Released fibroblast growth factor18 from a collagen membrane induces osteoblastic activity involved with downregulation of miR-133a and miR-135a.

We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.

In vivo evaluation of inter-operator reproducibility of digital dental and conventional impression techniques.

PURPOSE: The aim of this study was to evaluate and compare the inter-operator reproducibility of three-dimensional (3D) images of teeth captured by a digital impression technique to a conventional impression technique in vivo. MATERIALS AND METHODS: Twelve participants with complete natural dentition were included in this study. A digital impression of the mandibular molars of these participants was made by two operators with different levels of clinical experience, 3 or 16 years, using an intra-oral scanner (Lava COS, 3M ESPE). A silicone impression also was made by the same operators using the double mix impression technique (Imprint3, 3M ESPE). Stereolithography (STL) data were directly exported from the Lava COS system, while STL data of a plaster model made from silicone impression were captured by a three-dimensional (3D) laboratory scanner (D810, 3shape). The STL datasets recorded by two different operators were compared using 3D evaluation software and superimposed using the best-fit-algorithm method (least-squares method, PolyWorks, InnovMetric Software) for each impression technique. Inter-operator reproducibility as evaluated by average discrepancies of corresponding 3D data was compared between the two techniques (Wilcoxon signed-rank test). RESULTS: The visual inspection of superimposed datasets revealed that discrepancies between repeated digital impression were smaller than observed with silicone impression. Confirmation was forthcoming from statistical analysis revealing significantly smaller average inter-operator reproducibility using a digital impression technique (0.014± 0.02 mm) than when using a conventional impression technique (0.023 ± 0.01 mm). CONCLUSION: The results of this in vivo study suggest that inter-operator reproducibility with a digital impression technique may be better than that of a conventional impression technique and is independent of the clinical experience of the operator.

Effect of Nanofeatured Topography on Ceria-Stabilized Zirconia/Alumina Nanocomposite on Osteogenesis and Osseointegration.

PURPOSE: The objective of this study was to evaluate the osteogenic and osseointegration capability of the Ce-tetragonal zirconia polycrystal (TZP)-based nanostructured zirconia/alumina composite (Ce-TZP/Al2O3) that was treated with hydrofluoric acid (HF). MATERIALS AND METHODS: Osteogenic MC3T3-E1 cells were cultured on acid-etched titanium (AETi) disks and Ce-TZP/Al2O3 disks without HF treatment (Zr[0%]), with 4% HF treatment (Zr[4%]), or with 55% HF treatment (Zr[55%]) for 24 hours, and biologic responses were compared among four conditions in vitro. Miniature implants of AETi and Zr(55%) were surgically placed in the femora of rats. Osseointegration was evaluated by a biomechanical push-in test after 2 and 4 weeks of healing. RESULTS: The surface of Zr(55%) rendered nanofeatured topography with a greater surface area and roughness, and extensive geographical undercut as ceria-zirconia crystal disappeared from the superficial layer and was similar to the surface morphology of biomineralized matrices. Culture studies showed that the attachment, proliferation, spread, and functional phenotypes of osteogenic cells, such as alkaline phosphatase activity and bone-related gene expression, were remarkably increased on the Zr(55%) surface. The strength of osseointegration measured using the biomechanical push-in test in a rat model was stronger for Zr(55%) implants than for AETi implants by 1.6 fold. CONCLUSION: The nanostructured Ce-TZP/ Al2O3 surface substantially enhanced the osteogenic response in vitro and the osseointegration capability in vivo, which suggest its potential clinical application as a novel implant material.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration.

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration.

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using µCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative µCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts.

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-ß was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-ß to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.

Enhancement of bone morphogenetic protein-2-induced ectopic bone formation by transforming growth factor-ß1.

Bone morphogenetic proteins (BMPs) possess osteoinductive activities and are useful for clinical treatments, including bone regeneration. We found that transforming growth factor (TGF)-ß1 strongly enhances the osteoinductive activity of BMP-2. Collagen sponges containing 5 µg of BMP-2 were implanted into mouse muscle tissues, after which lump-like masses appeared and grew until day 7. Subsequently, calcification occurred in the lump-like masses by day 14. Addition of 50 ng of TGF-ß1 to the BMP-2-containing sponges markedly accelerated the growth of the lump-like masses and resulted in a fivefold increase in total bone volume as compared with BMP-2 alone. The number of osteoblasts in ectopic bone tissues at 14 days after implantation induced by BMP-2+TGF-ß1 was twofold greater than that with BMP-2 alone, whereas the number of osteoclasts was decreased by half. On the other hand, TGF-ß1 accelerated the differentiation of both osteoblasts and osteoclasts in the early stage (2-7 days after implantation) of ectopic bone formation. We also implanted collagen sponges into bone defects surgically created in mouse calvaria. Sponges containing 2.5 µg of BMP-2 and 25 ng of TGF-ß1 caused complete filling of the defects with orthotopic bone, whereas those containing 2.5 µg of BMP-2 alone caused only partial filling. These results suggest that TGF-ß1 enhances BMP-2-induced ectopic bone formation by accelerating the growth of lump-like masses, and regulates osteoblast and osteoclast generation. Our findings may contribute to the development of a new treatment method for skeletal disorders.

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts.

1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)(2)D(3) induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10(-9) M of 1,25(OH)(2)D(3). In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)(2)D(3) (greater than 10(-11) M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)(2)D(3). In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)(2)D(3) in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)(2)D(3), BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)(2)D(3).

Osteoclast differentiation induced by synthetic octacalcium phosphate through receptor activator of NF-kappaB ligand expression in osteoblasts.

Synthetic octacalcium phosphate (OCP) has a potential to enhance new bone formation and exhibits biodegradable characteristics when implanted in experimentally created bone defects. The precise mechanisms of OCP biodegradation remain unclear, though histological observations have revealed that bone-resorbing osteoclasts appear and resorb implanted OCP. To investigate how osteoclasts develop around implanted OCP, we examined osteoclast differentiation using OCP crystals in vitro. Coculturing of mouse bone marrow cells and osteoblasts in OCP-coated cell culture plates induced osteoclast differentiation, whereas that did not occur without coating. Further, addition of bone morphogenetic protein-2 significantly increased the number of osteoclasts in the OCP-coated wells. In the presence of OCP, osteoblasts expressed receptor activator of NF-kappaB ligand (RANKL), an osteoclast differentiation factor. In addition, when half of each culture well was coated with OCP, osteoclasts were formed in both coated and noncoated areas, suggesting that soluble factors mediate osteoclast differentiation induced by OCP. Also, calcium levels in culture medium were significantly decreased in the presence of OCP, while experimental reduction of calcium from 8.0 to 5.0 mg/dL significantly induced RANKL mRNA expression. These results suggest that OCP itself decreases calcium levels around implanted OCP, which induces osteoclast differentiation through RANKL expression by osteoblasts.