The development of non-resorbable bone allografts: Biological background and clinical perspectives.

Bone grafts are typically categorized into four categories: autografts, allografts, xenografts, and synthetic alloplasts. While it was originally thought that all bone grafts should be slowly resorbed and replaced with native bone over time, accumulating evidence has in fact suggested that the use of nonresorbable xenografts is favored for certain clinical indications. Thus, many clinicians take advantage of the nonresorbable properties/features of xenografts for various clinical indications, such as contour augmentation, sinus grafting, and guided bone regeneration, which are often combined with allografts (e.g., human freeze-dried bone allografts [FDBAs] and human demineralized freeze-dried bone allografts [DFDBAs]). Thus, many clinicians have advocated different 50/50 or 70/30 ratios of allograft/xenograft combination approaches for various grafting procedures. Interestingly, many clinicians believe that one of the main reasons for the nonresorbability or low substitution rates of xenografts has to do with their foreign animal origin. Recent research has indicated that the sintering technique and heating conducted during their processing changes the dissolution rate of hydroxyapatite, leading to a state in which osteoclasts are no longer able to resorb (dissolve) the sintered bone. While many clinicians often combine nonresorbable xenografts with the bone-inducing properties of allografts for a variety of bone augmentation procedures, clinicians are forced to use two separate products owing to their origins (the FDA/CE does not allow the mixture of allografts with xenografts within the same dish/bottle). This has led to significant progress in understanding the dissolution rates of xenografts at various sintering temperature changes, which has since led to the breakthrough development of nonresorbable bone allografts sintered at similar temperatures to nonresorbable xenografts. The advantage of the nonresorbable bone allograft is that they can now be combined with standard allografts to create a single mixture combining the advantages of both allografts and xenografts while allowing the purchase and use of a single product. This review article presents the concept with evidence derived from a 52-week monkey study that demonstrated little to no resorption along with in vitro data supporting this novel technology as a "next-generation" biomaterial with optimized bone grafting material properties.

Histological evaluation of nonsurgical periodontal treatment with and without the use of sodium hypochlorite / amino acids and cross-linked hyaluronic acid gels in dogs.

OBJECTIVES: To evaluate periodontal wound healing following scaling and root planing (SRP) in conjunction with the application of sodium hypochlorite/amino acids and cross-linked hyaluronic acid (xHyA) gels in dogs. MATERIALS AND METHODS: In four beagle dogs, 2-wall intrabony defects were created and metal strips were placed around the teeth. Clinical parameters were measured 4 weeks after plaque accumulation. The experimental root surfaces were subjected to SRP with either the subgingival application of a sodium hypochlorite/amino acid gel and a xHyA gel (test group) or SRP alone (control group) using a split-mouth design. Clinical parameters were re-evaluated at 6 weeks. The animals were sacrificed at 8 weeks for histological analysis. RESULTS: The test group showed significant improvements in all clinical parameters compared to the control group. Histologically, the test group exhibited statistically significantly greater new bone formation [i.e., length of newly formed bone, new bone area] compared with the control group (p < 0.05). Furthermore, statistically significantly greater formation of new attachment [i.e., linear length of new cementum adjacently to newly formed bone with inserting collagen fibers] and new cementum was detected in the test group compared with the control group at 8 weeks (p < 0.05 and p < 0.01, respectively). CONCLUSION: The adjunctive subgingival application of sodium hypochlorite/amino acid and xHyA gels to SRP offers an innovative novel approach to enhance periodontal wound healing/regeneration. CLINICAL RELEVANCE: The present findings have for the first-time shown histologic evidence for periodontal regeneration in support of this novel treatment modality.

Comparison of periodontal wound healing/regeneration by recombinant human fibroblast growth factor-2 combined with ß-tricalcium phosphate, carbonate apatite, or deproteinized bovine bone mineral in a canine one-wall intra-bony defect model.

AIM: To evaluate periodontal wound healing/regeneration of one-wall intra-bony defects treated with recombinant human fibroblast growth factor-2 (rhFGF-2) and beta-tricalcium phosphate (ß-TCP), carbonate apatite (CO3 Ap), or deproteinized bovine bone mineral (DBBM) in dogs. MATERIALS AND METHODS: The stability of rhFGF-2 adsorbed onto the bone substitutes was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). One-wall intra-bony defects (5 × 5 × 5 mm) created in five adult male beagle dogs were treated with rhFGF-2 alone (rhFGF-2), rhFGF-2 with ß-TCP (rhFGF-2/ß-TCP), rhFGF-2 with CO3 Ap (rhFGF-2/CO3 Ap), or rhFGF-2 with DBBM (rhFGF-2/DBBM). Histological outcomes (e.g., linear length of new cementum adjacent to the newly formed bone with inserting collagen fibres [NA] as the primary outcome) were evaluated at 10 weeks post surgery. RESULTS: Significantly higher amount of rhFGF-2 was adsorbed onto CO3 Ap compared with ß-TCP. Among the treatment groups, the rhFGF-2/DBBM group showed the highest amount of periodontal tissue regeneration. The rhFGF-2/DBBM group showed significantly greater formation of NA (3.22 ± 0.40 mm) compared with rhFGF-2 (1.17 ± 1.00 mm, p < .01) group. Additionally, new bone area in the rhFGF-2/DBBM group (9.78 ± 2.30 mm2 ) was significantly higher than that in the rhFGF-2 (5.08 ± 1.26 mm2 , p < .01), rhFGF-2/ß-TCP (5.91 ± 1.27 mm2 , p < .05), and rhFGF-2/CO3 Ap (6.51 ± 1.49 mm2 , p < .05) groups. Slight ankylosis was found in the rhFGF-2/ß-TCP (1/9 sites), rhFGF-2/CO3 Ap (3/10 sites), and rhFGF-2/DBBM (1/9 sites) groups. CONCLUSIONS: Within their limitations, the present data indicate that DBBM seems to be a suitable carrier for rhFGF-2 and that rhFGF-2/DBBM treatment promotes favourable periodontal regeneration compared with rhFGF-2, rhFGF-2/ß-TCP, and rhFGF-2/CO3 Ap treatments in one-wall intra-bony defects.

Cross-linked hyaluronic acid gel with or without a collagen matrix in the treatment of class III furcation defects: A histologic and histomorphometric study in dogs.

AIM: To histologically evaluate the effects of cross-linked hyaluronic acid (xHyA) with or without a collagen matrix (CM) on periodontal wound healing/regeneration in class III furcation defects in dogs. MATERIALS AND METHODS: Class III furcation defects were surgically created in the mandibular premolars in six beagle dogs. The defects were randomly treated as follows: open flap debridement (OFD) + CM (CM), OFD + xHyA (xHyA), OFD + xHyA + CM (xHyA/CM) and OFD alone (OFD). At 10 weeks, the animals were euthanized for histological evaluation. RESULTS: The newly formed bone areas in the xHyA (4.04 ± 1.51 mm2 ) and xHyA/CM (4.32 ± 1.14 mm2 ) groups were larger than those in the OFD (3.25 ± 0.81 mm2 ) and CM (3.31 ± 2.26 mm2 ) groups. The xHyA (6.25 ± 1.45 mm) and xHyA/CM (6.40 ± 1.35 mm) groups yielded statistically significantly (p < .05) greater formation of new connective tissue attachment (i.e., new cementum, with inserting connective tissue fibres) compared with the OFD (1.47 ± 0.85 mm) group. No significant differences were observed in any of the histomorphometric parameters between the xHyA and xHyA/CM groups. Complete furcation closure was not observed in any of the four treatment modalities. CONCLUSIONS: Within their limits, the present results suggest that the use of xHyA with or without CM positively influences periodontal wound healing in surgically created, acute-type class III furcation defects.

Healing of buccal gingival recessions following treatment with coronally advanced flap alone or combined with a cross-linked hyaluronic acid gel. An experimental study in dogs.

AIM: To clinically and histologically evaluate in dogs the healing of gingival recessions treated with coronally advanced flap (CAF) with or without cross-linked hyaluronic acid (HA). MATERIALS AND METHODS: Gingival recession defects were surgically created on the vestibular side of both maxillary canines in 8 dogs. After 8 weeks of plaque accumulation, the 16 chronic defects were randomly treated with either CAF alone or CAF and HA-gel (CAF/HA). Clinical and histological outcomes were evaluated at 10 weeks post-surgically. RESULTS: Compared to baseline, the clinical measurements at 10 weeks revealed a statistically significant decrease in gingival recession for both CAF (p < 0.01) and CAF/HA (p < 0.001) groups. Statistically significant differences were found in clinical attachment level (p < 0.05) and width of gingival recession (p < 0.01) favouring the CAF/HA group. Bone formation was statistically significantly greater in the CAF/HA group than in the CAF group (1.84 ± 1.16 mm vs., 0.72 ± 0.62 mm, respectively, p < 0.05). Formation of cementum and connective tissue attachment were statistically significantly higher in the CAF/HA group compared with the CAF group (i.e. 4.31 ± 1.78 mm versus 2.40 ± 1.35 mm and 1.69 ± 0.98 mm versus 0.74 ± 0.68 mm, respectively (p < 0.05)). CONCLUSIONS: The present data have for the first time provided histologic evidence for periodontal regeneration of gingival recession defects following treatment with CAF and HA. CLINICAL RELEVANCE: The use of HA in conjunction with CAF may represent a novel modality for treating gingival recession defects.

Effect of interleukin-1ß on bone morphogenetic protein-9-induced osteoblastic differentiation of human periodontal ligament fibroblasts.

Bone morphogenetic protein-9 (BMP-9) has been shown to potently induce osteoblastic differentiation of periodontal ligament fibroblasts (PDLFs) and may be a candidate therapeutic agent for periodontal tissue healing/regeneration, but the effect of the inflammatory environment of periodontitis on such approaches is unclear. We investigated whether interleukin-1ß (IL-1ß) affected BMP-9-mediated osteoblastic differentiation of human (h) PDLFs. IL-1ß suppressed BMP-9-induced osteogenic differentiation of hPDLFs, as evidenced by reduced alkaline phosphatase (ALP) activity and mineralization, and the downregulated expression of BMP-9-mediated bone-related genes, RUNX2, SP7, IBSP, and SPP1. In hPDLFs, with or without BMP-9, IL-1ß increased the protein expression of activin A, a BMP-9 antagonist, and decreased follistatin protein, an antagonist of activin A. Similarly, IL-1ß upregulated the expression of the activin A gene and downregulated that of the follistatin gene. Notably, follistatin re-established BMP-9-induced ALP activity suppressed by IL-1ß. Activin A inhibited the expression of BMP-9-responsive genes and BMP-9-induced ALP activity, while follistatin re-established them. Finally, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and nuclear factor-kappa B (NF-κB) inhibition significantly blocked IL-1ß-induced activin A gene expression. Our data indicate that IL-1ß inhibits BMP-9-induced osteoblastic differentiation of hPDLFs, possibly by promoting activin A production via the ERK1/2, p38, and NF-κB pathways.

Enhanced bone formation of calvarial bone defects by low-intensity pulsed ultrasound and recombinant human bone morphogenetic protein-9: a preliminary experimental study in rats.

OBJECTIVES: The aim of this study was to evaluate the combined effects of recombinant human bone morphogenetic protein - 9 (rhBMP-9) loaded onto absorbable collagen sponges (ACS) and low-intensity pulsed ultrasound (LIPUS) on bone formation in rat calvarial defects. MATERIALS AND METHODS: Circular calvarial defects were surgically created in 18 Wistar rats, which were divided into LIPUS-applied (+) and LIPUS-non-applied (-) groups. The 36 defects in each group received ACS implantation (ACS group), ACS with rhBMP-9 (rhBMP-9/ACS group), or surgical control (control group), yielding the following six groups: ACS (+/-), rhBMP-9/ACS (+/-), and control (+/-). The LIPUS-applied groups received daily LIPUS exposure starting immediately after surgery. At 4 weeks, animals were sacrificed and their defects were investigated histologically and by microcomputed tomography. RESULTS: Postoperative clinical healing was uneventful at all sites. More new bone was observed in the LIPUS-applied groups compared with the LIPUS-non-applied groups. Newly formed bone area (NBA)/total defect area (TA) in the ACS (+) group (46.49 ± 7.56%) was significantly greater than that observed in the ACS (-) (34.31 ± 5.68%) and control (-) (31.13 ± 6.74%) groups (p < 0.05). The rhBMP-9/ACS (+) group exhibited significantly greater bone volume, NBA, and NBA/TA than the rhBMP-9/ACS (-) group (2.46 ± 0.65 mm3 vs. 1.76 ± 0.44 mm3, 1.25 ± 0.31 mm2 vs. 0.88 ± 0.22 mm2, and 62.80 ± 11.87% vs. 42.66 ± 7.03%, respectively) (p < 0.05). Furthermore, the rhBMP-9/ ACS (+) group showed the highest level of bone formation among all groups. CONCLUSION: Within their limits, it can be concluded that LIPUS had osteopromotive potential and enhanced rhBMP-9-induced bone formation in calvarial defects of rats. CLINICAL RELEVANCE: The use of rhBMP-9 with LIPUS stimulation can be a potential bone regenerative therapy for craniofacial/peri-implant bone defects.

BMP9 directly induces rapid GSK3-ß phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

Bone morphogenetic protein (BMP)9 has been reported to be the most potent BMP to induce bone formation. However, the details of BMP9-transduced intracellular signaling remain ambiguous. Here, we have investigated signal transduction mechanisms of BMP9 in comparison to BMP2, another potent inducer of bone formation, in osteoblasts. In a mouse osteoblast cell line, BMP9 induced higher mRNA levels of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) than BMP2 within 2 h. Unlike BMP2, BMP9 induced rapid phosphorylation of glycogen synthase kinase 3-ß (GSK3-ß) and protein kinase B (Akt) and increased the cellular protein content of ß-catenin. BMP9 moderately increased mRNA levels of several canonical Wingless-related integration site to lower degrees than BMP2. Furthermore, BMP9-induced GSK3-ß phosphorylation was not inhibited by pretreatment with actinomycin D, cycloheximide, or Brefeldin A, indicating it is independent of Wnt protein secretion. BMP9-induced GSK3-ß phosphorylation was abrogated by Akt or class I PI3K-specific inhibitors. Moreover, inactivation of GSK3-ß by LiCl did not further promote ALP and Runx2 mRNA induction by BMP9 as significantly as that by BMP2. Notably, BMP9-induced GSK3-ß phosphorylation was inhibited by small interfering RNA against endoglin and GIPC PDZ domain-containing family, member 1. Taken together, our present findings have indicated that BMP9 directly activates GSK3ß-ß-catenin signaling pathway through class I PI3K-Akt Axis in osteoblasts, which may be essential for the potent osteoinductive activity of BMP9.-Eiraku, N., Chiba, N., Nakamura, T., Amir, M. S., Seong, C.-H., Ohnishi, T., Kusuyama, J., Noguchi, K., Matsuguchi, T. BMP9 directly induces rapid GSK3-ß phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Periodontal tissue regeneration after low-intensity pulsed ultrasound stimulation with or without intra-marrow perforation in two-wall intra-bony defects-A pilot study in dogs.

AIM: To evaluate the effects of low-intensity pulsed ultrasound (LIPUS) with/without intra-marrow perforation (IMP) on periodontal healing in two-wall intra-bony defects in dogs. MATERIALS AND METHODS: Two-wall intra-bony defects (5 mm wide, 5 mm deep) were created at the distal and mesial aspects of mandibular premolars in four beagle dogs (four defects per dog). The 16 defects were divided into four treatment groups: IMP, LIPUS, IMP + LIPUS (IMP/LIPUS) and control (open flap debridement). The LIPUS and IMP/LIPUS sites received daily LIPUS exposure for 3 weeks starting 1 week after surgery. The animals were euthanized 4 weeks after surgery for histologic evaluation. RESULTS: There was significantly greater new bone formation at LIPUS (2.93 ± 0.74 mm) and IMP/LIPUS (3.18 ± 0.52 mm) sites than at control sites (1.65 ± 0.46 mm). New bone area at LIPUS (6.36 ± 2.28 mm2 ) and IMP/LIPUS (6.13 ± 1.25 mm2 ) sites was significantly greater than that at control sites (2.15 ± 1.75 mm2 ). New cementum length at LIPUS sites (4.09 ± 0.75 mm) was significantly greater than that at control (2.29 ± 1.02 mm) and IMP (2.41 ± 0.41 mm) sites. No significant difference was observed between LIPUS and IMP/LIPUS sites in any histomorphometric parameter. CONCLUSIONS: These findings suggest that LIPUS effectively promotes periodontal regeneration in two-wall intra-bony defects in dogs.

Low-intensity pulsed ultrasound promotes bone morphogenic protein 9-induced osteogenesis and suppresses inhibitory effects of inflammatory cytokines on cellular responses via Rho-associated kinase 1 in human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low-intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9-induced osteogenic differentiation of PDLFs. In contrast, BMP2-induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS-induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS-PG), we also simultaneously treated PDLFs with LIPUS and LPS-PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK-binding kinase 1, and interferon regulatory factor 3 in LPS-PG-stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa-B ligand, and also diminished IL-1ß and tumor necrosis factor a (TNFa)-induced inflammatory reactions. Phosphorylation of Rho-associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1-specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS-PG-stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS-PG and IL-1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS-PG, IL-1ß, and TNFa and also promotes BMP9-induced osteogenesis through ROCK1 in PDLFs.

The mesenchymal transition subtype more responsive to dose dense taxane chemotherapy combined with carboplatin than to conventional taxane and carboplatin chemotherapy in high grade serous ovarian carcinoma: A survey of Japanese Gynecologic Oncology Group study (JGOG3016A1).

OBJECTIVE: Recently, we established new histopathological subtypes of high-grade serous ovarian cancer (HGSOC) that include the mesenchymal transition (MT) type, the immune reactive (IR) type, the solid and proliferative (SP) type and the papillo-glandular (PG) type. Furthermore, we identified that the mesenchymal transcriptome subtype might be sensitive to taxane. We investigated whether these different histopathological subtypes of HGSOC require individualized chemotherapy for optimal treatment. METHODS: We conducted the Japanese Gynecologic Oncology Group (JGOG) 3016A1 study, wherein we collected hematoxylin and eosin slides (total n = 201) and performed a histopathological analysis of patients with HGSOC registered in the JGOG3016 study, which compared the efficacy of conventional paclitaxel and carboplatin (TC) and dose-dense TC (ddTC). We analyzed the differences in progression-free survival (PFS) and overall survival (OS) among the four histopathological subtypes. We then compared the PFS between the TC group and the ddTC group for each histopathological subtype. RESULTS: There were significant differences in both PFS and OS among the four histopathological subtypes (p = 0.001 and p < 0.001, respectively). Overall, the MT subtype had the shortest PFS (median 1.4 y) and OS (median 3.6 y). In addition, the MT subtype had a longer PFS in the ddTC group (median 1.8 y) than in the TC group (median 1.2 y) (p = 0.01). Conversely, the other types had no significant difference in PFS when the two regimens were compared. CONCLUSIONS: The MT type of HGSOC is sensitive to taxane; therefore, the ddTC regimen is recommended for this histopathological subtype.

Influence of UGT1A1 polymorphism on etoposide plus platinum-induced neutropenia in Japanese patients with small-cell lung cancer.

BACKGROUND: The association between UGT1A1 polymorphism and etoposide-induced toxicities is still not clear. The aim of this study was to assess the association between uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene polymorphism and severe hematologic toxicities in Japanese patients receiving etoposide plus platinum chemotherapy for small-cell lung cancer. METHODS: This retrospective analysis included patients with small-cell lung cancer who had received their first-line chemotherapy with etoposide plus cisplatin or carboplatin, between October 2008 and April 2018, at the University of Fukui Hospital. The relationship between UGT1A1 polymorphisms and first-cycle neutropenia as well as thrombocytopenia was evaluated. RESULTS: A total of 55 patients were enrolled. The incidence of grade 4 neutropenia during the first cycle of etoposide-based chemotherapy was higher in patients with homozygous (hmz) polymorphisms for UGT1A1*28 and *6 (*28/*28, *6/*6, and *6/*28) than in patients with wild-type (wt) (*1/*1) and heterozygous (htz) (*1/*28 and *1/*6) polymorphisms (88% vs 43% P = 0.03). The incidence of febrile neutropenia and grade 4 thrombocytopenia, however, was not significantly different. Multivariate analysis suggested that grade 4 neutropenia associated significantly with an hmz UGT1A1 genotype [odds ratio (OR) 11.3; P = 0.04] and administration of granulocyte colony-stimulating factor (G-CSF) before the neutrophil counts dropped to < 500 cells/µL (OR; P = 0.01). CONCLUSIONS: UGT1A1*28 and UGT1A1*6 mutations might be regarded as predictors for etoposide-induced grade 4 neutropenia.

Whole-genome sequencing revealed novel prognostic biomarkers and promising targets for therapy of ovarian clear cell carcinoma.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is mostly resistant to standard chemotherapy that results in poor patient survival. To understand the genetic background of these tumours, we performed whole-genome sequencing of OCCC tumours. METHODS: Tumour tissue samples and matched blood samples were obtained from 55 Japanese women diagnosed with OCCC. Whole-genome sequencing was performed using the Illumina HiSeq platform according to standard protocols. RESULTS: Alterations to the switch/sucrose non-fermentable (SWI/SNF) subunit, the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway, and the receptor tyrosine kinase (RTK)/Ras signalling pathway were found in 51%, 42%, and 29% of OCCC tumours, respectively. The 3-year overall survival (OS) rate for patients with an activated PI3K/Akt signalling pathway was significantly higher than that for those with inactive pathway (91 vs 40%, hazard ratio 0.24 (95% confidence interval (CI) 0.10-0.56), P=0.0010). Similarly, the OS was significantly higher in patients with the activated RTK/Ras signalling pathway than in those with the inactive pathway (91 vs 53%, hazard ratio 0.35 (95% CI 0.13-0.94), P=0.0373). Multivariable analysis revealed that activation of the PI3K/Akt and RTK/Ras signalling pathways was an independent prognostic factor for patients with OCCC. CONCLUSIONS: The PI3K/Akt and RTK/Ras signalling pathways may be potential prognostic biomarkers for OCCC patients. Furthermore, our whole-genome sequencing data highlight important pathways for molecular and biological characterisations and potential therapeutic targeting in OCCC.

Healing of two-wall intra-bony defects treated with a novel EMD-liquid-A pre-clinical study in monkeys.

AIM: To investigate the effect of a novel enamel matrix derivative formulation (EMD-liquid or Osteogain) combined with an absorbable collagen sponge (ACS) on periodontal wound healing in intra-bony defects in monkeys. MATERIALS AND METHODS: Chronic two-wall intra-bony defects were created at the distal aspect of eight teeth in three monkeys (Macaca fascicularis). The 24 defects were randomly assigned to one of the following treatments: (i) open flap debridement (OFD) + ACS alone, (ii) OFD + Emdogain + ACS (Emdogain/ACS), (iii) OFD + Osteogain + ACS (Osteogain/ACS) or (iv) OFD alone. At 4 months, the animals were euthanized for histologic evaluation. RESULTS: Osteogain/ACS resulted in more consistent formation of cementum, periodontal ligament and bone with limited epithelial proliferation compared to OFD alone, Emdogain/ACS and OFD + ACS. Among the four treatment groups, the Osteogain/ACS group demonstrated the highest amount of regenerated tissues. However, complete periodontal regeneration was not observed in any of the defects in the four groups. CONCLUSIONS: The present findings indicate that in two-wall intra-bony defects, reconstructive surgery with Osteogain/ACS appears to be a promising novel approach for facilitating periodontal wound healing/regeneration, thus warranting further clinical testing.

Effects of EMD liquid (Osteogain) on periodontal healing in class III furcation defects in monkeys.

AIM: To evaluate the effect of a novel liquid carrier system of enamel matrix derivative (Osteogain) soaked on an absorbable collagen sponge (ACS) upon periodontal wound healing/regeneration in furcation defects in monkeys. MATERIALS AND METHODS: The stability of the conventional enamel matrix derivative (Emdogain) and Osteogain adsorbed onto ACS was evaluated by ELISA. Chronic class III furcation defects were created at teeth 36, 37, 46, 47 in three monkeys (Macaca fascicularis). The 12 defects were assigned to one of the following treatments: (1) open flap debridement (OFD) + ACS, (2) OFD+Emdogain/ACS, (3) OFD+Osteogain/ACS, and (4) OFD alone. At 16 weeks following reconstructive surgery, the animals were killed for histological evaluation. RESULTS: A 20-60% significantly higher amount of total adsorbed amelogenin was found for ACS-loaded Osteogain when compared to Emdogain. The histomorphometric analysis revealed that both approaches (OFD + Emdogain/ACS and OFD + Osteogain/ACS) resulted in higher amounts of connective tissue attachment and bone formation compared to treatment with OFD + ACS and OFD alone. Furthermore, OFD + Osteogain/ACS group showed higher new attachment formation, cementum, and new bone area. CONCLUSIONS: Within their limits, the present data indicate that Osteogain possesses favourable physicochemical properties facilitating adsorption of amelogenin on ACS and may additionally enhance periodontal wound healing/regeneration when compared to Emdogain.

Involvement of the phosphoinositide 3-kinase/Akt signaling pathway in bone morphogenetic protein 9-stimulated osteogenic differentiation and stromal cell-derived factor 1 production in human periodontal ligament fibroblasts.

Recent studies have shown that bone morphogenetic protein 9 (BMP-9) can induce osteogenic differentiation in human periodontal stem cells and human periodontal ligament fibroblasts (PDLFs). Bone morphogenetic protein 9 may be used in periodontal tissue regeneration because of its potent osteoinductive ability. Human periodontal ligament cells also have been demonstrated to produce stromal cell-derived factor 1 (SDF-1), which is important for stem-cell homing and recruitment to injured sites. In the present study, we examined the involvement of the phosphoinositide 3-kinase (PI3K)/Akt signaling axis in osteogenic differentiation and SDF-1 production in human PDLFs stimulated with BMP-9 in osteogenic medium supplemented with dexamethasone and ascorbic acid. Pretreatment of the cells with LY294002, a PI3K-specific inhibitor, suppressed not only BMP-9-enhanced alkaline phosphatase activity but also expression of a BMP-response gene (inhibitor of DNA binding 1) and osteogenic marker genes (runt-related transcription factor 2, osterix, bone sialoprotein, and osteopontin). In addition, BMP-9 up-regulated SDF-1 production, and the production of SDF-1 was suppressed by LY294002. The protein SDF-1-alpha was identified as a major isoform of SDF-1 that was regulated by BMP-9. Our data suggest involvement of the PI3K/Akt pathway in BMP-9-stimulated osteogenic differentiation and SDF-1 production in PDLFs cultured in osteogenic medium.

Osteogenic potential of recombinant human bone morphogenetic protein-9/absorbable collagen sponge (rhBMP-9/ACS) in rat critical size calvarial defects.

OBJECTIVES: It has been reported that bone morphogenetic protein (BMP)-9 has potent osteoinductive properties among the BMP family by adenovirus-transfection experiments. We very recently reported that absorbable collagen sponge (ACS) as a carrier for recombinant human (rh) BMP-9, compared with chitosan sponge, was suitable for inducing bone healing/regeneration by BMP-9 in a rat calvarial defect model. The aim of this study was to evaluate different doses of rhBMP-9/ACS on new bone formation in rat critical size calvarial defects. MATERIALS AND METHODS: Bilateral calvarial defects (n = 32) were surgically created in 16 wistar rats and randomly filled with one of the following materials: (1) absorbable collagen sponge (ACS) alone; (2) 1 µg-rhBMP-9/ACS (L-rhBMP-9/ACS); (3) 5 µg-rhBMP-9/ACS (H-rhBMP-9/ACS); and (4) blank defects (control). The animals were sacrificed 8 weeks postsurgery for radiographic and histomorphometric analyses. RESULTS: Bone volume and defect closure were statistically higher in the rhBMP-9/ACS-implanted (L-rhBMP-9/ACS and H-rhBMP-9/ACS) groups when compared with ACS-alone group (p < 0.05). Furthermore, defects filled with H-rhBMP-9/ACS showed the highest levels of newly formed bone area (NBA) and NBA/total defect area among all groups. No significant differences in any of the radiographic and histometric parameters could be observed between both concentrations of rhBMP-9. CONCLUSIONS: Within the limits of this study, it can be concluded that rhBMP-9/ACS-induced bone formation can be reached with as little as 1 µg/site in rat critical size calvarial defects. CLINICAL RELEVANCE: RhBMP-9 could be a potential therapeutic growth factor for future bone regenerative procedures.

Comparison of the effects of recombinant human bone morphogenetic protein-2 and -9 on bone formation in rat calvarial critical-size defects.

OBJECTIVES: Among bone morphogenetic protein (BMP) family members, BMP-2 and BMP-9 have demonstrated potent osteoinductive potential. However, in vivo differences in their potential for bone regeneration remain unclear. The present study aimed to compare the effects of recombinant human (rh) BMP-2 and rhBMP-9 on bone formation in rat calvarial critical-size defects (CSD). MATERIALS AND METHODS: Twenty-eight Wistar rats surgically received two calvarial defects bilaterally in each parietal bone. Defects (n = 56) were allocated into four groups: absorbable collagen sponge (ACS) alone, rhBMP-2 with ACS (rhBMP-2/ACS), rhBMP-9/ACS, or sham surgery (control), on the condition that the treatments of rhBMP-2/ACS and rhBMP-9/ACS, or the same treatments were not included in the same animal. Animals were sacrificed at 2 and 8 weeks post-surgery. The calvarial defects were analyzed for bone volume (BV) by micro-computed tomography and for percentages of defect closure (DC/DL), newly formed bone area (NBA/TA), bone marrow area (BMA/NBA), adipose tissue area (ATA/NBA), central bone height (CBH), and marginal bone height (MBH) by histomorphometric analysis. RESULTS: The BV in the rhBMP-2/ACS group (5.44 ± 3.65 mm3, n = 7) was greater than the other groups at 2 weeks post-surgery, and the rhBMP-2/ACS and rhBMP-9/ACS groups (18.17 ± 2.51 and 16.30 ± 2.46 mm3, n = 7, respectively) demonstrated significantly greater amounts of BV compared with the control and ACS groups (6.02 ± 2.90 and 9.30 ± 2.75 mm3, n = 7, respectively) at 8 weeks post-surgery. The rhBMP-2/ACS and rhBMP-9/ACS groups significantly induced new bone formation compared to the control and ACS groups at 8 weeks post-surgery. However, there were no statistically significant differences found between the rhBMP-2/ACS and rhBMP-9/ACS groups in any of the histomorphometric parameters. The ATA/NBA in the rhBMP-2/ACS group (9.24 ± 3.72%, n = 7) was the highest among the treatment groups at 8 weeks post-surgery. CONCLUSIONS: Within the limits of this study, it can be concluded that rhBMP-2/ACS induced a slight early increase in new bone formation at 2 weeks and that rhBMP-9/ACS provided comparable new bone formation to rhBMP-2/ACS with less adipose tissues after a healing period of 8 weeks in rat CSD. CLINICAL RELEVANCE: RhBMP-9/ACS treatment provided new bone formation with less adipose tissues compared with rhBMP-2/ACS.

The Association of Periodontopathic Bacteria Levels in Saliva and Tongue Coating with Oral Malodor in Periodontitis Patients.

PURPOSE: To elucidate the association oral malodor with the levels of periodontopathic bacteria in saliva and tongue coating of periodontitis patients with oral malodor. MATERIALS AND METHODS: In 25 periodontitis patients, the organoleptic test (OLT) was performed, the levels of volatile sulfur compounds (VSCs) were measured, tongue coating (TC) score was determined, and periodontal parameters and the proportions of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Tannerella forsythia and Prevotella intermedia in the saliva and the tongue coating were evaluated. RESULTS: The proportions of T. denticola and T. forsythia in the saliva of patients with an OLT score ≥ 2 were significantly higher than those with OLT scores < 2. The proportion of P. gingivalis in the saliva significantly correlated with periodontal parameters, whereas T. denticola and T. forsythia levels in the tongue coating correlated with VSC levels. However, the five periodontopathic bacteria were found in the tongue coating at levels approximately 12 times lower than in the saliva. CONCLUSIONS: The findings suggest that the levels of T. denticola and T. forsythia in the saliva of periodontitis patients correlate with oral malodor, and that the prevalence of P. gingivalis in the saliva is related to periodontitis. Periodontopathic bacteria in the tongue coating contribute minimally to oral malodor in periodontitis patients.