of patient satisfaction, little surgical discomfort, and that the patients would recommend the procedure and would undergo the treatment again. From the results obtained, it is concluded that the cranial calvarial site is an excellent donor area; calvarial grafts provided stability and maintenance of bone volume over the course of up to 11 years.

Effect of two bioabsorbable barrier membranes on bone regeneration of standardized defects in calvarial bone: a comparative histomorphometric study in pigs

BACKGROUND: The effect of two different bioabsorbable collagen membranes on bone regeneration was assessed in standardized, membrane-protected calvarial defects in pigs. METHODS: Two standardized defect types (6 x 6 x 6 mm and 9 x 9 x 9 mm) were produced in the calvaria of pigs: empty defects without a membrane (group 1; eight defects per size); defects filled with deproteinized bovine bone mineral (DBBM) without a membrane (group 2; eight defects per size); defects filled with DBBM and covered by a collagen membrane (group 3; eight defects per size); and defects filled with DBBM and covered by a cross-linked collagen membrane (CCM) (group 4; eight defects per size). Sacrifice took place 16 weeks after surgery, and the following parameters were analyzed: descriptive histology; semiquantitative histology (SQH), assessing bone regeneration in the whole defect area; and histomorphometric analysis of the percentage of bone and DBBM in the regenerated area at three different depth levels of the defect. RESULTS: Using SQH, both membrane types resulted in significantly better bone regeneration compared to groups 1 and 2, irrespective of the defect size (P <0.005), with no difference between the two membranes. In the histomorphometric analysis, the layer immediately below the surface exhibited a significantly higher percentage of bone in groups 3 (27%) and 4 (36%) versus the two other groups for the 9 x 9 x 9-mm defects. No such differences were apparent for the 6 x 6 x 6-mm defects or the other two depth levels (bottom and middle layer) for either defect size. CONCLUSIONS: The two collagen membranes tested significantly enhanced bone regeneration, especially in the superficial level of the calvarial bone defects. The prototype CCM did not provide any further advantage in the present animal model.

Utilization of surgical trepan for the obtainment of calvarial autogenous bone graft in maxillary reconstructions

Maxillomandibular reconstructions are traditionally performed by means of autogenous bone grafts collected from intraoral donor areas and extraoral donor areas such as clavicle, iliac bone, rib, and tibia. The calvarial bone has been studied as an alternative donor area, with a low incidence of complications and minimal postoperative morbidity. Complications such as dural lacerations associated with cerebrospinal fluid leakage and extradural and subdural bleeding were minimized due to the use of surgical trepan, allowing the diploic layer delimitation before the osteotomy, preserving the internal calvarial cortical. The purpose of this article is to suggest a new technique for the obtainment of calvarial bone grafts with surgical trepan.

Calvarial Autogenous Bone Graft for Maxillary Ridge and Sinus Reconstruction for Rehabilitation With Dental Implants

Autogenous bone grafting is the gold-standard technique for bone augmentation procedures prior to implant placement. If the amount of available intraoral donor bone is insufficient, it is necessary to harvest bone graft from extraoral sites, such as calvaria. Although this technique is well established, only a few case reports show the histological analysis of the grafted bone at the moment of implant placement. This article reports the case of a 48-year-old female patient with a critical atrophic maxillary ridge reconstructed using autogenous calvarial bone graft prior to implant placement, with clinical and histological evaluation. Bone was collected under general anesthesia from the parietal bone. The outer cortical originated the bone blocks, and the medullar bone layer between was collected to be used in the sinus augmentation procedure, together with 5 of the bone blocks triturated. Six months after bone augmentation, 8 implants were placed in the grafted area and 2 biopsies were retrieved (anterior and the posterior regions), allowing the visualization of the bone-remodeling process in the grafted areas. The patient had a stable recovery. Our results showed that although necrotic bone could still be seen in the outer layer of the grafted area, the interface between this necrotic bone and the already remodeled bone was consistent with biocompatibility. Two-year radiographic evaluation showed success of the grafts and the implants in supporting an esthetic and functionally stable prosthesis. Summarizing, calvarial bone grafts are a viable alternative for the attainment of adequate bone volume prior to implant placement.

Histologic and histometric evaluation of calvarial autogenous bone graft in reconstruction of atrophyc maxilla for dental implants

The calvarial bone is highlighted as a good donor area for large reconstructions of atrophic jaw for subsequent rehabilitation with implant-supported prosthesis. The aim of this study was to observe and measure through histological and histometric evaluation, the cellular events that occur at the interface of union from onlay parietal bone graft on the maxilla of 10 patients, after a period of 6 months of incorporation. The biopsies were performed at the time of installation of osseointegrated implants. The bone contact area represented 78.75% and connective contact 21.25%. The region of connective union between the bone graft to the maxillae presented new bone formation (41.26%), marrow bone (36.06%), osteoid tissue (15.86%) and connective tissue (6.80%). All samples had good graft incorporation to the receptor bed with osteogenic activity and absence of inflammatory cells.

Reconstruction of the severely atrophic mandible using calvarial split bone grafts for implant-supported oral rehabilitation

OBJECTIVES: This article describes reconstruction of the severely atrophic mandible using calvarial bone grafts for implant-supported prosthetic oral rehabilitation. The study aim was to evaluate the efficacy of the treatment by determining implant survival and complication rates, and the extent of the postoperative graft resorption. STUDY DESIGN: Ten patients who underwent the treatment were followed clinically and radiologically using panoramic radiographs and CT scans during a mean postoperative period of 30 months. RESULTS: Good bone healing was observable 6 months postoperatively. The height reduction measured on panoramic radiographs was insignificant (mean 0.68 mm). Only minor complications occurred. Implant survival was 95%. Prosthodontic treatment was successfully performed in all cases, resulting in an improvement of oral function. Histological analysis of 1 bone biopsy showed minimal resorptive changes in otherwise very dense bone. CONCLUSION: Augmentation using calvarial grafts is a promising treatment alternative for the severely atrophic mandible.

Development of Craniofacial Bone Defect Reconstruction Implant Based on Fibre-Reinforced Composite with Photopolymerisable Resin Systems. Experimental studies in vitro and in vivo.

Reconstruction of defects in the craniomaxillofacial (CMF) area has mainly been based on bone grafts or metallic fixing plates and screws. Particularly in the case of large calvarial and/or craniofacial defects caused by trauma, tumours or congenital malformations, there is a need for reliable reconstruction biomaterials, because bone grafts or metallic fixing systems do not completely fulfill the criteria for the best possible reconstruction methods in these complicated cases. In this series of studies, the usability of fibre-reinforced composite (FRC) was studied as a biostable, nonmetallic alternative material for reconstructing artificially created bone defects in frontal and calvarial areas of rabbits. The experimental part of this work describes the different stages of the product development process from the first in vitro tests with resin-impregnated fibrereinforced composites to the in vivo animal studies, in which this FRC was tested as an implant material for reconstructing different size bone defects in rabbit frontal and calvarial areas. In the first in vitro study, the FRC was polymerised in contact with bone or blood in the laboratory. The polymerised FRC samples were then incubated in water, which was analysed for residual monomer content by using high performance liquid chromatography (HPLC). It was found that this in vitro polymerisation in contact with bone and blood did not markedly increase the residual monomer leaching from the FRC. In the second in vitro study, different adhesive systems were tested in fixing the implant to bone surface. This was done to find an alternative implant fixing system to screws and pins. On the basis of this study, it was found that the surface of the calvarial bone needed both mechanical and chemical treatments before the resinimpregnated FRC could be properly fixed onto it. In three animal studies performed with rabbit frontal bone defects and critical size calvarial bone defect models, biological responses to the FRC implants were evaluated. On the basis of theseevaluations, it can be concluded that the FRC, based on E-glass (electrical glass) fibres forming a porous fibre veil enables the ingrowth of connective tissues to the inner structures of the material, as well as the bone formation and mineralization inside the fibre veil. Bone formation could be enhanced by using bioactive glass granules fixed to the FRC implants. FRC-implanted bone defects healed partly; no total healing of defects was achieved. Biological responses during the follow-up time, at a maximum of 12 weeks, to resin-impregnated composite implant seemed to depend on the polymerization time of the resin matrix of the FRC. Both of the studied resin systems used in the FRC were photopolymerised and the heat-induced postpolymerisation was used additionally.

Latex use as an occlusive membrane for guided bone regeneration

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

Osteoconductive Properties of beta-Tricalcium Phosphate Matrix, Polylactic and Polyglycolic Acid Gel, and Calcium Phosphate Cement in Bone Defects

Extensive bone defects in maxillofacial region can be corrected with autogenous grafts; otherwise, the disadvantages of the therapeutics modality take the research for new bone substitutes. The aim of the study was to evaluate and compare the osteoconductive properties of 3 commercial available biomaterials. A total of 30 calvarial defects (5-mm diameter) were randomly divided into 5 treatment groups, with a total of 6 defects per treatment group (n = 6). The treatment groups were as follows: 500 to 1000 Km beta-tricalcium phosphate (beta-TCP), polylactic and polyglycolic acid (PL/PG) gel, calcium phosphate cement, untreated control, and autograft control. The evaluations were based on histomorphometric analysis at 60 postoperative days. The results have shown that beta-TCP and autograft control supported bone formation at 60 postoperative days. beta-Tricalcium phosphate showed the highest amount of mineralized area per total area and statistically significant compared with PL/PG, calcium phosphate cement, and untreated control groups. The PL/PG gel does not have osteoconductive properties and performed similar to empty control. Calcium phosphate cement showed higher number of multinucleated giant cells around the sites of the biomaterial and showed newly formed bone only at the edges of the biomaterial, without bone formation within the biomaterial. The findings presented herein indicate that bone formation reached a maximum level when rat calvarial defects were filled with beta-TCP at 60 postoperative days. Further studies should be conducted with beta-TCP to understand the potential of this biomaterial in bone regeneration.

Management of pure medial orbital wall fracture with autogenous bone graft

The orbit is an irregular conical cavity formed from 7 bones including the frontal, sphenoid, zygomatic, maxillary, ethmoid, lacrimal, and palatine bones. Fractures of the internal orbit can cause a number of problems, including diplopia, ocular muscle entrapment, and enophthalmos. Although muscle entrapment is relatively rare, diplopia and enophthalmos are relatively common sequelae of internal orbital fractures. Medial orbital wall fracture is relatively uncommon and represents a challenge for its anatomical reconstruction. In this context, autogenous bone graft has been the criterion standard to provide framework for facial skeleton and orbital walls. Therefore, it is possible to harvest grafts of varying size and contour, and the operation is performed through the bicoronal incision, which is the usual approach to major orbital reconstruction. Thus, this article aimed to describe a patient with a pure medial orbital wall fracture, and it was causing diplopia and enophthalmos. The orbital fracture was treated using autogenous bone graft from calvarial bone. The authors show a follow-up of 12 months, with facial symmetry and without diplopia and enophthalmos. In addition, a computed tomography scan shows excellent bone healing at the anterior and posterior parts of the medial orbital wall reconstruction. Copyright © 2013 by Mutaz B. Habal, MD.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741-2358.2011.00526.x Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 mu g of pure rhBMP-2, (2) 5 mu g of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 mu g of pure P-1, (5) 5 mu g of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.