Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation.

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors.

The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization, the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here, we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments, we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors, leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development.

Quantitative control of Ets1 dosage by a multi-enhancer hub promotes Th1 cell differentiation and protects from allergic inflammation.

Multi-enhancer hubs are spatial clusters of enhancers present across numerous developmental programs. Here, we studied the functional relevance of these three-dimensional structures in T cell biology. Mathematical modeling identified a highly connected multi-enhancer hub at the Ets1 locus, comprising a noncoding regulatory element that was a hotspot for sequence variation associated with allergic disease in humans. Deletion of this regulatory element in mice revealed that the multi-enhancer connectivity was dispensable for T cell development but required for CD4+ T helper 1 (Th1) differentiation. These mice were protected from Th1-mediated colitis but exhibited overt allergic responses. Mechanistically, the multi-enhancer hub controlled the dosage of Ets1 that was required for CTCF recruitment and assembly of Th1-specific genome topology. Our findings establish a paradigm wherein multi-enhancer hubs control cellular competence to respond to an inductive cue through quantitative control of gene dosage and provide insight into how sequence variation within noncoding elements at the Ets1 locus predisposes individuals to allergic responses.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

netGO: R-Shiny package for network-integrated pathway enrichment analysis.

SUMMARY: We present an R-Shiny package, netGO, for novel network-integrated pathway enrichment analysis. The conventional Fisher's exact test (FET) considers the extent of overlap between target genes and pathway gene-sets, while recent network-based analysis tools consider only network interactions between the two. netGO implements an intuitive framework to integrate both the overlap and networks into a single score, and adaptively resamples genes based on network degrees to assess the pathway enrichment. In benchmark tests for gene expression and genome-wide association study (GWAS) data, netGO captured the relevant gene-sets better than existing tools, especially when analyzing a small number of genes. Specifically, netGO provides user-interactive visualization of the target genes, enriched gene-set and their network interactions for both netGO and FET results for further analysis. For this visualization, we also developed a standalone R-Shiny package shinyCyJS to connect R-shiny and the JavaScript version of cytoscape. AVAILABILITY AND IMPLEMENTATION: netGO R-Shiny package is freely available from github, https://github.com/unistbig/netGO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Glucosylceramide synthase regulates adipo-osteogenic differentiation through synergistic activation of PPARγ with GlcCer.

Dysregulation of the adipo-osteogenic differentiation balance of mesenchymal stem cells (MSCs), which are common progenitor cells of adipocytes and osteoblasts, has been associated with many pathophysiologic diseases, such as obesity, osteopenia, and osteoporosis. Growing evidence suggests that lipid metabolism is crucial for maintaining stem cell homeostasis and cell differentiation; however, the detailed underlying mechanisms are largely unknown. Here, we demonstrate that glucosylceramide (GlcCer) and its synthase, glucosylceramide synthase (GCS), are key determinants of MSC differentiation into adipocytes or osteoblasts. GCS expression was increased during adipogenesis and decreased during osteogenesis. Targeting GCS using RNA interference or a chemical inhibitor enhanced osteogenesis and inhibited adipogenesis by controlling the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ). Treatment with GlcCer sufficiently rescued adipogenesis and inhibited osteogenesis in GCS knockdown MSCs. Mechanistically, GlcCer interacted directly with PPARγ through A/B domain and synergistically enhanced rosiglitazone-induced PPARγ activation without changing PPARγ expression, thereby treatment with exogenous GlcCer increased adipogenesis and inhibited osteogenesis. Animal studies demonstrated that inhibiting GCS reduced adipocyte formation in white adipose tissues under normal chow diet and high-fat diet feeding and accelerated bone repair in a calvarial defect model. Taken together, our findings identify a novel lipid metabolic regulator for the control of MSC differentiation and may have important therapeutic implications.

Diffusible Signaling Factor, a Quorum-Sensing Molecule, Interferes with and Is Toxic Towards Bdellovibrio bacteriovorus 109J.

Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects two classes of bacterial signaling molecules (acyl homoserine lactones (AHLs) and diffusible signaling factor (DSF)) have on B. bacteriovorus 109J behavior and viability. While AHLs had a non-significant impact on predation rates, DSF considerably delayed predation and bdelloplast lysis. Subsequent experiments showed that 50 µM DSF also reduced the motility of attack-phase B. bacteriovorus 109J cells by 50% (38.2 ± 14.9 vs. 17 ± 8.9 µm/s). Transcriptomic analyses found that DSF caused genome-wide changes in B. bacteriovorus 109J gene expression patterns during both the attack and intraperiplasmic phases, including the significant downregulation of the flagellum assembly genes and numerous serine protease genes. While the former accounts for the reduced speeds observed, the latter was confirmed experimentally with 50 µM DSF completely blocking protease secretion from attack-phase cells. Additional experiments found that 30% of the total cellular ATP was released into the supernatant when B. bacteriovorus 109J was exposed to 200 µM DSF, implying that this QS molecule negatively impacts membrane integrity.

Mixed-Methods Study to Predict Upstaging of DCIS to Invasive Disease on Mammography.

BACKGROUND. The incidence of ductal carcinoma in situ (DCIS) has steadily increased, as have concerns regarding overtreatment. Active surveillance is a novel treatment strategy that avoids surgical excision, but identifying patients with occult invasive disease who should be excluded from active surveillance is challenging. Radiologists are not typically expected to predict the upstaging of DCIS to invasive disease, though they might be trained to perform this task. OBJECTIVE. The purpose of this study was to determine whether a mixed-methods two-stage observer study can improve radiologists' ability to predict upstaging of DCIS to invasive disease on mammography. METHODS. All cases of DCIS calcifications that underwent stereotactic biopsy between 2010 and 2015 were identified. Two cohorts were randomly generated, each containing 150 cases (120 pure DCIS cases and 30 DCIS cases upstaged to invasive disease at surgery). Nine breast radiologists reviewed the mammograms in the first cohort in a blinded fashion and scored the probability of upstaging to invasive disease. The radiologists then reviewed the cases and results collectively in a focus group to develop consensus criteria that could improve their ability to predict upstaging. The radiologists reviewed the mammograms from the second cohort in a blinded fashion and again scored the probability of upstaging. Statistical analysis compared the performances between rounds 1 and 2. RESULTS. The mean AUC for reader performance in predicting upstaging in round 1 was 0.623 (range, 0.514-0.684). In the focus group, radiologists agreed that upstaging was better predicted when an associated mass, asymmetry, or architectural distortion was present; when densely packed calcifications extended over a larger area; and when the most suspicious features were focused on rather than the most common features. Additionally, radiologists agreed that BI-RADS descriptors do not adequately characterize risk of invasion, and that microinvasive disease and smaller areas of DCIS will have poor prediction estimates. Reader performance significantly improved in round 2 (mean AUC, 0.765; range, 0.617-0.852; p = .045). CONCLUSION. A mixed-methods two-stage observer study identified factors that helped radiologists significantly improve their ability to predict upstaging of DCIS to invasive disease. CLINICAL IMPACT. Breast radiologists can be trained to better predict upstaging of DCIS to invasive disease, which may facilitate discussions with patients and referring providers.

Biclustering analysis of transcriptome big data identifies condition-specific microRNA targets.

We present a novel approach to identify human microRNA (miRNA) regulatory modules (mRNA targets and relevant cell conditions) by biclustering a large collection of mRNA fold-change data for sequence-specific targets. Bicluster targets were assessed using validated messenger RNA (mRNA) targets and exhibited on an average 17.0% (median 19.4%) improved gain in certainty (sensitivity + specificity). The net gain was further increased up to 32.0% (median 33.4%) by incorporating functional networks of targets. We analyzed cancer-specific biclusters and found that the PI3K/Akt signaling pathway is strongly enriched with targets of a few miRNAs in breast cancer and diffuse large B-cell lymphoma. Indeed, five independent prognostic miRNAs were identified, and repression of bicluster targets and pathway activity by miR-29 was experimentally validated. In total, 29 898 biclusters for 459 human miRNAs were collected in the BiMIR database where biclusters are searchable for miRNAs, tissues, diseases, keywords and target genes.

Proteomic evaluation of kimchi, a traditional Korean fermented vegetable, and comparison of kimchi manufactured in China and Korea.

Kimchi is a traditional Korean fermented vegetable, which is also widely consumed in Japan and China. However, little is known about the kimchi proteome. In this study, Korean and Chinese kimchi proteomes were evaluated by shotgun proteomics. A total of 250 proteins were annotated, and 29 of these were expressed at > 1% of the average relative abundance. Discrimination of the geographical origins of Korean and Chinese kimchi samples was possible using multivariate analysis of the proteomic data, and 23 proteins were expressed differently between the two types (p < 0.001), and represent possible markers to discriminate between Chinese and Korean kimchi. This study provides important insights into the kimchi proteome and illustrates the proteomic differences caused by geographical origin.

Efficient pathway enrichment and network analysis of GWAS summary data using GSA-SNP2.

Pathway-based analysis in genome-wide association study (GWAS) is being widely used to uncover novel multi-genic functional associations. Many of these pathway-based methods have been used to test the enrichment of the associated genes in the pathways, but exhibited low powers and were highly affected by free parameters. We present the novel method and software GSA-SNP2 for pathway enrichment analysis of GWAS P-value data. GSA-SNP2 provides high power, decent type I error control and fast computation by incorporating the random set model and SNP-count adjusted gene score. In a comparative study using simulated and real GWAS data, GSA-SNP2 exhibited high power and best prioritized gold standard positive pathways compared with six existing enrichment-based methods and two self-contained methods (alternative pathway analysis approach). Based on these results, the difference between pathway analysis approaches was investigated and the effects of the gene correlation structures on the pathway enrichment analysis were also discussed. In addition, GSA-SNP2 is able to visualize protein interaction networks within and across the significant pathways so that the user can prioritize the core subnetworks for further studies. GSA-SNP2 is freely available at https://sourceforge.net/projects/gsasnp2.

GScluster: network-weighted gene-set clustering analysis.

BACKGROUND: Gene-set analysis (GSA) has been commonly used to identify significantly altered pathways or functions from omics data. However, GSA often yields a long list of gene-sets, necessitating efficient post-processing for improved interpretation. Existing methods cluster the gene-sets based on the extent of their overlap to summarize the GSA results without considering interactions between gene-sets. RESULTS: Here, we presented a novel network-weighted gene-set clustering that incorporates both the gene-set overlap and protein-protein interaction (PPI) networks. Three examples were demonstrated for microarray gene expression, GWAS summary, and RNA-sequencing data to which different GSA methods were applied. These examples as well as a global analysis show that the proposed method increases PPI densities and functional relevance of the resulting clusters. Additionally, distinct properties of gene-set distance measures were compared. The methods are implemented as an R/Shiny package GScluster that provides gene-set clustering and diverse functions for visualization of gene-sets and PPI networks. CONCLUSIONS: Network-weighted gene-set clustering provides functionally more relevant gene-set clusters and related network analysis.

Machine learning-based prediction of future breast cancer using algorithmically measured background parenchymal enhancement on high-risk screening MRI.

BACKGROUND: Preliminary work has demonstrated that background parenchymal enhancement (BPE) assessed by radiologists is predictive of future breast cancer in women undergoing high-risk screening MRI. Algorithmically assessed measures of BPE offer a more precise and reproducible means of measuring BPE than human readers and thus might improve the predictive performance of future cancer development. PURPOSE: To determine if algorithmically extracted imaging features of BPE on screening breast MRI in high-risk women are associated with subsequent development of cancer. STUDY TYPE: Case-control study. POPULATION: In all, 133 women at high risk for developing breast cancer; 46 of these patients developed breast cancer subsequently over a follow-up period of 2 years. FIELD STRENGTH/SEQUENCE: 5 T or 3.0 T T1 -weighted precontrast fat-saturated and nonfat-saturated sequences and postcontrast nonfat-saturated sequences. ASSESSMENT: Automatic features of BPE were extracted with a computer algorithm. Subjective BPE scores from five breast radiologists (blinded to clinical outcomes) were also available. STATISTICAL TESTS: Leave-one-out crossvalidation for a multivariate logistic regression model developed using the automatic features and receiver operating characteristic (ROC) analysis were performed to calculate the area under the curve (AUC). Comparison of automatic features and subjective features was performed using a generalized regression model and the P-value was obtained. Odds ratios for automatic and subjective features were compared. RESULTS: The multivariate model discriminated patients who developed cancer from the patients who did not, with an AUC of 0.70 (95% confidence interval: 0.60-0.79, P < 0.001). The imaging features remained independently predictive of subsequent development of cancer (P < 0.003) when compared with the subjective BPE assessment of the readers. DATA CONCLUSION: Automatically extracted BPE measurements may potentially be used to further stratify risk in patients undergoing high-risk screening MRI. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2019;50:456-464.

Overaugmentation to compensate for postextraction ridge atrophy using a putty-type porcine bone substitute material with recombinant bone morphogenetic protein-2: 4 weeks of healing in a canine model.

OBJECTIVES: The aims of the present study were (i) to determine the short-term effects of putty-type porcine bone substitute material (PB) with recombinant bone morphogenetic protein-2 (rhBMP-2) for alveolar ridge preservation (ARP) and (ii) evaluate the early healing of labial overaugmentation. MATERIALS AND METHODS: Two groups were randomly assigned to the extraction sockets in mandibular incisors of eight dogs: labial overaugmentation with (i) PB (control) or (ii) rhBMP-2-loaded PB (BMP). Microcomputed tomography (micro-CT), and histologic and histomorphometric analyses were performed after 4 weeks. RESULTS: Micro-CT revealed that some of the overaugmented PB was dispersed in both groups. The new bone volume was significantly larger in the BMP group than in the control group (18.4 ± 3.3 vs. 15.5 ± 3.0 mm3, mean ± SD, P < 0.05). Labial bone resorption was generally found histologically. No signs of mineralization were observed in the overaugmented area despite significantly increased ridge width, as compared to the adjacent tooth area. The area of new bone formation was larger in the BMP group than in the control group overall (23.7 ± 18.8 vs. 18.3 ± 21.2 mm2) and in three parts (apical, middle, and coronal), although the difference was statistically significant only in the coronal part (7.7 ± 7.9 vs. 4.6 ± 6.4 mm2, P < 0.05). CONCLUSIONS: The addition of rhBMP-2 enhanced ossification in the coronal part of the extraction socket relative to using PB alone. Overaugmentation increased the ridge dimension with no evidence of ossification in situ at 4 weeks. CLINICAL RELEVANCE: In early healing following ARP, rhBMP-2 enhances bone formation within the socket, but ossification in the overaugmented area needs to be studied further.

Sinus floor elevation using implants coated with recombinant human bone morphogenetic protein-2: micro-computed tomographic and histomorphometric analyses.

OBJECTIVES: The objective of this study was to determine the validity of a graft-free sinus floor elevation (SFE) procedure with simultaneous placement of recombinant morphogenetic protein-2 (rhBMP-2)-coated implants compared to uncoated control implants. METHODS: In 10 rabbits, SFE was performed on both sides. Dental implants were randomly placed in the sinus filled with a blood clot. Test implants were coated with rhBMP-2, whereas in the control group, implants were uncoated. Micro-computed tomographic and histomophometric analyses were performed at 4 and 8 weeks, including measurement for newly formed bone height (NBHm). RESULTS: Bone formation was evident along the implant surfaces up to the apex in test, but limited in control implants at 4 weeks. NBHm amounted to 5.1 mm (Q1 = 4.1; Q3 = 5.3) for test implants and to 3.4 mm (2.6; 3.7) for control implants at 4 weeks. NBHm then decreased to 8 weeks (3.4 mm (3.3; 3.7)) for test implants, whereas in control sites, NBHm increased slightly to 4.4 mm (4.1; 4.5) (p = 0.1250; p = 0.6250). CONCLUSIONS: Implants coated with rhBMP-2 presented a strong osteogenic reaction at 4 weeks with more favorable outcomes in terms of bone formation along the implant surface up to the apex compared to uncoated control implants. Remodeling and resorption process between 4 and 8 weeks did not further improve the outcomes in the test, but in the control group. CLINICAL RELEVANCE: The use of rhBMP-2-coated implants in a graft-free SFE might show an advantage in early implant stability to prevent collapse of membrane. However, a potential clinical benefit still needs to be proven.

Gene dispersion is the key determinant of the read count bias in differential expression analysis of RNA-seq data.

BACKGROUND: In differential expression analysis of RNA-sequencing (RNA-seq) read count data for two sample groups, it is known that highly expressed genes (or longer genes) are more likely to be differentially expressed which is called read count bias (or gene length bias). This bias had great effect on the downstream Gene Ontology over-representation analysis. However, such a bias has not been systematically analyzed for different replicate types of RNA-seq data. RESULTS: We show that the dispersion coefficient of a gene in the negative binomial modeling of read counts is the critical determinant of the read count bias (and gene length bias) by mathematical inference and tests for a number of simulated and real RNA-seq datasets. We demonstrate that the read count bias is mostly confined to data with small gene dispersions (e.g., technical replicates and some of genetically identical replicates such as cell lines or inbred animals), and many biological replicate data from unrelated samples do not suffer from such a bias except for genes with some small counts. It is also shown that the sample-permuting GSEA method yields a considerable number of false positives caused by the read count bias, while the preranked method does not. CONCLUSION: We showed the small gene variance (similarly, dispersion) is the main cause of read count bias (and gene length bias) for the first time and analyzed the read count bias for different replicate types of RNA-seq data and its effect on gene-set enrichment analysis.

Recombinant bone morphogenetic protein-2 and platelet-derived growth factor-BB for localized bone regeneration. Histologic and radiographic outcomes of a rabbit study.

OBJECTIVES: Improvement in localized bone regeneration is needed to avoid the use of autogenous tissue. For that purpose, the use biologic mediators was proposed. The aim was to test whether or not one of two biologic mediators, recombinant human bone morphogenetic protein-2 (rhBMP-2) or recombinant platelet-derived growth factor (rhPDGF-BB), is superior to the other and to control groups for localized bone regeneration. MATERIALS AND METHODS: Four cylinders (height: 5 mm; diameter: 7 mm) were screwed on the parietal and frontal bones at the cranium in 12 rabbits. The cylinders either received (i) deproteinized bovine bone mineral (DBBM) mixed rhBMP-2 (DBBM/BMP-2), (ii) DBBM mixed with rhPDGF-BB (DBBM/PDGF), (iii) DBBM (DBBM), and (iv) empty control (control). Rabbits were euthanized at 2 and 8 weeks (n = 6, respectively). Conventional histomorphometric and micro-CT analyses were performed. Parametric linear mixed models were applied for the analyses with Bonferroni correction for the multiple group comparisons. RESULTS: The area of bone regeneration (histology; AAHisto ) at 2 weeks peaked for DBBM (41.91%) with statistically significantly greater values compared to DBBM/PDGF and the control group (P < 0.05). At 8 weeks, mean AAHisto values were 96.29% (DBBM/BMP-2), 46.37% (DBBM/PDFG), 39.66% (DBBM), and 35.98% (control) (DBBM/BMP-2 vs. all groups (P < 0.05)). At 8 weeks, bone regeneration was greatest for DBBM/BMP-2 (35.62%) with statistically significant differences compared to all other groups (P < 0.05). The area of bone regeneration (micro-CT; AAm-CT ) at 2 weeks amounted to 43.87% (DBBM/BMP-2), 42.81% (DBBM/PDFG), 48.71% (DBBM), and 0.96% (control). The control group demonstrated statistically significantly less AAm-CT compared to all groups (P < 0.05). At 8 weeks, mean AAm-CT values were 63.65% (DBBM/BMP-2), 50.21% (DBBM/PDFG), 44.81% (DBBM), and 4.57% (control) (P > 0.05). CONCLUSIONS: The use of rhBMP-2 significantly enhanced bone regeneration compared to all other groups including the group with rhPDGF-BB.

Sequential healing of onlay bone grafts using combining biomaterials with cross-linked collagen in dogs.

OBJECTIVES: The aim of this study was to observe the sequential healing of onlay grafts in terms of the volumetric and histologic changes using different bone substances and absorbable membranes according to the presence of collagen cross-linking. MATERIAL AND METHODS: Four groups involving onlay grafting with different materials were randomly assigned to both sides of the maxillae of 15 beagle dogs: (i) control group without any treatment; (ii) group NN, bovine hydroxyapatite incorporated into a non-cross-linked collagen matrix (BHC) + non-cross-linked collagen membrane (NCCM); (iii) group NC, BHC + cross-linked collagen membrane (CCM); and (iv) group CC, porcine hydroxyapatite incorporated into a cross-linked collagen matrix + CCM. Radiographic and histological analyses were performed after three different healing periods: 4, 8 and 12 weeks. RESULTS: At week 4, the bone substances were well localized under the barrier membrane in groups NC and CC, while the bone substances became spread out and flattened in group NN. Similarly, the augmented height was significantly greater in groups NC and CC (2.55 and 2.51 mm, respectively; median) than in group NN (1.96 mm, P < 0.001, both). The percentages of newly formed bone were significantly higher at week 12 than at weeks 4 and 8 in all of the groups. The NCCM showed an earlier angiogenesis pattern than the CCM; however, earlier degradation was observed at week 12. CONCLUSION: Combining biomaterials with cross-linked collagen might contribute to maintaining its initial morphology with excellent biocompatibility in early healing period of lateral onlay grafts.

Primary ridge augmentation with collagenated xenogenic block bone substitute in combination with collagen membrane and rhBMP-2: a pilot histological investigation.

OBJECTIVE: The aim of this pilot study was to test whether a porcine collagenated bone substitute block (PCBB) and collagen membrane (CM) loaded with bone morphogenetic protein-2 (BMP-2) used for horizontal ridge augmentation differ from PCBB and CM without BMP-2 regarding the osseointegration of the grafting material and the maintenance of the ridge contour. MATERIAL AND METHODS: Two semi-saddle bone defects were created in each side of the mandible of six dogs. The defects were randomly allocated to receive one of the following treatments: bone augmentation using (1) PCBB, (2) PCBB loaded with BMP-2 (PCBB-BMP2), (3) PCBB + CM and (4) PCBB + CM loaded with BMP-2 (PCBB + CM-BMP2). After 12 weeks, one titanium implant was inserted into every site. After 8 weeks, one central histological section of each site was prepared. Histomorphometrical assessments were performed evaluating the augmented area (AA), the area of new bone (NB) (primary outcome), residual bone substitute (BS) and non-mineralized tissue (NMT) within AA in mm2 . In addition, the most coronal and the most buccal localizations of new bone and residual bone substitute, and the most coronal bone-to-implant contact were measured in mm. RESULTS: Clinically, all PCBB were firmly integrated and permitted implant placement. All the implants osseointegrated and exhibited complete hard-tissue coverage of the buccal surface. Bone ingrowth always reached the central portions of PCBB. AA measured 10.4 ± 4.2 mm2 for PCBB, 11.8 ± 2.8 mm2 for PCBB-BMP2, 9.8 ± 2.9 mm2 for PCBB + CM and 8.5 ± 2.2 mm2 for PCBB + CM-BMP2. Only the difference between PCBB-BMP2 and PCBB + CM-BMP2 was statistically significant (P = 0.031). NB reached 2.3 ± 1.3 mm2 for PCBB, 2 ± 0.5 mm2 for PCBB-BMP2, 2.7 ± 1.2 mm2 for PCBB + CM and 1.8 ± 0.7 mm2 for PCBB + CM-BMP2. There were no statistically significant differences regarding NB, the most coronal and the most buccal localizations of new bone, residual bone substitute and bone-to-implant contact (P > 0.05). CONCLUSIONS: The addition of BMP-2 to PCBB or CM used for horizontal ridge augmentation did not render a statistically significant improvement in the maintenance of the augmented ridge contour and the new bone formation. PCBB with and without CM showed pronounced bone ingrowth and capacity to maintain the augmented ridge contour. In all the regions previously augmented with PCBB, the implants successfully integrated and presented with complete hard-tissue coverage.

De novo bone formation underneath the sinus membrane supported by a bone patch: a pilot experiment in rabbit sinus model.

AIMS: The objectives of this study were to evaluate bone regeneration beneath a newly devised bone substitute combined with collagen membrane (called a bone patch) lying over a concomitantly placed mini-implant following sinus floor elevation and verify its usefulness as a carrier system for recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbits. MATERIALS AND METHODS: The sinus floor elevation procedure was performed bilaterally in five rabbits. Either a plain bone patch (control group) or an rhBMP-2-loaded patch (experimental group) was randomly placed beneath the elevated sinus membrane (SM) of both sinuses, where the mini-implants were concomitantly placed. Micro-computed tomographic and histologic analyses were performed at 4 weeks post-surgery. RESULTS: In micro-computed tomography, the median values of the total augmented volume and the mineralized bone volume were significantly higher in the experimental group than in the control group (161 vs. 122 mm3 [P < 0.01] and 48 vs. 42 mm3 [P < 0.05], respectively). Histometric analysis revealed the same outcomes, with new bone areas of 6.41 and 2.97 mm2 in the experimental and control groups, respectively (P < 0.001), and bone-to-implant contact ratios of 22.6% and 5.2%, respectively (P < 0.001). CONCLUSION: The newly devised bone patch in this study can support the elevated SM and facilitate bone regeneration from the basal bone with a reduced amount of biomaterial. The addition of rhBMP-2 may shorten the healing time for multidirectional bone regeneration toward the implant.