Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.

Influence of periodontal tissue thickness on buccal plate remodelling on immediate implants with xenograft.

AIM: To evaluate the influence of gingival thickness and bone grafting on buccal bone plate remodelling after immediate implant placement in sockets with thin buccal bone, using a flapless approach. MATERIALS AND METHODS: The gingiva of eight dogs was thinned at one side of the mandible, mandibular premolars were extracted without flaps, and four implants were installed on each side at 1.5 mm from the buccal bone. The sites were randomly assigned into: TG (test group) = thin gingiva; TG + GM (TG with grafting material); CG (control group) = normal gingiva; and CG + GM (CG with grafting material). After 12 weeks the dogs were sacrificed and the samples were processed for histological analysis. RESULTS: All animals exhibited a thin buccal bone initially. In all the experimental groups the buccal gap was filled with newly formed bone and the buccal bone level was slightly apical to the implant shoulder. There were no statistically significant differences among the groups for the histomorphometric parameters. CONCLUSIONS: The thickness of the buccal bone was a fundamental factor in buccal bone plate resorption, even with flapless implantation. The gingival thickness or the addition of a biomaterial in the gap did not influence the results.

Buccal bone plate remodeling after immediate implants with or without synthetic bone grafting and flapless surgery: a histomorphometric and fluorescence study in dogs.

OBJECTIVE: The aim of this study was to evaluate the buccal bone plate remodeling after immediate implantation using the flapless approach with or without bone graft into the gap between the implant and the buccal bone. MATERIAL AND METHODS: Eight dogs had the mandibular bicuspids extracted without flaps, and four implants were installed on each side, totaling eight implants per animal. Randomly, in one side, the implants were positioned at the bone crest level (equicrestal), and on the opposite side, the implants were positioned 2 mm subcrestal. All the implants were positioned 2.0 mm from the buccal bone plate (gap) and associated or not with grafting material. Therefore, the following treatments were performed: implants subcrestal test (SCTG) with bone graft and control (SCCG) without bone graft, and equicrestal test (ECTG) with bone graft and control (ECCG) without bone graft. One week following the surgeries, metallic prostheses were installed. Bone markers were administered 1, 2, 4, and 12 weeks after implant placement for fluorescence analysis. Ground sections were prepared from 12-week healing biopsies, and histomorphometry was performed. RESULTS: The histomorphometric evaluation presents significant better results for the ECTG in the vertical crestal bone resorption, but the other parameters showed better results for the SCCG. The fluorescence evaluation in adjacent areas showed numerically different results between groups with a small decrease at 12 weeks, except for the SCCG, which was higher at this time. The distant area showed a continuous increase in the marked bone. CONCLUSION: The equicrestally placed implants presented little or no loss of the buccal bone wall. The subcrestally positioned implants presented loss of buccal bone, regardless of the use of bone graft. However, the buccal bone was always coronal to the implant shoulder. Both the equicrestal and subcrestal groups were benefited in the early stages of bone healing as evidenced by the fluorescence analysis.

Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects.

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Participation of MicroRNA-34a and RANKL on bone repair induced by poly(vinylidene-trifluoroethylene)/barium titanate membrane.

The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.

Bone Formation in a Local Defect around Dental Implants Coated with Extracellular Matrix Components.

PURPOSE: The coating of implant surfaces with components of the extracellular matrix offers an approach to influence peri-implant bone healing. In this study, bone healing around coated implants is analyzed in a peri-implant defect model. MATERIALS AND METHODS: Eight months after extraction of the premolar teeth, six dogs received 48 implants (eight per animal) in the mandible. Implant surfaces were sandblasted and acid-etched, and some were additionally coated with collagen type II and chondroitin sulfate (collagen/CS). On each side of the mandible, implants either had no peri-implant defect (control side) or a vertical defect of 5 mm in depth and 0.5, 1.0, or 2.0 mm in width. Implants healed submerged for 8 weeks. Fluorochrome staining, histology, and histomorphometry were used to analyze implant osseointegration. RESULTS: Fluorochrome labels showed an increased mineralization around collagen/CS-coated surfaces at 4 weeks (p = .031). Histomorphometry generally showed lower vertical and horizontal bone apposition with increasing gap size for both surface types. In gapless sites and 0.5-mm gaps, collagen/CS coated implants showed increased bone volume in areas directly adjacent to the implant, in comparison with uncoated implants (p < .05). CONCLUSION: The width of the peri-implant gap influences peri-implant bone formation. Complete filling of all gaps by newly formed bone could not be observed around either surface. In proximity to the surface, implant surface coating by collagen/CS positively influenced bone formation.

Aumento da espessura do rebordo ósseo com um novo enxerto sintético particulado. Caso clínico com avaliação microtomográfica / Horizontal alveolar ridge augmentation with a new particulate bone substitute: a case report with micro-CT evaluation

As limitações anatômicas do rebordo alveolar residual podem impedir a instalação de um implante osseointegrado. Nesses casos, procedimentos de regeneração óssea guiada são necessários para proporcionar osso alveolar suficiente em altura e/ou espessura para a inserção de implantes dentais. Este relato piloto de caso clínico apresenta um procedimento de aumento horizontal do rebordo ósseo usando um novo substituto ósseo aloplástico para proporcionar volume ósseo necessário para a colocação de um implante, avaliando também por meio de microtomografia o osso neoformado. O paciente do sexo masculino, 58 anos, não fumante, sem condições sistêmicas que pudessem afetar o procedimento cirúrgico, apresentava a ausência de um dente (primeiro pré-molar superior direito) e optou por instalar um implante para a reabilitação cirúrgico-protética desta área. A tomografia computadorizada pré-operatória mostrou que o osso residual tinha espessura insuficiente para a instalação de um implante, sendo necessária a realização de um procedimento cirúrgico para aumento ósseo horizontal. O paciente assinou um consentimento informado autorizando a realização dos procedimentos bem como a documentação científica do caso. Foi realizada cirurgia de regeneração óssea guiada (ROG) utilizando substituto ósseo particulado (Reprobone®) e uma membrana colágena (Biomend), para aumentar a espessura óssea vestíbulo-palatal. O paciente foi apropriadamente medicado e a cicatrização ocorreu sem intercorrências. Após 6 meses, a área foi reaberta e antes da instalação do implante uma biópsia óssea foi coletada para análise microtomográfica. A técnica de ROG proporcionou volume ósseo adequado para a colocação do implante. A análise microtomográfica da biópsia óssea resultou em 40,85% de volume ósseo cortical e 17,08% de biomaterial residual...

Anatomic limitations of the residual alveolar bone may impair implant placement. Alveolar ridge augmentation procedures are required in such cases to provide alveolar bone width and/or height for dental implant placement. This case report presents a horizontal ridge augmentation procedure using a new alloplastic bone substitute providing bone volume for implant placement, with micro-CT analysis of the newly formed bone. The patient was a 58-year-old male, non-smoker, with no systemic health conditions that could affect the surgical procedure, and reported the willingness of rehabilitating the edentulous area corresponding to the tooth 14 with an osseointegrated implant. The CBCT analysis revealed that residual alveolar bone width was too narrow for implant insertion, and therefore a bone augmentation procedure was necessary. The patient signed an informed consent form authorizing all procedures and scientific documentation. Guided bone regeneration was performed using ReproBone® granules and a collagen membrane (BioMend®) to increase the buccal-palatal bone width. The patient was properly medicated and healing was uneventful. After 6 months, the area was reopened and before placing an implant a bone biopsy was collected for micro-CT analysis. The bone augmentation procedure provided adequate bone volume for implant placement. The micro-CT results of the bone biopsy showed 40% of bone volume and 17% of remnant particles of the biomaterial after 6 months. It was concluded that this biomaterial may be used in such clinical situations as an alternative to autogenous bone blocks and still avoiding patient morbidity...

Desenvolvimento de bases ósseas padronizadas para avaliação da estabilidade primária de implantes / Development of standardized bone specimens to evaluate primary stability of dental implants

A qualidade óssea do sítio receptor é considerada um dos fatores relevantes para o sucesso de implantes orais. O objetivo deste estudo piloto foi avaliar comparativamente cilindros ósseos de origem suína, de alta e baixa densidades, em modelo in vitro, por meio de análises bidimensional (radiografia digital) e tridimensional (tomografia computadorizada). Os cilindros ósseos foram preparados a partir de osso suíno, retirados do côndilo mandibular ou da cabeça do fêmur, e separados em dois grupos: grupo A: cilindros removidos da cabeça do fêmur (alta densidade óssea); grupo B: cilindros removidos do côndilo mandibular (baixa densidade óssea). Para a certificação dos cilindros (análise 2D) foram realizadas tomadas radiográficas digitais para determinar a densidade radiográfica. Foram realizadas ainda análises tridimensionais, por meio de tomografias computadorizadas, avaliando os seguintes parâmetros tomográficos: área óssea do cilindro, volume ósseo do cilindro, densidade óssea tridimensional, volume total do espaço de poros, porcentagem da porosidade total. O teste t Student foi utilizado para verificar a significância da diferença entre os grupos A e B; em todas as análises foi considerado nível de significância de 5%. Os resultados da análise 2D dos blocos ósseos mostraram que a diferença entre os grupos A e B foi estatisticamente significante (p < 0,0001). As diferenças entre os grupos A e B também foram estatisticamente significantes para todos os parâmetros tomográficos avaliados (p < 0,05). Concluiu-se que a metodologia apresentada gera cilindros ósseos padronizados de alta e baixa densidades, que poderão ser utilizados em estudos futuros para avaliar comparativamente a estabilidade inicial de implantes.

Bone quality is one of most important factors for the success of dental implants. The purpose of this study was to comparatively evaluate porcine bone cylinders, of high and low bone density, using 2D- (digital radiograph) and three-dimensional (computerized tomography) analysis. Bone cylinders were prepared from porcine bone, removed from mandibular condyle or from the femur head, and separated in two groups: Group A: cylinders removed from femur head (high bone density), and Group B: cylinders removed from mandibular condyle (low bone density). Digital radiographs were taken for the certification of bone cylinders (2D analysis), to determine the radiographic bone density. Using computerized tomograms, tri-dimensional analysis were made, evaluating the following tomographic parameters: cylinder bone area, cylinder bone volume, tridimensional bone density, total volume of porosity, total percentage of porosity. The Student´s t test was used to verify the significance of differences between groups A and B (at 5% level). The results showed that for 2D analysis of bone cylinders, the difference between groups A and B was statistically significant (p < 0.0001). The differences between groups were also statistically significant for all the evaluated tomographic parameters (p < 0.05). It was concluded that the methodology evaluated can produce standardized bone cylinders of high and low bone density that should be used in future studies to comparatively evaluate the initial stability of dental implants